Thymol–Carvacrol (1:2) in Weaned Pigs: An In Silico–In Vitro–In Vivo Evaluation Linking Antibacterial Synergy, Microbiota Stabilization, and Performance Gains

Thymol–Carvacrol (1:2) in Weaned Pigs: An In Silico–In Vitro–In Vivo Evaluation Linking Antibacterial Synergy, Microbiota Stabilization, and Performance Gains | 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 Thymol–Carvacrol (1:2) in Weaned Pigs: An In Silico–In Vitro–In Vivo Evaluation Linking Antibacterial Synergy, Microbiota Stabilization, and Performance Gains Jiangtao Ao, Fangyan Yuan, Kai Wei, Xia Yang, Roujin Wang, Huanchun Chen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8095465/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 Phytogenic feed additives, owing to their antimicrobial and anti-inflammatory properties, are emerging as promising tools to mitigate weaning stress in piglets. This study evaluated the mechanisms and efficacy of thymol and carvacrol in weaned pigs. Consensus blind docking was used to predict and verify swine molecular targets of thymol/carvacrol; Checkerboard assays quantified antibacterial effects of each compound alone and in combination against E. coli, Salmonella, S. flexneri , and Staphylococcus ; finally, a 28-day feeding trial was conducted with 100 weaned piglets (5 pens/group × 10 pigs/pen): the control received a basal diet, and the treatment diet was supplemented with 25 mg/kg thymol and 50 mg/kg carvacrol. Results showed that docking prioritized MAOA, MAOB, PTGS1, PTGS2, ESR1, and VDR as candidate targets. In vitro, the thymol–carvacrol combination was additive overall and synergistic against S. flexneri (1:2; ΣFICI = 0.38). In vivo, supplementation increased final BW at d 28 ( P < 0.01) and ADFI and ADG during d 15–28 and d 1–28 ( P < 0.05), with a trend toward lower F/G over the whole period ( P = 0.09) and reduced early post-weaning diarrhea ( P < 0.01). While cecal α-diversity was unchanged, β-diversity dispersion decreased by d 28 ( P < 0.05). Serum DAO and ET decreased at d 28 ( P < 0.05), and TNF-α was lower at d 14 ( P < 0.05). Collectively, a 1:2 thymol:carvacrol blend appears to stabilize the intestinal microbiota and moderately modulate barrier and inflammatory responses, thereby lowering early diarrhea and improving late-phase and overall growth, supporting its feasibility as an antibiotic-free strategy for nursery pigs. weaned piglets growth performance systemic inflammation carvacrol thymol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction During the weaning transition, piglets face concurrent social, dietary, and environmental stressors that hinder intestinal barrier maturation (Yang et al. 2016 ) and increase the risk of post-weaning diarrhea and growth checks, leading to substantial economic losses (Moeser et al. 2017 ; Rhouma et al. 2017 ). With in-feed antibiotics and pharmacological zinc increasingly restricted, there is an urgent need for effective antibiotic-free strategies (WHO, 2017; EEA, 2024). Phytogenic feed additives have emerged as promising candidates owing to beneficial effects on growth and digestion, together with antimicrobial and antioxidant activities (Canibe et al. 2022 ; Rebucci et al. 2022 ; Shao et al. 2023 ). Thymol and carvacrol—widely used phenolic monoterpenes—exhibit clear in vitro antibacterial activity (Wei et al. 2017 ). Their delocalized phenolic systems and hydrophobicity disrupt bacterial membranes and interfere with biofilm formation, thereby inhibiting proliferation (Didry et al. 1994 ; Ultee et al. 1999 ; Cid-Pérez et al. 2024 ). Nevertheless, recommended inclusion levels for nursery pigs vary widely (approximately 13.50–55.00 mg/kg for thymol and 12.50–60.00 mg/kg for carvacrol), and reported outcomes on growth, gut health, microbiota, and inflammatory markers remain heterogeneous (Guarda et al. 2011 ; Wei et al. 2017 ). Such variability likely reflects differences in dose, ratio, formulation, and challenge context; moreover, an integrated, systems-level link along the “microbiota–barrier–inflammation–growth” axis is still lacking. To address these gaps, we implemented an integrated in silico–in vitro–in vivo workflow: swine targets relevant to weaning-stage inflammation/barrier homeostasis were first nominated via target prediction and consensus blind docking; antibacterial activity and synergy against representative pathogens were quantified by checkerboard assays; and a 28-day feeding trial in weaned piglets then evaluated a 1:2 (thymol:carvacrol) blend for effects on growth and health outcomes, with systematic assessment of cecal microbiota, intestinal morphology, and serum markers of barrier integrity and inflammation/immunity. Our aim was to define a rational ratio and inclusion level and to delineate mechanisms whereby thymol–carvacrol improves post-weaning performance through the microbiota–barrier–inflammation axis, thereby informing precision use in production settings. Material and methods In silico target prediction, pathway annotation, and consensus blind docking Protein targets of thymol and carvacrol were predicted from public resources (ChEMBL—Target/Mechanism; OpenTargets) and mapped to Sus scrofa orthologs via UniProt. Pathway annotations (not enrichment) were standardized to KEGG identifiers to support hypothesis-driven prioritization. Candidates were retained if they (i) were biologically relevant to weaning-stage intestinal inflammation and epithelial-barrier homeostasis, (ii) met an affinity threshold for both ligands (AutoDock Vina score ≤ − 6.0 kcal·mol⁻¹), and (iii) had a swine ortholog with a stable docking pocket. For the six selected targets (MAOA, MAOB, ESR1, PTGS1, PTGS2, VDR), protein structures were taken from Sus scrofa AlphaFold models (chain A); non-protein entities were removed, hydrogens were added, and protonation was set to pH 7.4. Thymol and carvacrol were standardized (tautomer/canonicalization) and protonated at pH 7.4. A consensus blind-docking workflow (CoBDock) was employed that detects candidate cavities using multiple detectors, integrates cavity and preliminary docking scores by machine learning, and returns a prioritized pocket; for each target, the prioritized site was binding_site_1. Pocket stability was evaluated in a ligand-agnostic manner by 100 sampling runs, yielding pocket-score mean/median/SD and defining the final grid center/size (Å). Ligand-specific docking was performed with AutoDock Vina (v1.2.7) using identical grids for thymol and carvacrol per target, and the best Vina score was recorded in kcal·mol⁻¹ for each target–ligand pair. Top poses were inspected in PyMOL (v3.1.6.1) to annotate key non-covalent interactions (Ugurlu et al. 2024 ). Antimicrobial susceptibility and synergy testing Carvacrol (≥ 97.0% purity) and thymol (≥ 99.0% purity) were obtained from Aldrich. Sterile stock solutions were prepared at 50% (v/v) in ethanol (Merck Millipore; endotoxin ≤ 0.1 EU/mL), aliquoted, protected from light, and stored at − 20°C. Test organisms were Escherichia coli (E. coli) C83715, Salmonella ATCC 14028, Shigella flexneri ( S. flexneri ) ATCC 12022, and Staphylococcus ST188 (weaned-pig isolates held in a private collection). Minimum inhibitory concentration (MIC) was determined in tryptic soy broth (TSB) by the broth microdilution micromethod in sterile polystyrene flat-bottom 96-well plates following Clinical and laboratory standards institute recommendations. Inocula were adjusted to ~ 5 × 10⁵ CFU/mL in each well (final volume 200 µL) and plates were incubated 24 h at 37°C under static conditions. MIC was defined as the lowest concentration with no visible growth and ΔOD₆₀₀ ≤ 0.05 versus the medium control. Vehicle controls were included (TSB and TSB containing ethanol at the highest well concentration); the final ethanol content in all assay wells was matched and kept ≤ 1% (v/v). Synergy was evaluated by the checkerboard method using two-fold dilution matrices spanning the ranges that bracketed single-agent MICs (typically 0.0625–512.00 µg/mL). Fractional inhibitory concentrations were calculated as FIC_T = MIC_T (combined) / MIC_T (alone) and FIC_C = MIC_C (combined) / MIC_C (alone), with ΣFICI = FIC_T + FIC_C; interaction was interpreted as synergy (≤ 0.50), additive/partial synergy (0.50–1.00), indifference (1.00–4.00), or antagonism (> 4.00). Each condition was tested in four independent replicates and the central tendency (median) was used for reporting and FICI computation. Thymol–carvacrol supplementation in weaned piglets Experimental animals and experimental design The trial was conducted on a commercial pig farm in Hubei Province, China (September–October 2022). A total of 100 weaned piglets (Duroc × Landrace × Yorkshire) were weaned at 26 d of age and randomly allocated to two treatments with 5 pens per treatment and 10 piglets per pen (n = 5 pens per group). Treatments were: Control (CON), basal diet formulated to meet or exceed NRC (2012) requirements for weanling piglets; and TC, basal diet plus a thymol-carvacrol blend (Xilaikang 150®, Hubei Refine Biotechnology Co., Ltd.) supplying 25 mg thymol/kg diet and 50 mg carvacrol/kg diet (thymol:carvacrol = 1:2). A two-phase feeding program was used: pre-starter (d 1–14) and starter (d 15–28); ingredient composition and calculated nutrients are provided in Table 1 . Piglets were housed in semi-enclosed concrete-floor pens (~ 10 m² per pen; 10 pigs per pen) with natural/mechanical ventilation. Feed and water were provided ad libitum. Routine health management and biosecurity procedures of the farm were followed throughout the study. Table 1 Ingredients and nutrient composition of diet Ingredients 1-14d 15-28d Calculated nutrients 1-14d 15-28d Maize (%) 28.57 47.93 Crude Protein (%) 18.50 17.98 Maize Extruded (%) 25.00 15.00 Sodium (mg/kg) 0.31 0.22 Low protein whey powder (%) 10.00 4.00 Calcium (mg/kg) 0.60 0.65 Soybean Meal Fermented (%) 10.80 5.50 Total P (mg/kg) 0.60 0.65 Soybean Meal Dehulled (%) 4.00 12.00 Digestible P (mg/kg) 0.44 0.46 Soybean Extruded (%) 5.00 5.00 Copper (mg/kg) 121.00 121.00 Peruvian Fish Meal (%) 5.00 2.00 Zinc (mg/kg) 110.00 110.00 Cane Sugar (%) 5.00 2.00 Digestive Energy (MJ/kg) 15.06 14.61 Homogenizing Oil Powder (%) 2.00 1.00 Net Energy (MJ/kg) 10.74 10.53 Soya Oil (%) 0.50 0.50 Digestible Lys (%) 1.35 1.25 Calcium Formate (%) 0.20 0.69 Digestible Met (%) 0.58 0.55 Monocalcium Phosphate (%) 0.67 1.31 Digestible Met + Cys (%) 0.81 0.79 Salt (%) 0.40 0.40 Digestible Thr (%) 0.92 0.83 Sodium Glutamate (%) 0.15 0.15 Digestible Trp (%) 0.28 0.26 Choline Chloride 50% (%) 0.10 0.08 Digestible Ile (%) 0.72 0.66 98% L-Lysine HCL (%) 0.59 0.57 Digestible Val (%) 0.93 0.86 DL-Methionine (%) 0.30 0.30 L-Threonine (%) 0.35 0.29 L-Tryptophan (%) 0.10 0.09 Isoleucine 90% (%) 0.05 0.02 Valine 98% (%) 0.20 0.16 BHT 60% (%) 0.02 0.02 Premix* (%) 1.00 1.00 Total (%) 100.00 100.00 * The premix provided per kg of diets: Vitamin A, 11972 IU; Vitamin D3, 2535IU; Vitamin E, 200IU; Vitamin K3, 3.4mg; Vitamin B1, 2.4mg; Vitamin B2,6.70mg; Vitamin B6, 3.80mg; Vitamin B12, 0.0225mg; Niacin, 40mg; Pantothenic,13.30mg; Folic acid, 1.06mg; Biotin, 0.122mg; Fe, 150mg; Cu, 115mg; Mn, 36mg;Zn, 85mg; I, 1.57mg; Se, 0.3mg; Sweetener, 200g; Flavor, 750g; Phytase, 5000FTU Growth performance and diarrhea assessment Body weight (BW) was recorded on d 1, 14, and 28. For each pen, feed offered and refusals were recorded daily to compute for d 1–14, d 15–28, and d 1–28. was calculated as (BW_end − BW_start)/days for each phase, and feed-to-gain ratio (F/G) as total feed intake/weight gain for the corresponding phase. Pen was considered the experimental unit for performance analyses. Mortality and clinical signs were monitored daily. Body weight (BW) was recorded on d 1, 15, and 29 to derive average daily gain (ADG) for d 1–14, d 15–28, and d 1–28. For each pen, the total feed offered was recorded and feed refusals were weighed daily at 17:30; these records were used to calculate average daily feed intake (ADFI) for the same three periods. ADG was computed as (BW end −BW start )/days, and the feed conversion ratio (F/G, feed-to-gain) as total feed intake/weight gain for each phase. The pen served as the experimental unit for performance analyses. Mortality and clinical signs were monitored daily. During the first 28 days post-weaning, fecal consistency of all piglets was assessed twice daily (08:00 and 16:00) using a 0–3 scale: 0 = formed, log-shaped; 1 = soft, poorly formed; 2 = loose/pasty; 3 = watery/jet-like. A score ≥ 2 was considered diarrhea; to reduce misclassification, diarrhea was confirmed when two consecutive assessments were ≥ 2 or any single assessment was 3. The diarrhea proportion (%) for each group was calculated as (number of diarrhea piglets / total number of piglets) × 100. Calculations were performed for d 1–14, d 15–28, and d 1–28. Cecal microbiome sequencing and analysis On d 15 (14 d post-weaning) and d 29 (28 d post-weaning), four piglets per group were randomly selected and humanely euthanized by intravenous chlorpromazine hydrochloride (2 mg/kg BW; Shanghai Hefeng Pharmaceutical Co., Ltd., Shanghai, China). Following Peng et al. (2019), cecal chyme was collected aseptically (~ 200 mg per aliquot), transferred into cryotubes, snap-frozen in liquid nitrogen, and stored at − 80°C. From each pig, ~ 200 mg chyme was used for DNA extraction (QIAamp Fast DNA Stool Mini Kit, Qiagen, Germany). The 16S rRNA V3–V4 region was amplified with 341F/806R and sequenced on an Illumina MiSeq platform (Shanghai Penosen Biotechnology Co., Ltd., Shanghai, China). Reads were processed in QIIME 2 (v2019.4) and denoised with DADA2 to generate ASVs. After rarefaction to a unified depth, α-diversity indices (Chao1, Shannon, Simpson, Pielou) and β-diversity (Bray–Curtis, PCoA) were computed. PERMANOVA was used to test between-group differences in community structure, with PERMDISP assessing dispersion. LEfSe (LDA ≥ 2.0, Kruskal–Wallis P < 0.05) identified discriminative taxa (Segata et al., 2011 ). Intestinal morphology HE-stained small-intestinal sections were prepared by routine histology. Images were acquired under a light microscope at 40× objective, keeping illumination constant and filling the field of view with tissue. Morphometry was performed in Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) after calibrating to the 40× scale bar in the lower-right corner of each image. For each slide (one per piglet), five well-oriented, intact villi with their corresponding crypts were chosen. Villus height was measured from the tip of the villus to the villus–crypt junction; crypt depth from the base of the crypt to the villus–crypt junction. Values (mm) were averaged to obtain one villus height and one crypt depth per slide, and the villus-to-crypt ratio was calculated accordingly. Measurements were performed blinded to treatment. Assessment of serum markers of barrier function and mucosal immunity On days 1, 15, and 29 of the trial at 06:30, one medium-weight piglet was randomly selected from each of the five pens per group (total n = 5 piglets per group) for fasted blood collection. Twenty milliliters of blood were drawn from the anterior vena cava into non-heparinized vacuum tubes, centrifuged at 3000 × g for 10 min at 4°C, and the supernatant (serum) was collected for analysis. According to the manufacturers’ manuals, serum diamine oxidase (DAO; colorimetric), endotoxin (ET; LAL method), and secretory IgA (sIgA; ELISA) were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All samples were analyzed in duplicate with blanks, standards, and internal controls; standard curves (four-parameter or linear) and concentration calculations followed the kit instructions. Serum inflammatory cytokine assays Serum samples were the same as those used for the serum markers of barrier function and mucosal immunity. Concentrations of IL-1β, IL-6, IL-8, and TNF-α were measured with sandwich ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. Each sample was analyzed in duplicate, with blank, negative/positive controls, and a standard curve fitted by four-parameter logistic (4-PL) regression (R² ≥ 0.99). Results were expressed as ng/L. Intra- and inter-assay CVs were maintained within the limits specified by the manufacturer (typically < 10%). Statistical analysis Data were analyzed using SPSS v27.0. The pen was the experimental unit for growth performance (n = 5 pens/group), whereas individual pigs were the unit for serum variables and intestinal morphology. Normality (Shapiro–Wilk) and homogeneity of variance (Levene’s test) were examined, with transformations applied when necessary. At each time point, growth traits and serum variables were compared using independent-samples t-tests (or Mann–Whitney U when normality was violated), and diarrhea incidence was compared using the chi-square test. Results are expressed as mean ± SD for growth performance and mean ± SEM for serum variables; P ≤ 0.05 was considered significant and P ≤ 0.01 highly significant. Results Predicted targets and consensus blind-docking outcomes for thymol and carvacrol We first predicted the targets of thymol and carvacrol and mapped the candidates to pathways using database annotations (ChEMBL, OpenTargets, KEGG/Reactome). Based on predefined criteria - (i) high relevance to weaning-stage intestinal inflammation and barrier homeostasis (e.g., tryptophan metabolism, arachidonic-acid metabolism, nuclear-receptor signaling); (ii) predicted affinity to both ligands meeting a threshold (Score ≤ − 6.0 kcal·mol-1); and (iii) availability of a swine ortholog together with a stable docking pocket—we prioritized six swine targets (MAOA, MAOB, ESR1, PTGS1, PTGS2, and VDR) for consensus blind docking (Table S1 ). CoBDock consistently identified binding_site_1 as the prioritized pocket for all six targets (Table S2). We then performed ligand-specific docking of thymol and carvacrol in this pocket and reported the best scores in Table 2 . Overall, thymol scored − 6.9 on MAOA/MAOB, − 6.8 on PTGS1, and − 6.0 to − 6.4 on ESR1/PTGS2/VDR. Carvacrol achieved the lowest score on MAOA (− 7.6) and showed moderate affinities on MAOB/ESR1/VDR (− 6.6 to − 7.4). An overview of the docking of thymol and carvacrol on selected key swine targets is shown in Fig. 1 . Together with pathway annotations, these results suggest that thymol and carvacrol may act along the tryptophan/monoamine metabolism axis (MAO A/B), the arachidonic-acid/prostaglandin axis (PTGS1/2), and nuclear-receptor signaling (ESR1, VDR), thereby indirectly modulating inflammation, oxidative stress, and epithelial-barrier/immune functions that shape the physiological status of weaned piglets. Table 2 Core targets and molecular docking results of carvacrol and thymol UniProt ID (pig) Gene symbol Protein name ChEMBL target ID Structure Thymol docking score (kcal·mol − ¹) Carvacrol docking score (kcal·mol − ¹) Q6Q2J0 MAOA Amine oxidase A CHEMBL1951 AlphaFold Q6Q2J0_A -6.9 -7.6 Q6PLK3 MAOB Amine oxidase B CHEMBL2039 AlphaFold Q6PLK3_A -6.9 -7.4 Q29040 ESR1 Estrogen receptor CHEMBL206 AlphaFold Q29040_A -6.4 -6.8 C9EF58 PTGS1 Prostaglandin G/H synthase 1 CHEMBL221 AlphaFold C9EF58_A -6.8 -6.3 Q8SPR3 PTGS2 Prostaglandin G/H synthase 2 CHEMBL230 AlphaFold Q8SPR3_A -6.4 -6.3 A3RGC1 VDR Vitamin D3 receptor CHEMBL1977 AlphaFold A3RGC1_A -6.0 -6.6 *Docking was performed in the CoBDock-identified prioritized pocket binding_site_1, using the same grid settings for thymol and carvacrol per target. Protein structures are Sus scrofa AlphaFold models (chain A) unless otherwise noted. Score is the best AutoDock Vina docking score reported in kcal·mol − ¹; more negative indicates higher predicted affinity and does not represent experimental ΔG. Representative docking pose of thymol or carvacrol in target gene within the prioritized pocket binding_site_1. The protein backbone is shown as a teal cartoon; the ligand is rendered in orange sticks. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. Blue dashed lines denote hydrogen bonds and green dashed lines indicate hydrophobic/π–π interactions according to the viewer’s default legend. The inset reports the best AutoDock Vina score (kcal·mol - ¹; more negative indicates higher predicted affinity) Antibacterial activity and synergy of thymol–carvacrol The checkerboard assay showed that, when used alone, both compounds had identical MICs against the tested bacteria (Gram-negative E. coli, Salmonella , and S. flexneri : 250 µg/mL; Gram-positive Staphylococcus : 500 µg/mL). In combination, MICs decreased for all strains, with interaction types varying by species. For E. coli and Salmonella , the MICs of both compounds were halved, yielding ΣFICI = 1.00 (additive). For S. flexneri , the combination produced the greatest reduction compared with monotherapy (thymol decreased 8-fold; carvacrol 4-fold), with ΣFICI = 0.38 (synergy). For Staphylococcus , MICs decreased to 125 µg/mL (thymol) and 250 µg/mL (carvacrol), with ΣFICI = 0.75 (additive/partial synergy, Table 3 ). Overall, these data suggest that the 1:2 thymol:carvacrol ratio can confer at least additive—and for certain pathogens (e.g., S. flexneri ) synergistic—antibacterial effects. Table 3 MICs of carvacrol and thymol alone and in combination against selected bacteria, and FICI-based interaction Bacterium MIC (µg/mL) FIC_T FIC_C ΣFICI Interaction Thymol (alone) Carvacrol (alone) Thymol (combined) Carvacrol (combined) E.coli 250.00 250.00 125.00 125.00 0.50 0.50 1.00 additive Salmonella 250.00 250.00 125.00 125.00 0.50 0.50 1.00 additive S.flexneri 250.00 250.00 31.25 62.50 0.125 0.25 0.38 synergy Staphylococcus 500.00 500.00 125.00 250.00 0.25 0.50 0.75 additive *MIC, minimal inhibitory concentration; FICI, fractional inhibitory concentration index. FIC_T = (MIC of thymol in combination)/(MIC of thymol alone); FIC_C = (MIC of carvacrol in combination)/(MIC of carvacrol alone); ΣFICI = FIC_T + FIC_C. The interaction was interpreted as synergy (≤ 0.50), additive/partial synergy (0.50–1.00), indifference (1.00–4.00), or antagonism (> 4.00). Each condition was tested in four independent replicates and the central tendency (median) was used for reporting and FICI computation. Thymol-carvacrol mixture improves late and overall growth in weaned piglets We formulated a thymol-carvacrol mixture at a 1:2 ratio (thymol 25 mg/kg diet, carvacrol 50 mg/kg diet) and supplemented it to weaned piglets to evaluate effects on growth performance. As shown in Table 4 , compared with the basal diet, the mixture improved late-phase and overall growth. No significant differences were detected between groups at d 1 or d 14 in BW, ADFI, ADG, or F/G ( P > 0.05). However, relative to controls, the TC group exhibited a higher final body weight at d 28 ( P < 0.01); during d 15–28 and d 1–28, ADFI ( P < 0.05) and ADG ( P < 0.05) increased, and the overall F/G showed a decreasing trend ( P = 0.09). In addition, compared with the control, piglets in the TC group showed a significantly lower diarrhea incidence during d 1–14 and d 1–28 post-weaning ( P < 0.01). Taken together, these results indicate that dietary supplementation with the thymol-carvacrol mixture reduces early post-weaning diarrhea and enhances feed intake and growth rate in the later phase, thereby increasing final body weight at d 28. Table 4 Growth performance of weaned piglets fed a basal diet or a carvacrol-thymol mixture Items Controp group TC group P -value BW (kg) 1 d 6.26 ± 0.20 6.26 ± 0.15 0.97 14 d 9.03 ± 0.12 9.10 ± 0.10 0.30 28 d 13.77 ± 0.20 B 14.48 ± 0.30 A < 0.01 ADFI (g/d) 1–14 d 260.26 ± 14.92 263.21 ± 21.45 0.81 15–28 d 530.57 ± 34.68 b 587.93 ± 22.80 a < 0.05 1–28 d 395.42 ± 19.18 b 425.57 ± 17.74 a < 0.05 ADG (g/d) 1–14 d 198.14 ± 9.74 203.14 ± 9.84 0.44 15–28 d 338.29 ± 18.60 B 383.86 ± 22.25 A < 0.01 1–28 d 268.21 ± 11.76 b 293.50 ± 13.77 a < 0.05 F/G (g/g) 1–14 d 1.31 ± 0.04 1.30 ± 0.06 0.60 15–28 d 1.57 ± 0.04 1.53 ± 0.05 0.24 1–28 d 1.47 ± 0.03 1.45 ± 0.01 0.09 Diarrhea incidence (%) 1–14 d 5.57 2.57 < 0.01 15–28 d 2.86 1.86 0.29 1–28 d 4.21 2.21 < 0.01 *Control group: basal diet; TC: basal diet plus thymol-carvacrol mixture (1:2), day ranges refer to post-weaning periods. BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; F/G, feed conversion ratio, feed-to-gain ratio. Values are means ± SD (n = 5 pens per group). a, b values in rows with different letters differ significantly, P ≤ 0.05; A, B as above for P ≤ 0.01. Thymol-carvacrol diet remodels the cecal microbiota of weaned piglets To examine whether the growth benefits of the TC diet were associated with gut microbiota, 16S rRNA sequencing was performed on cecal chyme from weaned piglets. Across time points and diets, α-diversity showed no differences on any index (Chao1, Shannon, Simpson, Pielou), indicating limited effects of diet on overall richness and evenness (Fig. 2 A-D). In terms of β-diversity, PCoA revealed clear between-diet separation; at d 14 the TC group showed a trend toward lower within-group Bray–Curtis pairwise distances than the control ( P = 0.06), and at d 28 this reduction was significant ( P < 0.01, Fig. 2 E), suggesting a time-dependent decrease in dispersion and a more stable community structure under TC group. At the phylum level, communities were dominated by Firmicutes and Bacteroidetes , while their relative abundances shifted with time and diet (Fig. 2 F). At the genus level, Lactobacillus , Prevotella , and other dominant genera prevailed, with diet- and time-dependent adjustments in abundance (Fig. 2 G). LEfSe identified diet-specific biomarkers (LDA ≥ 2): At d14, the TC group was significantly enriched for the family BS11 ( Bacteroidetes ) and for the lineages Spirochaetes , Treponema, and Pseudomonadales , whereas the control group was enriched for Blautia and Faecalibacterium (Fig. 2 H). By d28, the TC group remained enriched for the Pseudomonas lineage, while the control group was enriched for Mogibacterium (Fig. 2 I). Taken together, the TC diet progressively reshaped and stabilized the cecal community without altering overall α-diversity, providing a microbiological basis for improved nutrient utilization and mucosal homeostasis. (A-D) α-diversity indices: Chao1, Shannon, Simpson, and Pielou’s evenness index of piglets in CON and CP groups at 14 and 28 days. Bars show mean ± SEM per group; (E) PCoA based on Bray-Curtis distances illustrating community structure at d 14 and d 28 (left). The bar plot (right) shows mean pairwise Bray-Curtis distance between CON and TC at each time point; Taxonomic composition at the phylum level (F, stacked relative abundances) and the genus level (G, top taxa shown); LEfSe biomarkers at d 14 (H) and d 28 (I). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values are means ± SEM (n = 3–4 piglets per group). * differ significantly, P ≤ 0.05; ** as above for P ≤ 0.01. Thymol-carvacrol diet maintains small-intestinal mucosal morphology HE staining showed intact small-intestinal mucosa in both groups at d 14 and d 28, with orderly villi and well-defined crypts. Quantitative analysis indicated no significant differences in villus height or crypt depth between groups at either time point ( P > 0.05, Fig. 3 ). However, at d 28 the TC group exhibited numerically higher villus height and villus-to-crypt ratio than the control. Assessment of representative serum markers of intestinal barrier and mucosal immunity showed that at 28 d post-weaning, the TC group exhibited significantly lower serum DAO ( P < 0.05, Fig. 4 A) and ET levels ( P 0.05, Figure. 4C). The results suggesting that the thymol–carvacrol diet may promote epithelial renewal and villus remodeling during the later post-weaning period. Left: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 µm. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean ± SEM; n = 4 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Left: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 µm. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean ± SEM; n = 4–5 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Thymol-carvacrol diet mitigates early post-weaning systemic inflammation As shown, serum IL-1β, IL-6, and IL-8 did not differ between groups at either d 14 or d 28 ( P > 0.05, Fig. 5 A, B and D). However, at d 14 post-weaning, TNF-α was significantly lower in the TC group than in the control group ( P < 0.05, Fig. 5 C). These findings indicate that the thymol–carvacrol diet exerts a modest anti-inflammatory effect by attenuating the early peripheral TNF-α response, while having minimal impact on overall IL-1β, IL-6, and IL-8 levels. CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values were showed as mean ± SEM (n = 4–5 per group). * indicated that the difference between CON group and TC group was statistically significant ( P < 0.05). Discission This study used an integrated in silico–in vitro–in vivo approach to evaluate a thymol–carvacrol blend (1:2). In vitro, the combination showed at least additive antibacterial activity against E. coli , Salmonella , S. flexneri , and Staphylococcus , with clear synergy for S. flexneri . In vivo, weaned piglets fed the blend exhibited higher 28 d final BW and improved ADG and ADFI in the late and overall phases, along with a reduction in early diarrhea. At the microecological level, α-diversity was unchanged, whereas β-diversity dispersion declined over time, indicating a more stabilized community. Concurrent reductions in serum DAO and endotoxin and a decrease in TNF-α suggest alleviated epithelial barrier disruption and systemic inflammatory burden. In the consensus blind docking (CoBDock) analysis, the thymol–carvacrol blend showed high predicted affinity for the swine targets MAOA, MAOB, PTGS1, PTGS2, ESR1, and VDR, which were mapped to three key signaling axes: the tryptophan/monoamine metabolism axis (MAO A/B), the arachidonic-acid/prostaglandin axis (PTGS1/2), and the nuclear-receptor axis (ESR1, VDR). Prior work indicates that MAO A/B catalyze monoamine oxidation and generate H₂O₂, thereby amplifying oxidative-stress and inflammatory signaling while perturbing tryptophan/monoamine balance with consequences for gut immunity and barrier function (Bortolato et al. 2008 ; Agus et al. 2018 ). PTGS1/2 govern prostaglandin biosynthesis, integrating acute and chronic inflammatory-mediator networks and shaping the epithelial microenvironment, closely linked to mucosal inflammatory phenotypes (Ricciotti and FitzGerald 2011 ). ESR1/VDR, as nuclear receptors, regulate genes involved in immunity and barrier integrity, sustaining mucosal integrity, antimicrobial-peptide expression, and host control of the microbiota (Kong et al. 2008 ; Ooi et al. 2013 ; Paterni et al. 2014 ; Kovats 2015 ). These findings suggest that thymol and carvacrol, by anchoring to swine molecular targets, modulate three interconnected axes that collectively link oxidative stress, inflammation, and mucosal homeostasis during the weaning period. The in vitro antibacterial assays further showed that the thymol-carvacrol combination produced additive effects overall against both Gram-negative and Gram-positive bacteria and synergy against S. flexneri . This is consistent with the classical mechanism whereby phenolic compounds inhibit bacteria via membrane permeabilization and interference with biofilm formation and energy metabolism (Didry et al. 1994 ; Ultee et al. 1999 ), and aligns with more recent evidence that thymol-carvacrol combinations can exhibit synergy against enteric pathogens (Cid-Pérez et al. 2024 ). Notably, the magnitude of synergy is species/strain dependent, which helps explain variability across studies (Michiels et al. 2010 ; Wei et al. 2017 ). Taken together, these data support a model in which thymol and carvacrol improve post-weaning physiology in piglets through antibacterial activity coupled with attenuation of inflammatory pathways. In line with recent work on essential oils and phytogenic additives in weaned piglets, we observed improvements in late-phase and overall ADG and ADFI together with a reduction in early diarrhea, despite the absence of a strong challenge—an outcome that is broadly consistent with reports of performance and gut-health benefits under commercial conditions (Shao et al. 2023 ; Wang et al. 2022 ). Notably, our data connect these phenotypes to a time-ordered cascade spanning the microbiota–barrier–inflammation axis: a progressive stabilization of β-diversity was accompanied by lower serum DAO and endotoxin and an early attenuation of TNF-α, suggesting that early-phase (d 1–14) dampening of inflammatory expenditure and barrier leakage is followed by late-phase (d 15–28) gains in feed intake and nutrient utilization that translate into higher weight gain. The heterogeneity seen across prior studies likely reflects differences in dose, thymol and carvacrol ratio, formulation (e.g., free vs. microencapsulated), and challenge context (Michiels et al. 2010 ; Canibe et al. 2022 ). By applying an evidence-driven 1:2 (thymol:carvacrol) ratio within a single in silico–in vitro–in vivo framework, the present work helps reconcile some of this variability and provides a mechanistic bridge from microbial community dynamics to barrier and cytokine readouts. The lack of statistical differences in villus height, crypt depth, and villus-to-crypt ratio, alongside numerical improvements at d 28, is compatible with a scenario in which the blend maintains mucosal architecture under non-severe challenge rather than inducing overt remodeling (Rebucci et al. 2022 ; Shao et al. 2023 ). Taken together, the 1:2 thymol:carvacrol blend appears to be a feasible antibiotic-free strategy for the weaning period: it improves performance primarily by stabilizing the microbiota and modulating the barrier/inflammation axis in a moderate manner, rather than by bluntly suppressing the community. Future work should adopt factorial designs (dose × ratio ×formulation) and challenge models, and integrate metabolomics with mucosal transcriptomics and tight-junction protein assays to strengthen the causal chain from docking predictions-in vivo chemical evidence-phenotypes. In parallel, attention should be paid to the cost–benefit and robustness of thymol and carvacrol under different feeding systems and health-risk scenarios (Canibe et al. 2022 ; EEA 2024). These efforts will advance the precision application of thymol and carvacrol in large-scale production while clarifying its mechanisms. Conclusion Supplementing weaned pig diets with 25 mg/kg thymol and 50 mg/kg carvacrol stabilizes the intestinal microbiota, lowers early post-weaning serum inflammatory cytokines, enhances immune function, reduces early post-weaning diarrhea, and improves late-phase and overall growth performance. Declarations Ackowledgments We gratefully acknowledge Hailin Pig Farm, Huanggang, Hubei Province, China, for providing the experimental facilities and on-farm support. We also thank Chongqing Kangzhou Big Data (Group) Co., Ltd., for performing the molecular docking services of thymol and carvacrol. Funding This work was supported by the China Agriculture Research System of the Ministry of Finance and the Ministry of Agriculture and Rural Affairs (CARS-35), the Hubei Provincial Special Project for Central Government-Guided Local Science and Technology Development (2024CSA070), and the Hubei Provincial Key Research and Development Program (2023BBB069). Competing interests The authors declare no competing interests. Author contributions Conceptualization, Supervision and Funding acquisition: Weicheng Bei and Huanchun Chen; Investigation, Methodology and Data curation: Jiangtao Ao, Fangyan Yuan, Kai Wei, and Xia Yang; Formal analysis and Visualization: Jiangtao Ao, Kai Wei, Xia Yang, and Ruojin Wang; Project administration: Ruojin Wang and Weicheng Bei; Writing-review and editing: Jiangtao Ao and Weicheng Bei. All authors read and approved the final manuscript. Data availability Raw 16S rRNA sequencing reads are hosted on the Paisen Gene Cloud platform under project ID MD2022120718050WT4.Other data available on reasonable request. Ethics approval All animal husbandry, euthanasia, experimental procedures, and biosecurity measures complied with the protocols approved by the Institutional Animal Care and Use Committee (IACUC 202401140009) of the College of Animal Science and Technology, Huazhong Agricultural University. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish The manuscript does not contain any individual person’s data. Supplementary material List supplementary tables S1 and S2. References Agus A, Planchais J, Sokol H (2018) Gut microbiota regulation of tryptophan metabolism in health and disease. Cell Host Microbe 23:716–724. Bortolato M, Chen K, Shih JC (2008) Monoamine oxidase inactivation: from pathophysiology to therapeutics. Adv Drug Deliv Rev 60:1527–1533. Canibe N, Højberg O, Kongsted H, Vodolazska D, Lauridsen C, Nielsen TS, Schönherz AA (2022) Review on preventive measures to reduce post-weaning diarrhoea in piglets. Animals 12:2585. Cid-Pérez TS, Munguía-Pérez R, Nevárez-Moorillón GV, Ochoa-Velasco CE, Navarro-Cruz AR, Ávila-Sosa R (2024) Carvacrol and thymol effect in vapor phase on Escherichia coli and Salmonella Typhimurium inoculated in fresh salad. Heliyon 10:e29638. 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Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12:R60. Shao Y, Peng Q, Wu Y, Peng C, Wang S, Zou L, et al. (2023) The effect of an essential oil blend on growth performance, intestinal health and microbiota in early-weaned piglets. Nutrients 15:450. Ultee A, Kets EPW, Smid EJ (1999) Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus. Appl Environ Microbiol 65:4606–4610. Ugurlu SY, McDonald D, Lei H, Jones AM, Li S, Tong HY, Butler MS, He S (2024) Cobdock: an accurate and practical machine learning-based consensus blind docking method. J Cheminform 16:5. Wang Y, Chen F, Gao W, Yang H, Lu S, Zhang C, et al. (2022) Effects of different amino acid levels and a carvacrol–thymol blend on growth performance and intestinal health of weaned pigs. J Anim Sci Biotechnol 13:67. Wei HK, Xue HX, Zhou ZX, Peng J (2017) A carvacrol–thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger RNA levels of tight junction proteins in jejunal mucosa of weaning piglets. Animal 11:193–201. World Health Organization (WHO) (2017) WHO guidelines on use of medically important antimicrobials in food-producing animals. WHO, Geneva. Yang H, Xiong X, Wang X, Li T, Yin Y (2016) Effects of weaning on intestinal crypt epithelial cells in piglets. Sci Rep 6:36939. Supplementary Files Supplementarymaterial.docx 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-8095465","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":563030562,"identity":"85dcf1c9-5f35-48e2-bb15-aecdc0fcd0e6","order_by":0,"name":"Jiangtao Ao","email":"","orcid":"","institution":"Huazhong Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Jiangtao","middleName":"","lastName":"Ao","suffix":""},{"id":563030563,"identity":"559d4ff5-f09c-4a6a-8984-c03a17cfe84e","order_by":1,"name":"Fangyan Yuan","email":"","orcid":"","institution":"Huazhong Agriculture 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2","display":"","copyAsset":false,"role":"figure","size":658255,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dietary thymol-carvacrol on cecal chyme microbiota of weaned piglets at d 14 and d 28\u003c/p\u003e\n\u003cp\u003e(A-D) α-diversity indices: Chao1, Shannon, Simpson, and Pielou’s evenness index of piglets in CON and CP groups at 14 and 28 days. Bars show mean ± SEM per group; (E) PCoA based on Bray-Curtis distances illustrating community structure at d 14 and d 28 (left). The bar plot (right) shows mean pairwise Bray-Curtis distance between CON and TC at each time point; Taxonomic composition at the phylum level (F, stacked relative abundances) and the genus level (G, top taxa shown); LEfSe biomarkers at d 14 (H) and d 28 (I). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values are means ± SEM (n=3 - 4 piglets per group). \u003csup\u003e*\u003c/sup\u003e differ significantly, \u003cem\u003eP\u003c/em\u003e ≤ 0.05; \u003csup\u003e**\u003c/sup\u003e as above for \u003cem\u003eP \u003c/em\u003e≤ 0.01.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/b2dc8baa0f15f1454d8e097e.png"},{"id":98803217,"identity":"adb417a7-c94f-4452-9113-8eadaeef82eb","added_by":"auto","created_at":"2025-12-22 14:20:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":313639,"visible":true,"origin":"","legend":"\u003cp\u003eIntestinal morphology in weaning piglets\u003c/p\u003e\n\u003cp\u003eLeft: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 μm. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean ± SEM; n = 4 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/430527bd377bdf118866a5af.png"},{"id":98802896,"identity":"8650c28b-2a58-41f7-b16f-3de1fb64f459","added_by":"auto","created_at":"2025-12-22 14:19:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123517,"visible":true,"origin":"","legend":"\u003cp\u003eSerum DAO, ET and sIgA levels in weaning piglets\u003c/p\u003e\n\u003cp\u003eLeft: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 μm. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean ± SEM; n = 4-5 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/bfd33e4745801cc49ccc32f7.png"},{"id":98803263,"identity":"e3246954-7ea5-41bc-988d-09d9a6e2f14c","added_by":"auto","created_at":"2025-12-22 14:20:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":163034,"visible":true,"origin":"","legend":"\u003cp\u003eSerum levels of (A) IL-1β, (B) IL-6, (C) TNF-α, and (D) IL-8 of piglets on days 14 and days 28 of the trail\u003c/p\u003e\n\u003cp\u003eCON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values were showed as mean ± SEM (n = 4-5 per group). * indicated that the difference between CON group and TC group was statistically significant (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/fa5eaf13fcfce864393c9cf0.png"},{"id":99322170,"identity":"7b1f7383-ef8e-4c21-8849-5787baa1eea0","added_by":"auto","created_at":"2025-12-31 16:43:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2863302,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/ddd115a9-5604-4461-a935-b9393c1d73e2.pdf"},{"id":98803388,"identity":"273925d1-b232-4ba4-9602-fa48f7446159","added_by":"auto","created_at":"2025-12-22 14:20:25","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":23547,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8095465/v1/b43b306f1bc2d1083770c89b.docx"}],"financialInterests":"","formattedTitle":"Thymol–Carvacrol (1:2) in Weaned Pigs: An In Silico–In Vitro–In Vivo Evaluation Linking Antibacterial Synergy, Microbiota Stabilization, and Performance Gains","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring the weaning transition, piglets face concurrent social, dietary, and environmental stressors that hinder intestinal barrier maturation (Yang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and increase the risk of post-weaning diarrhea and growth checks, leading to substantial economic losses (Moeser et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rhouma et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). With in-feed antibiotics and pharmacological zinc increasingly restricted, there is an urgent need for effective antibiotic-free strategies (WHO, 2017; EEA, 2024). Phytogenic feed additives have emerged as promising candidates owing to beneficial effects on growth and digestion, together with antimicrobial and antioxidant activities (Canibe et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Rebucci et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThymol and carvacrol\u0026mdash;widely used phenolic monoterpenes\u0026mdash;exhibit clear in vitro antibacterial activity (Wei et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Their delocalized phenolic systems and hydrophobicity disrupt bacterial membranes and interfere with biofilm formation, thereby inhibiting proliferation (Didry et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ultee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Cid-P\u0026eacute;rez et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, recommended inclusion levels for nursery pigs vary widely (approximately 13.50\u0026ndash;55.00 mg/kg for thymol and 12.50\u0026ndash;60.00 mg/kg for carvacrol), and reported outcomes on growth, gut health, microbiota, and inflammatory markers remain heterogeneous (Guarda et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Such variability likely reflects differences in dose, ratio, formulation, and challenge context; moreover, an integrated, systems-level link along the \u0026ldquo;microbiota\u0026ndash;barrier\u0026ndash;inflammation\u0026ndash;growth\u0026rdquo; axis is still lacking.\u003c/p\u003e \u003cp\u003eTo address these gaps, we implemented an integrated in silico\u0026ndash;in vitro\u0026ndash;in vivo workflow: swine targets relevant to weaning-stage inflammation/barrier homeostasis were first nominated via target prediction and consensus blind docking; antibacterial activity and synergy against representative pathogens were quantified by checkerboard assays; and a 28-day feeding trial in weaned piglets then evaluated a 1:2 (thymol:carvacrol) blend for effects on growth and health outcomes, with systematic assessment of cecal microbiota, intestinal morphology, and serum markers of barrier integrity and inflammation/immunity. Our aim was to define a rational ratio and inclusion level and to delineate mechanisms whereby thymol\u0026ndash;carvacrol improves post-weaning performance through the microbiota\u0026ndash;barrier\u0026ndash;inflammation axis, thereby informing precision use in production settings.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn silico target prediction, pathway annotation, and consensus blind docking\u003c/h2\u003e \u003cp\u003eProtein targets of thymol and carvacrol were predicted from public resources (ChEMBL\u0026mdash;Target/Mechanism; OpenTargets) and mapped to Sus scrofa orthologs via UniProt. Pathway annotations (not enrichment) were standardized to KEGG identifiers to support hypothesis-driven prioritization. Candidates were retained if they (i) were biologically relevant to weaning-stage intestinal inflammation and epithelial-barrier homeostasis, (ii) met an affinity threshold for both ligands (AutoDock Vina score\u0026thinsp;\u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;6.0 kcal\u0026middot;mol⁻\u0026sup1;), and (iii) had a swine ortholog with a stable docking pocket. For the six selected targets (MAOA, MAOB, ESR1, PTGS1, PTGS2, VDR), protein structures were taken from Sus scrofa AlphaFold models (chain A); non-protein entities were removed, hydrogens were added, and protonation was set to pH 7.4. Thymol and carvacrol were standardized (tautomer/canonicalization) and protonated at pH 7.4. A consensus blind-docking workflow (CoBDock) was employed that detects candidate cavities using multiple detectors, integrates cavity and preliminary docking scores by machine learning, and returns a prioritized pocket; for each target, the prioritized site was binding_site_1. Pocket stability was evaluated in a ligand-agnostic manner by 100 sampling runs, yielding pocket-score mean/median/SD and defining the final grid center/size (\u0026Aring;). Ligand-specific docking was performed with AutoDock Vina (v1.2.7) using identical grids for thymol and carvacrol per target, and the best Vina score was recorded in kcal\u0026middot;mol⁻\u0026sup1; for each target\u0026ndash;ligand pair. Top poses were inspected in PyMOL (v3.1.6.1) to annotate key non-covalent interactions (Ugurlu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntimicrobial susceptibility and synergy testing\u003c/h3\u003e\n\u003cp\u003eCarvacrol (\u0026ge;\u0026thinsp;97.0% purity) and thymol (\u0026ge;\u0026thinsp;99.0% purity) were obtained from Aldrich. Sterile stock solutions were prepared at 50% (v/v) in ethanol (Merck Millipore; endotoxin\u0026thinsp;\u0026le;\u0026thinsp;0.1 EU/mL), aliquoted, protected from light, and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Test organisms were \u003cem\u003eEscherichia coli (E. coli)\u003c/em\u003e C83715, \u003cem\u003eSalmonella\u003c/em\u003e ATCC 14028, \u003cem\u003eShigella flexneri\u003c/em\u003e (\u003cem\u003eS. flexneri\u003c/em\u003e) ATCC 12022, and \u003cem\u003eStaphylococcus\u003c/em\u003e ST188 (weaned-pig isolates held in a private collection). Minimum inhibitory concentration (MIC) was determined in tryptic soy broth (TSB) by the broth microdilution micromethod in sterile polystyrene flat-bottom 96-well plates following Clinical and laboratory standards institute recommendations. Inocula were adjusted to ~\u0026thinsp;5 \u0026times; 10⁵ CFU/mL in each well (final volume 200 \u0026micro;L) and plates were incubated 24 h at 37\u0026deg;C under static conditions. MIC was defined as the lowest concentration with no visible growth and ΔOD₆₀₀ \u0026le; 0.05 versus the medium control. Vehicle controls were included (TSB and TSB containing ethanol at the highest well concentration); the final ethanol content in all assay wells was matched and kept\u0026thinsp;\u0026le;\u0026thinsp;1% (v/v). Synergy was evaluated by the checkerboard method using two-fold dilution matrices spanning the ranges that bracketed single-agent MICs (typically 0.0625\u0026ndash;512.00 \u0026micro;g/mL). Fractional inhibitory concentrations were calculated as FIC_T\u0026thinsp;=\u0026thinsp;MIC_T (combined) / MIC_T (alone) and FIC_C\u0026thinsp;=\u0026thinsp;MIC_C (combined) / MIC_C (alone), with ΣFICI\u0026thinsp;=\u0026thinsp;FIC_T\u0026thinsp;+\u0026thinsp;FIC_C; interaction was interpreted as synergy (\u0026le;\u0026thinsp;0.50), additive/partial synergy (0.50\u0026ndash;1.00), indifference (1.00\u0026ndash;4.00), or antagonism (\u0026gt;\u0026thinsp;4.00). Each condition was tested in four independent replicates and the central tendency (median) was used for reporting and FICI computation.\u003c/p\u003e\n\u003ch3\u003eThymol–carvacrol supplementation in weaned piglets\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals and experimental design\u003c/h2\u003e \u003cp\u003eThe trial was conducted on a commercial pig farm in Hubei Province, China (September\u0026ndash;October 2022). A total of 100 weaned piglets (Duroc \u0026times; Landrace \u0026times; Yorkshire) were weaned at 26 d of age and randomly allocated to two treatments with 5 pens per treatment and 10 piglets per pen (n\u0026thinsp;=\u0026thinsp;5 pens per group). Treatments were: Control (CON), basal diet formulated to meet or exceed NRC (2012) requirements for weanling piglets; and TC, basal diet plus a thymol-carvacrol blend (Xilaikang 150\u0026reg;, Hubei Refine Biotechnology Co., Ltd.) supplying 25 mg thymol/kg diet and 50 mg carvacrol/kg diet (thymol:carvacrol\u0026thinsp;=\u0026thinsp;1:2). A two-phase feeding program was used: pre-starter (d 1\u0026ndash;14) and starter (d 15\u0026ndash;28); ingredient composition and calculated nutrients are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Piglets were housed in semi-enclosed concrete-floor pens (~\u0026thinsp;10 m\u0026sup2; per pen; 10 pigs per pen) with natural/mechanical ventilation. Feed and water were provided ad libitum. Routine health management and biosecurity procedures of the farm were followed throughout the study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIngredients and nutrient composition of diet\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1-14d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15-28d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCalculated nutrients\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1-14d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15-28d\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaize (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCrude Protein (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e18.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaize Extruded (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSodium (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow protein whey powder (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCalcium (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean Meal Fermented (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal P (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean Meal Dehulled (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible P (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean Extruded (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCopper (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e121.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e121.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeruvian Fish Meal (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZinc (mg/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e110.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e110.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCane Sugar (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestive Energy (MJ/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHomogenizing Oil Powder (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNet Energy (MJ/kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoya Oil (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Lys (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium Formate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Met (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocalcium Phosphate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Met\u0026thinsp;+\u0026thinsp;Cys (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalt (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Thr (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium Glutamate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Trp (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCholine Chloride 50% (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Ile (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e98% L-Lysine HCL (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDigestible Val (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDL-Methionine (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Threonine (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Tryptophan (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsoleucine 90% (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine 98% (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBHT 60% (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremix* (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e* The premix provided per kg of diets: Vitamin A, 11972 IU; Vitamin D3, 2535IU; Vitamin E, 200IU; Vitamin K3, 3.4mg; Vitamin B1, 2.4mg; Vitamin B2,6.70mg; Vitamin B6, 3.80mg; Vitamin B12, 0.0225mg; Niacin, 40mg; Pantothenic,13.30mg; Folic acid, 1.06mg; Biotin, 0.122mg; Fe, 150mg; Cu, 115mg; Mn, 36mg;Zn, 85mg; I, 1.57mg; Se, 0.3mg; Sweetener, 200g; Flavor, 750g; Phytase, 5000FTU\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth performance and diarrhea assessment\u003c/h3\u003e\n\u003cp\u003eBody weight (BW) was recorded on d 1, 14, and 28. For each pen, feed offered and refusals were recorded daily to compute for d 1\u0026ndash;14, d 15\u0026ndash;28, and d 1\u0026ndash;28. was calculated as (BW_end\u0026thinsp;\u0026minus;\u0026thinsp;BW_start)/days for each phase, and feed-to-gain ratio (F/G) as total feed intake/weight gain for the corresponding phase. Pen was considered the experimental unit for performance analyses. Mortality and clinical signs were monitored daily.\u003c/p\u003e \u003cp\u003eBody weight (BW) was recorded on d 1, 15, and 29 to derive average daily gain (ADG) for d 1\u0026ndash;14, d 15\u0026ndash;28, and d 1\u0026ndash;28. For each pen, the total feed offered was recorded and feed refusals were weighed daily at 17:30; these records were used to calculate average daily feed intake (ADFI) for the same three periods. ADG was computed as (BW\u003csub\u003eend\u003c/sub\u003e\u0026minus;BW\u003csub\u003estart\u003c/sub\u003e)/days, and the feed conversion ratio (F/G, feed-to-gain) as total feed intake/weight gain for each phase. The pen served as the experimental unit for performance analyses. Mortality and clinical signs were monitored daily.\u003c/p\u003e \u003cp\u003eDuring the first 28 days post-weaning, fecal consistency of all piglets was assessed twice daily (08:00 and 16:00) using a 0\u0026ndash;3 scale: 0\u0026thinsp;=\u0026thinsp;formed, log-shaped; 1\u0026thinsp;=\u0026thinsp;soft, poorly formed; 2\u0026thinsp;=\u0026thinsp;loose/pasty; 3\u0026thinsp;=\u0026thinsp;watery/jet-like. A score\u0026thinsp;\u0026ge;\u0026thinsp;2 was considered diarrhea; to reduce misclassification, diarrhea was confirmed when two consecutive assessments were \u0026ge;\u0026thinsp;2 or any single assessment was 3. The diarrhea proportion (%) for each group was calculated as (number of diarrhea piglets / total number of piglets) \u0026times; 100. Calculations were performed for d 1\u0026ndash;14, d 15\u0026ndash;28, and d 1\u0026ndash;28.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCecal microbiome sequencing and analysis\u003c/h2\u003e \u003cp\u003eOn d 15 (14 d post-weaning) and d 29 (28 d post-weaning), four piglets per group were randomly selected and humanely euthanized by intravenous chlorpromazine hydrochloride (2 mg/kg BW; Shanghai Hefeng Pharmaceutical Co., Ltd., Shanghai, China). Following Peng et al. (2019), cecal chyme was collected aseptically (~\u0026thinsp;200 mg per aliquot), transferred into cryotubes, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. From each pig, ~\u0026thinsp;200 mg chyme was used for DNA extraction (QIAamp Fast DNA Stool Mini Kit, Qiagen, Germany). The 16S rRNA V3\u0026ndash;V4 region was amplified with 341F/806R and sequenced on an Illumina MiSeq platform (Shanghai Penosen Biotechnology Co., Ltd., Shanghai, China). Reads were processed in QIIME 2 (v2019.4) and denoised with DADA2 to generate ASVs. After rarefaction to a unified depth, α-diversity indices (Chao1, Shannon, Simpson, Pielou) and β-diversity (Bray\u0026ndash;Curtis, PCoA) were computed. PERMANOVA was used to test between-group differences in community structure, with PERMDISP assessing dispersion. LEfSe (LDA\u0026thinsp;\u0026ge;\u0026thinsp;2.0, Kruskal\u0026ndash;Wallis P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) identified discriminative taxa (Segata et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntestinal morphology\u003c/h3\u003e\n\u003cp\u003eHE-stained small-intestinal sections were prepared by routine histology. Images were acquired under a light microscope at 40\u0026times; objective, keeping illumination constant and filling the field of view with tissue. Morphometry was performed in Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) after calibrating to the 40\u0026times; scale bar in the lower-right corner of each image. For each slide (one per piglet), five well-oriented, intact villi with their corresponding crypts were chosen. Villus height was measured from the tip of the villus to the villus\u0026ndash;crypt junction; crypt depth from the base of the crypt to the villus\u0026ndash;crypt junction. Values (mm) were averaged to obtain one villus height and one crypt depth per slide, and the villus-to-crypt ratio was calculated accordingly. Measurements were performed blinded to treatment.\u003c/p\u003e\n\u003ch3\u003eAssessment of serum markers of barrier function and mucosal immunity\u003c/h3\u003e\n\u003cp\u003eOn days 1, 15, and 29 of the trial at 06:30, one medium-weight piglet was randomly selected from each of the five pens per group (total n\u0026thinsp;=\u0026thinsp;5 piglets per group) for fasted blood collection. Twenty milliliters of blood were drawn from the anterior vena cava into non-heparinized vacuum tubes, centrifuged at 3000 \u0026times; g for 10 min at 4\u0026deg;C, and the supernatant (serum) was collected for analysis. According to the manufacturers\u0026rsquo; manuals, serum diamine oxidase (DAO; colorimetric), endotoxin (ET; LAL method), and secretory IgA (sIgA; ELISA) were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All samples were analyzed in duplicate with blanks, standards, and internal controls; standard curves (four-parameter or linear) and concentration calculations followed the kit instructions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSerum inflammatory cytokine assays\u003c/h2\u003e \u003cp\u003eSerum samples were the same as those used for the serum markers of barrier function and mucosal immunity. Concentrations of IL-1β, IL-6, IL-8, and TNF-α were measured with sandwich ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer\u0026rsquo;s instructions. Each sample was analyzed in duplicate, with blank, negative/positive controls, and a standard curve fitted by four-parameter logistic (4-PL) regression (R\u0026sup2; \u0026ge; 0.99). Results were expressed as ng/L. Intra- and inter-assay CVs were maintained within the limits specified by the manufacturer (typically\u0026thinsp;\u0026lt;\u0026thinsp;10%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS v27.0. The pen was the experimental unit for growth performance (n\u0026thinsp;=\u0026thinsp;5 pens/group), whereas individual pigs were the unit for serum variables and intestinal morphology. Normality (Shapiro\u0026ndash;Wilk) and homogeneity of variance (Levene\u0026rsquo;s test) were examined, with transformations applied when necessary. At each time point, growth traits and serum variables were compared using independent-samples t-tests (or Mann\u0026ndash;Whitney U when normality was violated), and diarrhea incidence was compared using the chi-square test. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD for growth performance and mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for serum variables; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered significant and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 highly significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePredicted targets and consensus blind-docking outcomes for thymol and carvacrol\u003c/h2\u003e \u003cp\u003eWe first predicted the targets of thymol and carvacrol and mapped the candidates to pathways using database annotations (ChEMBL, OpenTargets, KEGG/Reactome). Based on predefined criteria - (i) high relevance to weaning-stage intestinal inflammation and barrier homeostasis (e.g., tryptophan metabolism, arachidonic-acid metabolism, nuclear-receptor signaling); (ii) predicted affinity to both ligands meeting a threshold (Score\u0026thinsp;\u0026le;\u0026thinsp;\u0026minus;\u0026thinsp;6.0 kcal\u0026middot;mol-1); and (iii) availability of a swine ortholog together with a stable docking pocket\u0026mdash;we prioritized six swine targets (MAOA, MAOB, ESR1, PTGS1, PTGS2, and VDR) for consensus blind docking (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). CoBDock consistently identified binding_site_1 as the prioritized pocket for all six targets (Table S2). We then performed ligand-specific docking of thymol and carvacrol in this pocket and reported the best scores in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Overall, thymol scored \u0026minus;\u0026thinsp;6.9 on MAOA/MAOB, \u0026minus;\u0026thinsp;6.8 on PTGS1, and \u0026minus;\u0026thinsp;6.0 to \u0026minus;\u0026thinsp;6.4 on ESR1/PTGS2/VDR. Carvacrol achieved the lowest score on MAOA (\u0026minus;\u0026thinsp;7.6) and showed moderate affinities on MAOB/ESR1/VDR (\u0026minus;\u0026thinsp;6.6 to \u0026minus;\u0026thinsp;7.4). An overview of the docking of thymol and carvacrol on selected key swine targets is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Together with pathway annotations, these results suggest that thymol and carvacrol may act along the tryptophan/monoamine metabolism axis (MAO A/B), the arachidonic-acid/prostaglandin axis (PTGS1/2), and nuclear-receptor signaling (ESR1, VDR), thereby indirectly modulating inflammation, oxidative stress, and epithelial-barrier/immune functions that shape the physiological status of weaned piglets.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCore targets and molecular docking results of carvacrol and thymol\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUniProt ID (pig)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene symbol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProtein name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChEMBL\u003c/p\u003e \u003cp\u003etarget ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStructure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eThymol docking score (kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCarvacrol docking score (kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ6Q2J0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMAOA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmine oxidase A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL1951\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold Q6Q2J0_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ6PLK3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMAOB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmine oxidase B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL2039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold Q6PLK3_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-7.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ29040\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eESR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEstrogen receptor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold Q29040_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC9EF58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTGS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProstaglandin G/H synthase 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold C9EF58_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQ8SPR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePTGS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProstaglandin G/H synthase 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold Q8SPR3_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-6.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA3RGC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVitamin D3 receptor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCHEMBL1977\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAlphaFold A3RGC1_A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-6.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Docking was performed in the CoBDock-identified prioritized pocket binding_site_1, using the same grid settings for thymol and carvacrol per target. Protein structures are Sus scrofa AlphaFold models (chain A) unless otherwise noted. Score is the best AutoDock Vina docking score reported in kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;; more negative indicates higher predicted affinity and does not represent experimental ΔG.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRepresentative docking pose of thymol or carvacrol in target gene within the prioritized pocket binding_site_1. The protein backbone is shown as a teal cartoon; the ligand is rendered in orange sticks. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. Blue dashed lines denote hydrogen bonds and green dashed lines indicate hydrophobic/π\u0026ndash;π interactions according to the viewer\u0026rsquo;s default legend. The inset reports the best AutoDock Vina score (kcal\u0026middot;mol\u003csup\u003e-\u003c/sup\u003e\u0026sup1;; more negative indicates higher predicted affinity)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial activity and synergy of thymol\u0026ndash;carvacrol\u003c/h2\u003e \u003cp\u003eThe checkerboard assay showed that, when used alone, both compounds had identical MICs against the tested bacteria (Gram-negative \u003cem\u003eE. coli, Salmonella\u003c/em\u003e, and \u003cem\u003eS. flexneri\u003c/em\u003e: 250 \u0026micro;g/mL; Gram-positive \u003cem\u003eStaphylococcus\u003c/em\u003e: 500 \u0026micro;g/mL). In combination, MICs decreased for all strains, with interaction types varying by species. For \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e, the MICs of both compounds were halved, yielding ΣFICI\u0026thinsp;=\u0026thinsp;1.00 (additive). For \u003cem\u003eS. flexneri\u003c/em\u003e, the combination produced the greatest reduction compared with monotherapy (thymol decreased 8-fold; carvacrol 4-fold), with ΣFICI\u0026thinsp;=\u0026thinsp;0.38 (synergy). For \u003cem\u003eStaphylococcus\u003c/em\u003e, MICs decreased to 125 \u0026micro;g/mL (thymol) and 250 \u0026micro;g/mL (carvacrol), with ΣFICI\u0026thinsp;=\u0026thinsp;0.75 (additive/partial synergy, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, these data suggest that the 1:2 thymol:carvacrol ratio can confer at least additive\u0026mdash;and for certain pathogens (e.g., \u003cem\u003eS. flexneri\u003c/em\u003e) synergistic\u0026mdash;antibacterial effects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMICs of carvacrol and thymol alone and in combination against selected bacteria, and FICI-based interaction\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eBacterium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFIC_T\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFIC_C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eΣFICI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003cp\u003e(alone)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarvacrol\u003c/p\u003e \u003cp\u003e(alone)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThymol\u003c/p\u003e \u003cp\u003e(combined)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCarvacrol\u003c/p\u003e \u003cp\u003e(combined)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e125.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e125.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eadditive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e125.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e125.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eadditive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS.flexneri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e62.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003esynergy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e500.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e500.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e125.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e250.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eadditive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*MIC, minimal inhibitory concentration; FICI, fractional inhibitory concentration index. FIC_T = (MIC of thymol in combination)/(MIC of thymol alone); FIC_C = (MIC of carvacrol in combination)/(MIC of carvacrol alone); ΣFICI\u0026thinsp;=\u0026thinsp;FIC_T\u0026thinsp;+\u0026thinsp;FIC_C. The interaction was interpreted as synergy (\u0026le;\u0026thinsp;0.50), additive/partial synergy (0.50\u0026ndash;1.00), indifference (1.00\u0026ndash;4.00), or antagonism (\u0026gt;\u0026thinsp;4.00). Each condition was tested in four independent replicates and the central tendency (median) was used for reporting and FICI computation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThymol-carvacrol mixture improves late and overall growth in weaned piglets\u003c/h2\u003e \u003cp\u003eWe formulated a thymol-carvacrol mixture at a 1:2 ratio (thymol 25 mg/kg diet, carvacrol 50 mg/kg diet) and supplemented it to weaned piglets to evaluate effects on growth performance. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, compared with the basal diet, the mixture improved late-phase and overall growth. No significant differences were detected between groups at d 1 or d 14 in BW, ADFI, ADG, or F/G (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, relative to controls, the TC group exhibited a higher final body weight at d 28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); during d 15\u0026ndash;28 and d 1\u0026ndash;28, ADFI (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and ADG (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increased, and the overall F/G showed a decreasing trend (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09). In addition, compared with the control, piglets in the TC group showed a significantly lower diarrhea incidence during d 1\u0026ndash;14 and d 1\u0026ndash;28 post-weaning (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Taken together, these results indicate that dietary supplementation with the thymol-carvacrol mixture reduces early post-weaning diarrhea and enhances feed intake and growth rate in the later phase, thereby increasing final body weight at d 28.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance of weaned piglets fed a basal diet or a carvacrol-thymol mixture\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControp group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTC group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBW (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADFI (g/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;14 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e260.26\u0026thinsp;\u0026plusmn;\u0026thinsp;14.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e263.21\u0026thinsp;\u0026plusmn;\u0026thinsp;21.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e530.57\u0026thinsp;\u0026plusmn;\u0026thinsp;34.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e587.93\u0026thinsp;\u0026plusmn;\u0026thinsp;22.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e395.42\u0026thinsp;\u0026plusmn;\u0026thinsp;19.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e425.57\u0026thinsp;\u0026plusmn;\u0026thinsp;17.74\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADG (g/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;14 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198.14\u0026thinsp;\u0026plusmn;\u0026thinsp;9.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e203.14\u0026thinsp;\u0026plusmn;\u0026thinsp;9.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e338.29\u0026thinsp;\u0026plusmn;\u0026thinsp;18.60\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e383.86\u0026thinsp;\u0026plusmn;\u0026thinsp;22.25\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e268.21\u0026thinsp;\u0026plusmn;\u0026thinsp;11.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e293.50\u0026thinsp;\u0026plusmn;\u0026thinsp;13.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF/G (g/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;14 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDiarrhea incidence (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;14 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;28 d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Control group: basal diet; TC: basal diet plus thymol-carvacrol mixture (1:2), day ranges refer to post-weaning periods. BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; F/G, feed conversion ratio, feed-to-gain ratio. Values are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5 pens per group). \u003csup\u003ea, b\u003c/sup\u003e values in rows with different letters differ significantly, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; \u003csup\u003eA, B\u003c/sup\u003e as above for \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eThymol-carvacrol diet remodels the cecal microbiota of weaned piglets\u003c/h2\u003e \u003cp\u003eTo examine whether the growth benefits of the TC diet were associated with gut microbiota, 16S rRNA sequencing was performed on cecal chyme from weaned piglets. Across time points and diets, α-diversity showed no differences on any index (Chao1, Shannon, Simpson, Pielou), indicating limited effects of diet on overall richness and evenness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). In terms of β-diversity, PCoA revealed clear between-diet separation; at d 14 the TC group showed a trend toward lower within-group Bray\u0026ndash;Curtis pairwise distances than the control (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06), and at d 28 this reduction was significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), suggesting a time-dependent decrease in dispersion and a more stable community structure under TC group. At the phylum level, communities were dominated by \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e, while their relative abundances shifted with time and diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). At the genus level, \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and other dominant genera prevailed, with diet- and time-dependent adjustments in abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). LEfSe identified diet-specific biomarkers (LDA\u0026thinsp;\u0026ge;\u0026thinsp;2): At d14, the TC group was significantly enriched for the family \u003cem\u003eBS11\u003c/em\u003e (\u003cem\u003eBacteroidetes\u003c/em\u003e) and for the lineages \u003cem\u003eSpirochaetes\u003c/em\u003e, Treponema, and \u003cem\u003ePseudomonadales\u003c/em\u003e, whereas the control group was enriched for \u003cem\u003eBlautia\u003c/em\u003e and \u003cem\u003eFaecalibacterium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). By d28, the TC group remained enriched for the \u003cem\u003ePseudomonas\u003c/em\u003e lineage, while the control group was enriched for \u003cem\u003eMogibacterium\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Taken together, the TC diet progressively reshaped and stabilized the cecal community without altering overall α-diversity, providing a microbiological basis for improved nutrient utilization and mucosal homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e(A-D) α-diversity indices: Chao1, Shannon, Simpson, and Pielou\u0026rsquo;s evenness index of piglets in CON and CP groups at 14 and 28 days. Bars show mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM per group; (E) PCoA based on Bray-Curtis distances illustrating community structure at d 14 and d 28 (left). The bar plot (right) shows mean pairwise Bray-Curtis distance between CON and TC at each time point; Taxonomic composition at the phylum level (F, stacked relative abundances) and the genus level (G, top taxa shown); LEfSe biomarkers at d 14 (H) and d 28 (I). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 piglets per group). \u003csup\u003e*\u003c/sup\u003e differ significantly, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05; \u003csup\u003e**\u003c/sup\u003e as above for \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThymol-carvacrol diet maintains small-intestinal mucosal morphology\u003c/h2\u003e \u003cp\u003eHE staining showed intact small-intestinal mucosa in both groups at d 14 and d 28, with orderly villi and well-defined crypts. Quantitative analysis indicated no significant differences in villus height or crypt depth between groups at either time point (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, at d 28 the TC group exhibited numerically higher villus height and villus-to-crypt ratio than the control. Assessment of representative serum markers of intestinal barrier and mucosal immunity showed that at 28 d post-weaning, the TC group exhibited significantly lower serum DAO (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and ET levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). No significant changes were observed for other markers or time points (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05, Figure. 4C). The results suggesting that the thymol\u0026ndash;carvacrol diet may promote epithelial renewal and villus remodeling during the later post-weaning period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeft: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 \u0026micro;m. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; n\u0026thinsp;=\u0026thinsp;4 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLeft: representative HE-stained sections of the small intestine from both groups at each time point; scale bar, 100 \u0026micro;m. Right: quantitative results for villus height, crypt depth, and villus-to-crypt ratio (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM; n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5 per group per time point). CON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eThymol-carvacrol diet mitigates early post-weaning systemic inflammation\u003c/h2\u003e \u003cp\u003eAs shown, serum IL-1β, IL-6, and IL-8 did not differ between groups at either d 14 or d 28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B and D). However, at d 14 post-weaning, TNF-α was significantly lower in the TC group than in the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These findings indicate that the thymol\u0026ndash;carvacrol diet exerts a modest anti-inflammatory effect by attenuating the early peripheral TNF-α response, while having minimal impact on overall IL-1β, IL-6, and IL-8 levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCON, Control group, basal diet; TC, basal diet plus thymol-carvacrol mixture (1:2). Values were showed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;4\u0026ndash;5 per group). * indicated that the difference between CON group and TC group was statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discission","content":"\u003cp\u003eThis study used an integrated in silico\u0026ndash;in vitro\u0026ndash;in vivo approach to evaluate a thymol\u0026ndash;carvacrol blend (1:2). In vitro, the combination showed at least additive antibacterial activity against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eS. flexneri\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e, with clear synergy for \u003cem\u003eS. flexneri\u003c/em\u003e. In vivo, weaned piglets fed the blend exhibited higher 28 d final BW and improved ADG and ADFI in the late and overall phases, along with a reduction in early diarrhea. At the microecological level, α-diversity was unchanged, whereas β-diversity dispersion declined over time, indicating a more stabilized community. Concurrent reductions in serum DAO and endotoxin and a decrease in TNF-α suggest alleviated epithelial barrier disruption and systemic inflammatory burden.\u003c/p\u003e \u003cp\u003eIn the consensus blind docking (CoBDock) analysis, the thymol\u0026ndash;carvacrol blend showed high predicted affinity for the swine targets MAOA, MAOB, PTGS1, PTGS2, ESR1, and VDR, which were mapped to three key signaling axes: the tryptophan/monoamine metabolism axis (MAO A/B), the arachidonic-acid/prostaglandin axis (PTGS1/2), and the nuclear-receptor axis (ESR1, VDR). Prior work indicates that MAO A/B catalyze monoamine oxidation and generate H₂O₂, thereby amplifying oxidative-stress and inflammatory signaling while perturbing tryptophan/monoamine balance with consequences for gut immunity and barrier function (Bortolato et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Agus et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). PTGS1/2 govern prostaglandin biosynthesis, integrating acute and chronic inflammatory-mediator networks and shaping the epithelial microenvironment, closely linked to mucosal inflammatory phenotypes (Ricciotti and FitzGerald \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). ESR1/VDR, as nuclear receptors, regulate genes involved in immunity and barrier integrity, sustaining mucosal integrity, antimicrobial-peptide expression, and host control of the microbiota (Kong et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ooi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Paterni et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kovats \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These findings suggest that thymol and carvacrol, by anchoring to swine molecular targets, modulate three interconnected axes that collectively link oxidative stress, inflammation, and mucosal homeostasis during the weaning period.\u003c/p\u003e \u003cp\u003eThe in vitro antibacterial assays further showed that the thymol-carvacrol combination produced additive effects overall against both Gram-negative and Gram-positive bacteria and synergy against \u003cem\u003eS. flexneri\u003c/em\u003e. This is consistent with the classical mechanism whereby phenolic compounds inhibit bacteria via membrane permeabilization and interference with biofilm formation and energy metabolism (Didry et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ultee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), and aligns with more recent evidence that thymol-carvacrol combinations can exhibit synergy against enteric pathogens (Cid-P\u0026eacute;rez et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Notably, the magnitude of synergy is species/strain dependent, which helps explain variability across studies (Michiels et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Taken together, these data support a model in which thymol and carvacrol improve post-weaning physiology in piglets through antibacterial activity coupled with attenuation of inflammatory pathways.\u003c/p\u003e \u003cp\u003eIn line with recent work on essential oils and phytogenic additives in weaned piglets, we observed improvements in late-phase and overall ADG and ADFI together with a reduction in early diarrhea, despite the absence of a strong challenge\u0026mdash;an outcome that is broadly consistent with reports of performance and gut-health benefits under commercial conditions (Shao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Notably, our data connect these phenotypes to a time-ordered cascade spanning the microbiota\u0026ndash;barrier\u0026ndash;inflammation axis: a progressive stabilization of β-diversity was accompanied by lower serum DAO and endotoxin and an early attenuation of TNF-α, suggesting that early-phase (d 1\u0026ndash;14) dampening of inflammatory expenditure and barrier leakage is followed by late-phase (d 15\u0026ndash;28) gains in feed intake and nutrient utilization that translate into higher weight gain. The heterogeneity seen across prior studies likely reflects differences in dose, thymol and carvacrol ratio, formulation (e.g., free vs. microencapsulated), and challenge context (Michiels et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Canibe et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). By applying an evidence-driven 1:2 (thymol:carvacrol) ratio within a single in silico\u0026ndash;in vitro\u0026ndash;in vivo framework, the present work helps reconcile some of this variability and provides a mechanistic bridge from microbial community dynamics to barrier and cytokine readouts. The lack of statistical differences in villus height, crypt depth, and villus-to-crypt ratio, alongside numerical improvements at d 28, is compatible with a scenario in which the blend maintains mucosal architecture under non-severe challenge rather than inducing overt remodeling (Rebucci et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, the 1:2 thymol:carvacrol blend appears to be a feasible antibiotic-free strategy for the weaning period: it improves performance primarily by stabilizing the microbiota and modulating the barrier/inflammation axis in a moderate manner, rather than by bluntly suppressing the community. Future work should adopt factorial designs (dose \u0026times; ratio \u0026times;formulation) and challenge models, and integrate metabolomics with mucosal transcriptomics and tight-junction protein assays to strengthen the causal chain from docking predictions-in vivo chemical evidence-phenotypes. In parallel, attention should be paid to the cost\u0026ndash;benefit and robustness of thymol and carvacrol under different feeding systems and health-risk scenarios (Canibe et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; EEA 2024). These efforts will advance the precision application of thymol and carvacrol in large-scale production while clarifying its mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eSupplementing weaned pig diets with 25 mg/kg thymol and 50 mg/kg carvacrol stabilizes the intestinal microbiota, lowers early post-weaning serum inflammatory cytokines, enhances immune function, reduces early post-weaning diarrhea, and improves late-phase and overall growth performance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAckowledgments\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge Hailin Pig Farm, Huanggang, Hubei Province, China, for providing the experimental facilities and on-farm support. We also thank Chongqing Kangzhou Big Data (Group) Co., Ltd., for performing the molecular docking services of thymol and carvacrol.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the China Agriculture Research System of the Ministry of Finance and the Ministry of Agriculture and Rural Affairs (CARS-35), the Hubei Provincial Special Project for Central Government-Guided Local Science and Technology Development (2024CSA070), and the Hubei Provincial Key Research and Development Program (2023BBB069).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization, Supervision and Funding acquisition: Weicheng Bei and Huanchun Chen; Investigation, Methodology and Data curation: Jiangtao Ao, Fangyan Yuan, Kai Wei, and Xia Yang; Formal analysis and Visualization: Jiangtao Ao, Kai Wei, Xia Yang, and Ruojin Wang; Project administration: Ruojin Wang and Weicheng Bei; Writing-review and editing: Jiangtao Ao and Weicheng Bei. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eData availability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw 16S rRNA sequencing reads are hosted on the Paisen Gene Cloud platform under project ID MD2022120718050WT4.Other data available on reasonable request.\u003c/p\u003e\n\u003cp\u003eEthics approval\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal husbandry, euthanasia, experimental procedures, and biosecurity measures complied with the protocols approved by the Institutional Animal Care and Use Committee (IACUC 202401140009) of the College of Animal Science and Technology, Huazhong Agricultural University.\u003c/p\u003e\n\u003cp\u003eConsent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003eConsent to publish\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe manuscript does not contain any individual person\u0026rsquo;s data.\u003c/p\u003e\n\u003cp\u003eSupplementary material List supplementary tables S1 and S2.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgus A, Planchais J, Sokol H (2018) Gut microbiota regulation of tryptophan metabolism in health and disease. 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J Cheminform 16:5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Chen F, Gao W, Yang H, Lu S, Zhang C, et al. (2022) Effects of different amino acid levels and a carvacrol\u0026ndash;thymol blend on growth performance and intestinal health of weaned pigs. J Anim Sci Biotechnol 13:67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei HK, Xue HX, Zhou ZX, Peng J (2017) A carvacrol\u0026ndash;thymol blend decreased intestinal oxidative stress and influenced selected microbes without changing the messenger RNA levels of tight junction proteins in jejunal mucosa of weaning piglets. Animal 11:193\u0026ndash;201.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization (WHO) (2017) WHO guidelines on use of medically important antimicrobials in food-producing animals. WHO, Geneva.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang H, Xiong X, Wang X, Li T, Yin Y (2016) Effects of weaning on intestinal crypt epithelial cells in piglets. Sci Rep 6:36939.\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"weaned piglets, growth performance, systemic inflammation, carvacrol, thymol","lastPublishedDoi":"10.21203/rs.3.rs-8095465/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8095465/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhytogenic feed additives, owing to their antimicrobial and anti-inflammatory properties, are emerging as promising tools to mitigate weaning stress in piglets. This study evaluated the mechanisms and efficacy of thymol and carvacrol in weaned pigs. Consensus blind docking was used to predict and verify swine molecular targets of thymol/carvacrol; Checkerboard assays quantified antibacterial effects of each compound alone and in combination against \u003cem\u003eE. coli, Salmonella, S. flexneri\u003c/em\u003e, and \u003cem\u003eStaphylococcus\u003c/em\u003e; finally, a 28-day feeding trial was conducted with 100 weaned piglets (5 pens/group \u0026times; 10 pigs/pen): the control received a basal diet, and the treatment diet was supplemented with 25 mg/kg thymol and 50 mg/kg carvacrol. Results showed that docking prioritized MAOA, MAOB, PTGS1, PTGS2, ESR1, and VDR as candidate targets. In vitro, the thymol\u0026ndash;carvacrol combination was additive overall and synergistic against \u003cem\u003eS. flexneri\u003c/em\u003e (1:2; ΣFICI\u0026thinsp;=\u0026thinsp;0.38). In vivo, supplementation increased final BW at d 28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and ADFI and ADG during d 15\u0026ndash;28 and d 1\u0026ndash;28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a trend toward lower F/G over the whole period (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09) and reduced early post-weaning diarrhea (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). While cecal α-diversity was unchanged, β-diversity dispersion decreased by d 28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Serum DAO and ET decreased at d 28 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and TNF-α was lower at d 14 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, a 1:2 thymol:carvacrol blend appears to stabilize the intestinal microbiota and moderately modulate barrier and inflammatory responses, thereby lowering early diarrhea and improving late-phase and overall growth, supporting its feasibility as an antibiotic-free strategy for nursery pigs.\u003c/p\u003e","manuscriptTitle":"Thymol–Carvacrol (1:2) in Weaned Pigs: An In Silico–In Vitro–In Vivo Evaluation Linking Antibacterial Synergy, Microbiota Stabilization, and Performance Gains","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 14:07:57","doi":"10.21203/rs.3.rs-8095465/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":"0b234a14-5872-4b69-9e4d-abd2791e9e73","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-30T12:16:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 14:07:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8095465","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8095465","identity":"rs-8095465","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}