Dietary thyme essential oil supplementation improves production performance, egg quality, antioxidant status, and intestinal health in late-phase laying hens

Abstract

Thyme essential oil (TEO), a phytogenic feed additive extracted from Thymus vulgaris L., has been shown to enhance poultry performance; however, information regarding its effects in late-phase laying hens remains limited. This study evaluated the effects of dietary TEO supplementation on laying performance, antioxidant status, intestinal morphology, and cecal microbiota in late-phase Hy-Line Brown hens. A total of 160 hens were randomly assigned to four dietary treatments containing 0, 100, 300, or 500 mg/kg TEO and fed the experimental diets for 42 d. Dietary TEO supplementation significantly increased egg production compared with the control group (P < 0.05). Haugh unit values were higher in TEO-treated groups during days 22 to 42 (P < 0.05). Supplementation with 300 and 500 mg/kg TEO increased serum total antioxidant capacity (T-AOC) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), while decreasing malondialdehyde (MDA) concentrations (P < 0.05). Villus height and the villus height-to-crypt depth ratio in the jejunum and ileum were improved in TEO-fed hens (P < 0.05). Cecal microbiota analysis conducted in the control and TEO 300 mg/kg groups showed increased microbial richness and altered community composition following TEO supplementation, characterized by a higher relative abundance of Bacteroidota and a lower relative abundance of Bacillota. Collectively, these results indicate that dietary TEO supplementation improves laying performance and antioxidant status and modulates intestinal morphology and cecal microbial structure, suggesting its potential as a nutritional strategy to support health and productivity in aging laying hens.

Introduction

In recent years, the sustained growth in global egg consumption has increased interest in nutritional strategies aimed at extending the productive lifespan of laying hens while maintaining stable performance (Bain et al., 2016). Under commercial conditions, hens typically exhibit declines in egg production, antioxidant capacity, and intestinal function between 60 and 70 wk of age, and individuals older than approximately 450 d are considered late-lay or physiologically aged (Wang et al., 2024). Following peak production, laying rate gradually decreases, accompanied by deterioration in egg quality parameters, including shell thickness and strength, shell uniformity, albumen height, and Haugh unit (HU), reflecting reproductive senescence and progressive physiological decline in aged hens (Gao et al., 2025).

Age-related reductions in laying performance are closely associated with weakening endogenous antioxidant defenses (Liu et al., 2024). Activities of key antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), decline with age (Haan et al., 1995), promoting the accumulation of reactive oxygen species (ROS), lipid peroxidation, and oxidative cellular damage. Excess ROS increases the susceptibility of ovarian follicles and oocytes to apoptosis, impairing follicular development and ovulation, thereby accelerating age-related declines in egg production (Čejková et al., 2004). Oxidative stress is often accompanied by chronic low-grade inflammation, further compromising ovarian microenvironment and oocyte quality (Orisaka et al., 2023). Serum malondialdehyde (MDA), a widely used biomarker of lipid peroxidation, is elevated in late-laying hens compared with peak-lay birds, reflecting increased oxidative burden and impaired ovarian steroidogenic capacity (Mas-Bargues et al., 2021; Soto-Heras and Paramio, 2020). Age-related deterioration of intestinal structure and function further constrains productive performance. Reductions in villus height and the villus height-to-crypt depth ratio decrease absorptive surface area, impairing digestion and nutrient availability. Ngunjiri et al. demonstrated that, in commercial laying hens, rearing stage, age, and sampling site dominantly affect the quantity, taxonomic composition, and dynamics of the respiratory and intestinal microbiota (Ngunjiri et al., 2019). With advancing age, shifts in the intestinal microbial community are commonly observed, with a tendency toward increased relative abundance of opportunistic or potentially pathogenic bacteria (Choi and Kim, 2025; Li et al., 2024). These shifts are associated with impaired mucosal immunity and elevated intestinal inflammation, which may indirectly accelerate reproductive aging through increased metabolic and inflammatory stress (Cotton et al., 2023; Gan et al., 2020). Intestinal barrier integrity is also compromised with age, characterized by villus shortening, altered crypt architecture, and a reduced number and secretory activity of goblet cells, resulting in increased intestinal permeability and susceptibility to pathogenic invasion (Branca et al., 2019; Gu et al., 2021a).

Phytogenic feed additives have attracted increasing attention because of their natural origin, safety, and broad-spectrum biological activities (Gorlov et al., 2023). Thyme essential oil (TEO), derived from Thymus species, is rich in monoterpenes and their oxygenated derivatives, with thymol and carvacrol as the principal bioactive compounds. These constituents exhibit potent antioxidant activity by enhancing endogenous SOD and CAT activities and maintaining redox homeostasis (Sun et al., 2024a, 2025). Dietary supplementation with plant-derived essential oils has also been shown to improve intestinal histomorphology in laying hens by increasing villus height and the villus height-to-crypt depth ratio (Shao et al., 2023; Youssef et al., 2020), and to modulate the intestinal microbial community by promoting beneficial bacteria while suppressing potentially pathogenic taxa, thereby contributing to improved intestinal health and metabolic function (Gopalsamy et al., 2025).

Despite these findings, the effects of dietary TEO supplementation in aged laying hens remain incompletely understood. Accordingly, the present study employed late-phase Hy-Line Brown hens to systematically investigate the effects of graded dietary TEO supplementation on laying performance, egg quality, serum antioxidant capacity, intestinal morphology, and cecal microbiota composition. The study aims to provide a mechanistic and empirical basis for mitigating age-related physiological decline and supporting sustainable egg production in late-phase flocks.

Materials and methods

Experimental design, animals, and dietary treatments

Thyme essential oil (TEO; purity 98 %) was obtained from a commercial manufacturer in Ji’an, China. To minimize oxidative degradation, the essential oil was stored in airtight amber glass bottles at −20 °C and protected from light until use. During diet preparation, TEO was freshly incorporated into the basal diet to minimize exposure to oxygen and light. Although the peroxide value of the essential oil was not directly measured, the storage and handling procedures followed previously validated protocols demonstrating the chemical stability of thyme essential oil under low-temperature and light-protected conditions, thereby minimizing the risk of oxidative deterioration prior to dietary inclusion (Qiang et al., 2024).

A completely randomized design was adopted. A total of 160 Hy-Line Brown laying hens at 466 d of age were randomly assigned to four dietary treatments, with four replicates per treatment and 10 hens per replicate. The experiment included a 7-d adaptation period followed by a 42-d feeding trial, which was divided into two phases (days 0–21 and 22–42). All hens were housed in a naturally ventilated laying facility under ambient environmental conditions and subjected to a 16 h light regimen. Birds were individually housed in battery cages measuring 45 cm × 45 cm × 45 cm, providing approximately 2,025 cm² per hen. Each cage was equipped with a feeder and a nipple drinker. Feed and water were provided ad libitum throughout the experimental period. Routine management practices, including daily manure removal and regular sanitation, were implemented. Bird health status was monitored daily, and environmental conditions were maintained within recommended ranges. During the experimental period, ambient temperature was maintained at 22–24 °C, and relative humidity was maintained at 50–60 %.

The basal diet was formulated using a corn–soybean meal system to meet or exceed the nutrient requirements for laying hens recommended by the NRC (Table 1). Four dietary treatments consisted of a basal diet without TEO supplementation (CON) and the basal diet supplemented with 100 mg/kg (TEO 100), 300 mg/kg (TEO 300), or 500 mg/kg (TEO 500) TEO. The supplementation levels were selected based on previous studies demonstrating the biological efficacy and safety of thyme essential oil in poultry at comparable inclusion rates (Feng et al., 2021; Gao et al., 2022; Xiao et al., 2022), and were intended to evaluate potential dose-dependent responses in aged laying hens.

Table 1.

Ingredient composition and nutrient levels of the basal diet (as-fed basis, %).

Ingredient Composition	Content (%)
Corn	62.00
Soybean meal	22.00
Wheat bran	3.00
Limestone	8.00
Premix1	5.00
Total	100.00
Nutrient composition
Metabolizable energy2 (MJ/kg)	11.10
Crude protein (%)	17.00
Total phosphorus (%)	0.48
Non-phytate phosphorus (%)	0.35
Calcium (%)	3.80
Methionine (%)	0.38
Lysine (%)	0.84

¹ The vitamin–mineral premix provided the following per kilogram of premix: vitamin A (retinol), 150,000 IU; vitamin D₃ (cholecalciferol), 350,000 IU; vitamin E (tocopherol), 400 mg; vitamin K (phylloquinone), 360 mg; thiamine, 50 mg; riboflavin, 170 mg; pyridoxine, 80 mg; cyanocobalamin, 0.5 mg; nicotinic acid, 600 mg; calcium pantothenate, 280 mg; folic acid, 16 mg; biotin, 1.8 mg; and choline chloride, 8,000 mg. Trace minerals supplied per kilogram of premix included: copper, 100 mg; manganese, 2,000 mg; iron, 1,600 mg; zinc, 1,500 mg; iodine, 16 mg; and selenium, 12 mg.

The vitamin–mineral premix was identical across all dietary treatments to avoid confounding nutritional effects.

² Metabolizable energy was calculated according to NRC; other nutrient levels were determined by chemical analysis.

Feed intake and egg production were recorded daily for each replicate, and all eggs were individually weighed. At the end of each experimental phase (d 21 and 42), fifteen eggs per replicate were randomly collected for egg quality analysis. These data were subsequently used to calculate the egg production rate, average daily feed intake (ADFI), and feed conversion ratio (FCR) for each replicate.

Blood sampling

On d 21 and 42, following a 12 h overnight feed withdrawal, six hens from each dietary treatment were randomly selected for blood collection via the wing vein. Approximately 2 mL of blood was collected into serum tubes without anticoagulant and allowed to clot at room temperature for 2 h. Samples were then centrifuged at 4,000 × g for 15 min to separate the serum. The resulting serum was visually inspected for hemolysis, and no hemolysis was observed in any of the collected samples; only non-hemolyzed serum was used for subsequent biochemical analyses. Serum aliquots were prepared and stored at −80 °C until analysis.

Intestinal tissue and cecal content collection

At the end of the experiment (d 42), six hens from each dietary treatment were randomly selected and euthanized in accordance with the Guidelines for the Ethical Review of Laboratory Animal Welfare (GB/T 35892–2018, People’s Republic of China; MacArthur Clark and Sun). Birds were rendered unconscious by cervical dislocation and subsequently exsanguinated. The entire small intestine was excised, and adherent mesenteric and connective tissues were carefully removed. Approximately 2-cm segments from the mid-jejunum and mid-ileum were collected and fixed in 4 % paraformaldehyde according to standard histological procedures (Goodarzi et al., 2021) for subsequent histomorphological analysis.

Cecal digesta were aseptically collected from both the left and right ceca of each hen and pooled within each bird. Samples were transferred into sterile 2 mL cryogenic tubes, snap-frozen in liquid nitrogen, and stored at −80 °C until further processing. All sample collection, handling, and storage procedures followed best-practice recommendations for poultry microbiota studies to minimize environmental contamination and post-collection microbial alterations (Lyte et al., 2025).

Egg quality measurements

On d 21 and 42, fifteen eggs were randomly collected from each dietary treatment, with no more than one egg obtained from any individual hen, to ensure that each egg represented a different bird within the treatment. Egg shape index was calculated as the ratio of longitudinal to transverse diameter using a digital caliper (Mitutoyo, Kawasaki, Japan). Eggshell thickness was measured with a dial thickness gauge (Mitutoyo, Kawasaki, Japan). Albumen height, Haugh unit, and yolk color were determined using an automatic egg quality analyzer (Toukyo Rhythm, Tokyo, Japan). Eggshell breaking strength was assessed using a shell strength tester (KQ-1A, Beijing Tianxiang Feiyu Technology, Beijing, China). Yolk color was further quantified with a chromameter (CR-400, Konica Minolta, Tokyo, Japan). Yolk was separated from albumen using a commercial egg separator (Shanghai Specimen and Model Factory, Shanghai, China), and yolk weight was subsequently recorded.

Serum antioxidant indices

Serum antioxidant indices were quantified using commercial colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. Total antioxidant capacity was determined by the ferric reducing antioxidant power method (T-AOC, FRAP; Cat. No. A015-2-1). Superoxide dismutase activity was measured based on the xanthine oxidase method (Cat. No. A001-3-2). Glutathione peroxidase activity was determined from the rate of NADPH oxidation (Cat. No. A005-1-2). Catalase activity was assessed using the ammonium molybdate method (Cat. No. A007-1-1), and malondialdehyde concentration was quantified by the thiobarbituric acid reactive substances method (TBARS; Cat. No. A003-1-2).

Intestinal morphometry

Segments from the mid-jejunum and mid-ileum were collected, gently rinsed with phosphate-buffered saline, and fixed in 4 % paraformaldehyde. After dehydration through a graded ethanol series and clearing, tissues were embedded in paraffin. Transverse sections with a thickness of 5 μm were prepared and stained with hematoxylin and eosin according to standard histological procedures as previously described (Souza et al., 2021).

Histological sections were examined under a light microscope. From each section, six well-oriented and intact villus–crypt units were randomly selected for morphometric analysis. Measurements were obtained from two birds per replicate, with four replicates per treatment. Villus height was defined as the distance from the tip of the villus to the villus–crypt junction, and crypt depth was measured from the base of the crypt to the same junction. The villus height to crypt depth ratio was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA) in combination with a Nikon Eclipse Ci-L light microscope (Nikon, Tokyo, Japan).

16S rRNA gene sequencing and microbiota analysis

Genomic DNA extraction

Cecal digesta samples were used for total microbial DNA extraction with the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. DNA integrity was checked on 1 % agarose gels, and concentration and purity were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Only samples with DNA concentrations ≥20 ng/μL and A260/A280 ratios of 1.8–2.0 were used for downstream PCR and library preparation. DNA was stored at −80 °C until further use (Lyte et al., 2025; Nene et al., 2025).

PCR amplification and sequencing library construction

The V3–V4 region of the bacterial 16S rRNA gene was amplified using barcoded primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) on a T100 Thermal Cycler (Bio-Rad, USA). Each 20 µL PCR reaction contained 10 µL of 2 × Pro Taq Mix (YuHua, Shanghai, China), 0.8 µL of each primer (5 µM), 10 ng template DNA, and nuclease-free water. Thermal cycling consisted of 95 °C for 3 min; 27 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s; and a final extension at 72 °C for 10 min. PCR products were held at 4 °C until further processing, verified on 2 % agarose gels, purified (PCR Clean-Up Kit, YuHua, Shanghai, China), and quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA).

Sequencing libraries were constructed using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, TX, USA) following the manufacturer’s protocol, including adapter ligation, size selection with magnetic beads, PCR enrichment, and final purification. Library quality and fragment size were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Negative and positive controls were included. Libraries were sequenced on an Illumina NextSeq 2000 platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China). Raw data were deposited in NCBI SRA under BioProject PRJNA1424997.

High-throughput sequencing data analysis

High-quality sequences were clustered into OTUs at 97 % similarity using UPARSE version11.0 with chimeras removed, and an OTU-based approach rather than ASVs was used to ensure consistency with existing poultry microbiota datasets and facilitate meta-analytical comparisons. All samples were rarefied to 20,000 sequences per sample, achieving an average Good’s coverage of 99.09 %. OTUs were assigned taxonomy using the RDP classifier v2.11 against the SILVA 16S database (release 138.2) with a confidence threshold of 0.7 (Caporaso et al., 2010). Community composition was summarized at phylum, family, and genus levels. Alpha diversity (Sobs, Chao1, ACE) and beta diversity (PCoA based on Bray–Curtis distances) were calculated. LEfSe identified taxa differing between groups (LDA >3.0, P < 0.05) (García-López et al., 2021). Functional profiles were predicted using PICRUSt2 version2.2.0 (Douglas et al., 2020).

All procedures for cecal microbiota sample collection, DNA extraction, library preparation, sequencing, and bioinformatic analysis were conducted following established best-practice recommendations for poultry microbiota research to minimize environmental contamination and technical variability (Lyte et al., 2025).

Statistical analysis

All statistical analyses were performed using SPSS Statistics version 24.0 (IBM Corp., Armonk, NY, USA). Data were analyzed by one-way analysis of variance (ANOVA), and when a significant treatment effect was detected, differences among means were further compared using Duncan’s multiple range test. Statistical significance was declared at P < 0.05. Results are presented as means ± standard deviation (SD). Graphs were generated using Prism 2024 (GraphPad Software, San Diego, CA, USA), and different lowercase letters indicate statistically significant differences among treatment groups (P < 0.05).

Results

Laying performance

Production performance parameters are presented in Fig. 1. Compared with the control group, hens receiving dietary TEO at 100, 300, or 500 mg/kg exhibited significantly higher egg production rates during days 0–21 and 22–42 (P < 0.05). Among the supplemented groups, hens receiving 300 mg/kg TEO showed the greatest numerical increase in egg production among the supplemented groups (Fig. 1A and E). In contrast, no significant differences were detected among treatments for average egg weight, average daily feed intake (ADFI), or feed conversion ratio (FCR) in either experimental period (P > 0.05; Fig. 1B–D, 1F–H).

Fig. 1.

Effects of dietary thyme essential oil supplementation on production performance during the late laying period (d 0–21 and 22–42). (A, E) Egg production rate (%); (B, F) average egg weight (AEW, g); (C, G) average daily feed intake (ADFI, g/hen); and (D, H) feed conversion ratio (FCR) in the control (CON) and TEO-supplemented groups (TEO 100, TEO 300, and TEO 500). Data are expressed as mean ± SD (n = 40). Different lowercase letters above bars indicate significant differences among treatments (P < 0.

Egg quality

Egg quality parameters are presented in Fig. 2. Haugh unit (HU) values were not significantly affected by dietary treatments during days 0–21 (P > 0.05), but were significantly increased during days 22–42 in the TEO 100, TEO 300, and TEO 500 groups compared with the control (P < 0.05; Fig. 2N), with the highest value observed in the TEO 300 group (P < 0.05). In contrast, no significant differences were detected among treatments for egg shape index, eggshell thickness, eggshell strength, albumen height, yolk color, or yolk weight in either experimental period (P > 0.05).

Fig. 2.

Effects of dietary thyme essential oil supplementation on egg quality during the late laying period (d 0–21 and 22–42). (A, H) Egg shape index; (B, I) Eggshell thickness (mm); (C, J) Eggshell strength (kg·cm⁻²); (D, K) albumen height (mm); (E, L) yolk color; (F, M) yolk weight (g); and (G, N) Haugh unit (HU) in the control (CON) and TEO-supplemented groups (TEO 100, TEO 300, and TEO 500). Data are presented as mean ± SD (n = 15). Different lowercase letters above bars indicate significant differences among treatments (P < 0.05).

Serum antioxidant capacity

The effects of dietary TEO supplementation on serum antioxidant parameters are presented in Fig. 3. Serum malondialdehyde (MDA) concentrations were significantly reduced in the TEO 300 and TEO 500 groups during days 0–21, whereas during days 22–42, all TEO-supplemented groups exhibited lower MDA levels compared with the control, with the greatest reduction observed in the TEO 500 group (P < 0.05; Figs. 3A, F). Serum total antioxidant capacity (T-AOC) was significantly increased in the TEO 300 and TEO 500 groups during both days 0–21 and 22–42, with the highest values observed in the TEO 500 group (P < 0.05; Figs. 3B, G).Superoxide dismutase (SOD) activity was significantly elevated in the TEO 100, TEO 300, and TEO 500 groups compared with the control during days 0–21. During days 22–42, significant increases in SOD activity were observed in the TEO 300 and TEO 500 groups, with the greatest enhancement consistently detected in the TEO 500 group (P < 0.05; Figs. 3C, H).Glutathione peroxidase (GSH-Px) activity was significantly higher in the TEO 300 and TEO 500 groups during days 0–21, with the largest increase observed in the TEO 500 group. On days 22–42, GSH-Px activity was significantly elevated in the TEO 100, TEO 300, and TEO 500 groups relative to the control, with the greatest increase consistently detected in the TEO 500 group (P < 0.05; Figs. 3D, I).Catalase (CAT) activity was significantly enhanced in the TEO 300 and TEO 500 groups during days 0–21, with the highest activity observed in the TEO 500 group. During days 22–42, CAT activity was significantly increased in the TEO 100, TEO 300, and TEO 500 groups compared with the control, with the maximal activity consistently detected in the TEO 500 group (P < 0.05; Figs. 3E, J).

Fig. 3.

Effects of dietary thyme essential oil supplementation on serum antioxidant indices in laying hens during the late laying period (d 0–21 and 22–42). (A, F) T-AOC, U/mL; (B, G) SOD, U/mL; (C, H) GSH-Px, U/mL; (D, I) CAT, U/mL; and (E, J) MDA, nmol/mL in the control (CON) and TEO-supplemented groups (TEO 100, TEO 300, and TEO 500). Data are presented as mean ± SD (n = 6). Different lowercase letters above bars indicate significant differences among treatments (P < 0.05).

Intestinal morphology

The effects of dietary TEO supplementation on jejunal and ileal morphology are presented in Fig. 4. In the jejunum, villus height (VH) was significantly increased in the TEO 300 group compared with the control (P < 0.05; Fig. 4A). In addition, all TEO-supplemented groups exhibited significantly higher villus height to crypt depth ratios (VH:CD) than the control group (P < 0.05; Fig. 4C).

Fig. 4.

Effects of dietary thyme essential oil supplementation on intestinal morphology in laying hens during the late laying period. (A–C) Jejunal villus height (VH, μm), crypt depth (CD, μm), and the villus height to crypt depth ratio (VH:CD). (D–F) Ileal villus height (VH, μm), crypt depth (CD, μm), and VH:CD ratio in the control (CON) and TEO-supplemented groups (TEO 100, TEO 300, and TEO 500). Data are presented as mean ± SD (n = 6). Different lowercase letters above bars indicate significant differences among treatments (P < 0.05).

In the ileum, VH was significantly increased in the TEO 100, TEO 300, and TEO 500 groups relative to the control (P < 0.05; Fig. 4E), with greater increases observed in the TEO 300 and TEO 500 treatments. Consistent with the jejunal findings, the VH:CD ratio in the ileum was significantly higher in all TEO-supplemented groups compared with the control group (P < 0.05; Fig. 4F).

Intestinal microbial diversity

Analysis of alpha and beta diversity and venn diagram

The effects of dietary TEO supplementation on cecal microbial diversity are presented in Fig. 5. Alpha diversity analysis showed that the ACE, Chao, and Sobs indices were significantly higher in the TEO 300 group than in the control group (P < 0.05; Figs. 5A–C), indicating enhanced cecal microbial richness in response to dietary TEO supplementation. Beta diversity assessed by principal coordinates analysis (PCoA) revealed a clear separation between the CON and TEO 300 groups along the first principal component (Fig. 5D), suggesting a marked alteration in the overall structure of the cecal microbial community associated with TEO inclusion.Venn diagram analysis demonstrated that the CON and TEO 300 groups shared 738 operational taxonomic units (OTUs), accounting for 71.37 % of the total detected OTUs (Fig. 5E). In addition, 117 OTUs (11.31 %) were unique to the CON group, whereas 179 OTUs (17.31 %) were unique to the TEO 300 group, indicating that dietary TEO supplementation was associated with the enrichment of distinct microbial taxa while maintaining a substantial shared core microbiota.

Fig. 5.

(A–C) α-diversity indices of cecal microbiota, including Ace (A), Chao (B), and Sobs (C). Blue bars indicate the control group (CON, without thyme essential oil), and red bars indicate the TEO 300 group (supplemented with 300 mg/kg thyme essential oil). Significance between groups is indicated by asterisks (P < 0.05; P < 0.01; P < 0.001). (D) β-diversity was assessed using principal component analysis (PCA) at the phylum level. Blue dots represent samples from the CON group, and red dots represent samples from the TEO 300 group. (E) Venn diagram showing shared and unique OTUs between the CON and TEO 300 groups.

The top 15 microbial phyla and genera by relative abundance

The effects of dietary TEO supplementation on cecal microbial composition at the phylum and genus levels are presented in Fig. 6. At the phylum level, the cecal microbiota in both groups was predominantly composed of Bacteroidota, Bacillota, Spirochaetota, Actinomycetota, and unclassified taxa. Compared with the control group, hens in the TEO 300 group exhibited significantly higher relative abundances of Bacteroidota, Spirochaetota, and several low-abundance phyla, whereas the relative abundances of Bacillota, Fusobacteriota, and Patescibacteria were significantly reduced (P < 0.05).At the genus level, the predominant taxa included Bacteroides, Rikenellaceae_RC9_gut_group, Christensenellaceae_R-7_group, Mediterraneibacter, and Faecalibacterium. The relative abundances of Bacteroides, Rikenellaceae_RC9_gut_group, and Christensenellaceae_R-7_group tended to increase in the TEO 300 group compared with the control group, whereas Mediterraneibacter and Faecalibacterium showed a decreasing trend; however, these differences did not reach statistical significance (P > 0.05).

Fig. 6.

Effects of dietary thyme essential oil supplementation on cecal microbial composition in Hy-Line Brown laying hens. (A) Relative abundance of the 15 most prevalent bacterial phyla in the cecum. (B) Relative abundance of the 15 most prevalent bacterial genera in the cecum. Groups include the control (CON) and the TEO-supplemented group receiving 300 mg/kg TEO (TEO 300). Different colors represent distinct taxa, as indicated in the legend.

The LEfSe analysis of the cecal microbiota

Linear discriminant analysis effect size (LEfSe) analysis identified distinct microbial biomarkers that differentiated the CON and TEO 300 groups across multiple taxonomic levels (LDA score > 3.0; Fig. 7). The cladogram revealed clear phylogenetic separation between the two groups, indicating pronounced differences in the overall composition of the cecal microbial community.

Fig. 7.

LefSe identify differential cecal microbial taxa between the control (CON) and TEO-supplemented (TEO 300) groups. (A) LDA scores of taxa showing significant differences between groups (LDA score > 3.0). (B) Cladogram depicting taxa significantly enriched in each group.

In the CON group, taxa affiliated with Actinomycetota, Bacillota, and the order Lactobacillales were significantly enriched. Discriminative biomarkers included the families Lactobacillaceae, Peptostreptococcaceae, Atopobiaceae, and Christensenellaceae, along with the genera Ligilactobacillus, Blautia, and Megamonas, as well as several unclassified taxa belonging to the Clostridia vadinBB60 group.

In contrast, the TEO 300 group was characterized by a higher relative abundance of taxa associated with Campylobacterota, Spirochaetota, and the class Desulfovibrionia. The principal biomarkers identified in this group included the families Campylobacteraceae, Helicobacteraceae, Desulfovibrionaceae, and Prevotellaceae. At the genus level, Helicobacter, Desulfovibrio, and Barnesiella were significantly enriched, together with multiple unclassified taxa affiliated with Eremiobacteria, Bacteroidales, and Clostridia UCG lineages.

Discussion

TEO, a volatile bioactive extract derived from Thymus vulgaris L., is rich in phenolic monoterpenes, primarily thymol and carvacrol, which exhibit potent antioxidant and antimicrobial activities (Galovičová et al., 2021). These compounds efficiently scavenge reactive oxygen species (ROS), mitigate oxidative stress, and help maintain systemic redox homeostasis (Sun et al., 2024b). Moreover, TEO supports intestinal barrier integrity, improves gut morphology, and contributes to intestinal homeostasis, often accompanied by alterations in the cecal microbial community (Liu et al., 2023).

The functional efficacy of TEO has been demonstrated in various poultry species. In broilers, dietary supplementation with 200 mg/kg TEO enhances body weight gain, feed efficiency, and survival rate (Noruzi et al., 2021). In laying hens, 50 mg/kg TEO improves egg production, eggshell thickness, and yolk index, while reducing the feed-to-egg ratio (Yalçın et al., 2020). Similarly, supplementation with 200 mg/kg TEO in Muscovy ducks increases SOD and GSH-Px activities, elevates total antioxidant capacity (T-AOC), reinforces intestinal barrier function, promotes SCFA-producing bacteria, and suppresses opportunistic pathogens (Ge et al., 2023).

In the present study, dietary TEO significantly enhanced egg production during both days 0–21 and 22–42. Other production indices, including average egg weight (AEW), feed conversion ratio (FCR), and average daily feed intake (ADFI), remained unaffected. This pattern likely reflects age-related reductions in digestive and absorptive efficiency, impaired metabolic flexibility, and decreased ovarian responsiveness in late-phase laying hens, which may limit changes in AEW, FCR, and eggshell formation despite increased egg production (Abdelnour et al., 2022; Qiang et al., 2025). Consequently, dietary TEO supplementation appears to support laying persistence, without exerting significant effects on feed conversion efficiency or egg weight–related traits.

HU is a widely accepted indicator of internal egg quality and albumen freshness (Khaleel, 2019; Sheidaee and Bazyar, 2021). Essential oils have been reported to improve egg quality parameters, with peppermint oil, alone or combined with TEO, enhancing egg production, egg weight, eggshell thickness, and HU (Akbari et al., 2016). Dietary supplementation with 50 mg/kg TEO also significantly improved laying performance and HU in partridges (Baser et al., 2025). In the present study, dietary TEO supplementation significantly increased HU, while no notable changes were observed in other egg quality traits, including egg weight, eggshell thickness, and eggshell strength. This selective response may be attributed to the higher sensitivity of protein-associated indices, such as albumen quality and HU, to nutritional interventions and modulation of the gut microbiota. In contrast, structural or production-related traits, including shell characteristics and egg weight, are largely constrained by physiological maturity, laying stage, and genetic potential, and therefore respond more slowly (Portune et al., 2016; Wu et al., 2022). Furthermore, modulation of the intestinal microbiota and its downstream effects on protein metabolism generally occur gradually, resulting in a lag between improvements in albumen quality and changes in production performance. Consequently, sustained dietary intervention is typically required before significant enhancements in protein-associated egg quality traits become evident (Guo et al., 2020; Sopian et al., 2025).

Excessive ROS accumulation is a major driver of oxidative stress and age-related functional decline (Yang et al., 2024; Zia et al., 2021). When ROS production exceeds endogenous antioxidant capacity, oxidative damage occurs, manifested as lipid peroxidation, protein oxidation, and DNA injury (Gu et al., 2021b). Maintenance of redox homeostasis depends on antioxidant enzymes such as SOD, GSH-Px, and CAT, with MDA serving as a biomarker of lipid peroxidation (Hajam et al., 2022; Moldogazieva et al., 2019). In this study, TEO supplementation markedly increased serum T-AOC and significantly enhanced SOD, GSH-Px, and CAT activities, while reducing MDA levels. These effects are likely attributable to the high phenolic content of TEO, particularly thymol and carvacrol, which act through direct radical scavenging and the activation of endogenous antioxidant pathways (Gumus et al., 2017; Soobrattee et al., 2005). Furthermore, the antioxidant effects of TEO were dose dependent, in agreement with earlier reports in laying poultry and with the progressive enhancement of antioxidant indices observed in the present study as the dietary inclusion level of TEO increased (Hashemipour et al., 2013; Su et al., 2021).

Intestinal villus architecture is critical for nutrient absorption, pathogen exclusion, and immune regulation (Garic et al., 2025; Wang et al., 2025b). Compounds from Thymus species preferentially stimulate villus elongation rather than crypt expansion, increasing the villus height-to-crypt depth (VH/CD) ratio and absorptive surface area (Hernández-García et al., 2024; Orzuna-Orzuna and Lara-Bueno, 2023). In this study, supplementation with 300 or 500 mg/kg TEO significantly increased villus height and VH/CD ratios in both jejunum and ileum, indicating partial mitigation of age-related intestinal deterioration. These findings are in agreement with previous studies reporting that dietary supplementation with essential oils, which contain the bioactive compounds thymol or carvacrol, leads to improvements in intestinal morphology in both laying hens and broilers (Moharreri et al., 2022; Wang et al., 2022).

The cecal microbial community is central to nutrient metabolism and intestinal barrier function. Phytogenic feed additives can stabilize gut microbial communities and mitigate dysbiosis (Molinari et al., 2021; Santhiravel et al., 2022). Previous studies have shown that the bioactive phenolic monoterpenes in TEO, particularly thymol and carvacrol, can retain biological activity in the distal gut and may selectively modulate cecal microbial composition through antimicrobial and antioxidant effects (Lyte et al., 2024).

Dietary supplementation with 300 mg/kg TEO significantly influenced the cecal microbial community. Microbial richness indices (ACE, Chao, and Sobs) increased, and β-diversity analyses indicated compositional alterations rather than simple abundance shifts. Similar effects have been reported with other plant-derived feed additives, including polyphenol-rich extracts and fermentation-based products (Chen et al., 2020, 2025), supporting the capacity of TEO to modulate microbial community composition.

At the phylum level, TEO increased the relative abundance of Bacteroidota while decreasing Bacillota and Actinomycetota, reducing the Firmicutes/Bacteroidota ratio. Bacteroidota members possess extensive carbohydrate-active enzyme repertoires, facilitating degradation of complex polysaccharides and production of SCFAs, including acetate, propionate, and butyrate, indicative of enhanced fermentative activity and improved metabolic function (Mamun et al., 2025).

At the genus level, Bacteroides remained dominant. TEO selectively enriched SCFA-producing genera, including Rikenellaceae_RC9_gut_group, Mediterraneibacter, and unclassified Bacteroidales, consistent with phylum-level patterns. Mediterraneibacter, a major butyrate-producing genus within Lachnospiraceae, enhances energy supply to colonic epithelial cells, reinforces tight junctions, and mitigates intestinal inflammation via the NF-κB and NLRP3 pathways (Singh et al., 2023; Xiong et al., 2023; Zhu et al., 2021; ). These changes likely reflect selective antimicrobial activity of thymol and carvacrol, suppressing susceptible taxa while permitting metabolically active fermentative bacteria to expand (Sharma et al., 2022; Yang et al., 2023).

LEfSe analysis revealed reductions in several Bacillota taxa, including Lactobacillus, Ligilactobacillus, and Christensenellaceae_R-7_group, consistent with Gram-positive susceptibility to phenolic monoterpenes (Speranza et al., 2023). Conversely, the relative abundance of Treponema and Eremiobacterota increased, alongside enrichment of Helicobacter and Desulfovibrio, taxa often considered potential pathobionts (Liu et al., 2021). Helicobacter is associated with gastrointestinal inflammation, whereas Desulfovibrio produces hydrogen sulfide, a metabolite linked to mucosal irritation and inflammatory signaling (Huang et al., 2024).

Collectively, these results suggest that TEO exerts selective yet broad-spectrum effects on the cecal microbial community. Reductions in beneficial taxa likely result from direct antimicrobial activity, whereas proliferation of potential pathobionts may arise from reduced microbial competition and altered ecological niches (Sorbara and Pamer, 2019). Expansion of SCFA-producing genera, including Rikenellaceae_RC9_gut_group, Mediterraneibacter, and unclassified Bacteroidales, suggests enhanced fermentative capacity, improved energy metabolism, and reinforced intestinal barrier function (Huang et al., 2024). The concurrent enrichment of potential pathobionts highlights the complex ecological responses of the gut microbiota, indicating that beneficial and potentially adverse effects may coexist in late-phase laying hens.

Overall, dietary TEO supplementation promoted a cecal microbial community characterized by enhanced fermentative capacity and metabolic integration. These findings are consistent with previous studies showing that plant-derived essential oils, such as oregano and cinnamon, influence microbial pathways involved in carbohydrate metabolism, SCFA synthesis, and energy utilization in poultry (Bajagai et al., 2022; Oni and Oke, 2025). Considering the dual effects of TEO, further studies incorporating functional metagenomics and metabolomics are warranted to determine optimal dietary inclusion levels that maximize benefits while minimizing potential microbial risks.

Conclusions

Collectively, the present findings demonstrate that dietary supplementation with thyme essential oil (TEO) enhances antioxidant capacity, intestinal health, laying performance, and cecal microbial composition in late-phase laying hens. Among the tested inclusion levels, supplementation with 300 mg/kg TEO produced the most consistent and pronounced improvements. These included enhanced laying performance, improved intestinal morphology, and a more favorable cecal microbiota, characterized by increased richness and enrichment of SCFA-producing genera. Both 300 and 500 mg/kg TEO significantly elevated systemic antioxidant enzyme activities and reduced oxidative stress markers, with the higher inclusion level showing a slightly greater effect. Together, these results indicate that appropriate dietary TEO supplementation can partially mitigate age-related functional decline, modulate gut microbial ecology, and help maintain productive efficiency in late-phase laying hens. Overall, the study provides robust experimental evidence supporting the application of TEO as a natural nutritional strategy to sustain egg productivity, egg quality, and intestinal homeostasis in commercial layer production systems.

Ethical statement

All experimental procedures involving animals were reviewed and approved by the Animal Care and Use Committee of Nanjing Agricultural University. The study was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals and complied with the principles of Replacement, Reduction, and Refinement (3Rs). Throughout the experimental period, proper management practices were implemented to minimize animal stress and discomfort, including appropriate housing, careful handling, routine health monitoring, and humane euthanasia during sample collection.

Data availability

The raw 16S rRNA gene sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1424997, and will be publicly accessible upon publication. All other datasets are available from the corresponding author upon reasonable request.

CRediT authorship contribution statement

Wenze Song: Writing – original draft, Writing – review & editing. Jingbo Ma: Data curation. Chao Jia: Writing – review & editing. Yaomei Wang: Resources. Xinyue Yang: Investigation. Liuyang Han: Validation. Ziheng Zhou: Supervision. Debing Yu: Conceptualization, Methodology.

Disclosures

The authors declare that there are no conflicts of interest related to this study.