Front. Vet. Sci. Frontiers in Veterinary Science Front. Vet. Sci. 2297-1769 Frontiers Media S.A. 10.3389/fvets.2025.1739666 Original Research Effects of dietary supplementation with a thymol-carvacrol blend on growth performance and intestinal health of poultry Liu Xiaoxia 1 2 * † Visualization Formal analysis Writing – original draft Methodology Conceptualization Investigation Li Xiang 1 3 † Software Resources Writing – original draft Validation Visualization Conceptualization Chen Ruiying 3 Writing – review & editing Formal analysis Liu Jing 2 Validation Writing – review & editing Liu Rui 4 Writing – review & editing Resources Zhao Ruting 2 Project administration Writing – review & editing Luo Aiguo 1 Data curation Writing – review & editing Zhao Jia 1 Writing – review & editing Methodology Hao Jianwei 1 Conceptualization Writing – review & editing Yang Shuming 2 Supervision Writing – review & editing Chen Ailiang 2 * Supervision Funding acquisition Writing – review & editing Department of Biological Science and Technology, Shanxi Center of Technology Innovation for Compound Condiment, Jinzhong University, Jinzhong, China Key Laboratory of Agro-product Quality and Safety, Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences, Beijing, China School of Food Science and Engineering, Shanxi Agricultural University, Jinzhong, China School of Investigation, People's Public Security University of China, Beijing, China Correspondence: Xiaoxia Liu, lxxiagood@163.com; Ailiang Chen, ailiang.chen@gmail.com

These authors have contributed equally to this work and share first authorship

 12 01 2026 2025 12 1739666 05 11 2025 19 12 2025 19 12 2025 Copyright © 2026 Liu, Li, Chen, Liu, Liu, Zhao, Luo, Zhao, Hao, Yang and Chen. 2026 Liu, Li, Chen, Liu, Liu, Zhao, Luo, Zhao, Hao, Yang and Chen https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Background

Antibiotic resistance has intensified the search for alternatives in poultry production. Essential oils (EOs), particularly blends of carvacrol and thymol, have shown potential as natural growth promoters and antimicrobials. This study evaluated a composite carvacrol-thymol EO as an antibiotic substitute in broiler production, focusing on growth performance, serum biochemistry, intestinal morphology, and gut microbiota.

 Materials and methods

A total of 672 Aibayi-Yijia broilers were randomly assigned to seven treatment groups: control (CK), EO1 (200 g/t feed), EO2 (600 g/t feed), EO3 (1200 g/t feed), EO1+AG (EO 200 g/t + FON 0.15 g/kg feed), EO3+AG (EO 1200 g/t + FON 0.15 g/kg feed), and AG (FON 0.15 g/kg feed). Growth performance, serum biochemistry (TP, ALB, GLB, GLU, AST, GGT, CHOL, TG, IL), jejunal histology (villus height, crypt depth, V:C ratio), and cecal microbiota (16S rRNA sequencing) were assessed.

 Results

Supplementation with EO (600 g/t) or florfenicol followed by 1200 g/t EO significantly increased ABW and ADFI (p < 0.05). EO (200 g/t or 1200 g/t) supplementation after antibiotics reduced serum TP, ALB, GLB, and CHOL (p < 0.05). Histological analysis showed increased villus height and V:C ratios with 1200 g/t EO. Cecal microbiota shifted, with increased Bacteroidetes and decreased Firmicutes.

 Discussion

The composite carvacrol-thymol EO blend showed promise as an antibiotic alternative, improving growth performance, supporting intestinal health, and modulating the gut microbiota. Further research is needed to optimize dosing, assess long-term safety, and explore EO interactions for scalable use.

 antibiotics carvacrol gut microbiota plant essential oils thymol The author(s) declared that financial support was received for this work and/or its publication. Financial support was provided by Special PhD Funding Project of Jinzhong University (JUD2023021); the National Key Research and Development Program (EU: SFS-46-2017; China: 2017YFE0114400). section-at-acceptance Animal Nutrition and Metabolism Introduction

Intensification of livestock production has increased demands for faster growth, improved feed conversion efficiency, and higher meat quality in broiler chickens (1). In broiler production, antibiotics are commonly added to feed to enhance growth and prevent disease (2). However, indiscriminate use has promoted antimicrobial resistance and generated drug residues. It has also caused environmental contamination, thereby threatening animal health and food safety (3, 4). Recent national regulations have restricted or banned antibiotic growth promoters in feed. Consequently, researchers and producers are pursuing environmentally friendly, effective alternatives.

Plant essential oils (EOs) are volatile, bioactive phytochemicals proposed as alternatives to antibiotics because of their antibacterial, anti-inflammatory, antioxidant and digestion promoting properties (5, 6). Wińska and colleagues reviewed the antibacterial activities and mechanisms of multiple EOs and their constituents (7). Vlaicu et al. summarized experimental evidence that EOs affect digestion, intestinal morphology, immunity and growth performance, and discussed their potential as antibiotic substitutes (6, 8). In particular, the monoterpenoid phenols carvacrol and thymol exhibit pronounced antimicrobial activity, modulate the gut microbiota and improve intestinal health, thereby contributing to enhanced feed efficiency and growth in poultry (9, 10). Several reports indicate that a thymol-carvacrol eutectic markedly improves intestinal morphology and barrier function and significantly enhances growth and health in broilers (11, 12). EO efficacy is influenced by inclusion level, component ratio and timing of administration. Consequently, the precise mechanisms and optimal application protocols required to replace antibiotics remain to be determined.

Arbor Acres Plus broilers were used to evaluate a composite essential oil (carvacrol + thymol) as an alternative to antibiotics. Different inclusion levels and feeding schedules of the blend were compared with a conventional florfenicol regimen. Effects on production performance (ABW, ADG, ADFI, F/G) and serum biochemistry (TP, ALB, GLB, CHOL) were assessed. Small intestinal histomorphology (villus height, crypt depth, V: C) and cecal microbiota (16S rRNA) were also examined. The study aimed to provide theoretical support and practical guidance for the use of plant essential oils in antibiotic free broiler diets.

 Materials and methods Experimental materials

Male day-old Arbor Acres Plus (AA) broiler chicks were obtained from Beijing Dafa Chia Tai Co., Ltd. (also listed as Beijing Dafa Zhengda Co., Ltd.). The essential oil feed additive (total effective essential-oil content ≥3.5%; carvacrol ≥2.3%; thymol ≥1.2%) was supplied by Guangzhou Wisdom Bio-Technology Co., Ltd. (also listed as Guangzhou Zhiteqi Bio-technology Co., Ltd.). Florfenicol soluble powder (30% w/w) was purchased from Henan Muxiang Biological Co., Ltd. (Henan Muxiang Animal Pharmaceutical/Biotech).

 Experimental design and grouping

A total of 700 one-day-old male Arbor Acres (AA) broiler (initial body weight, 45.75 ± 0.30 g) were selected and housed in cages under continuous lighting. The house temperature was maintained at 34 ± 1 °C for 3 days before broiler placement; comprehensive disinfection was performed prior to arrival. Thereafter, temperature was reduced by 2 °C per week until it reached 24 °C, and this temperature was kept until the end of the trial. Feed and water were provided ad libitum throughout the experiment. Ventilation, hygiene and manure removal were routinely maintained, and regular disinfection was performed. Diets were formulated for two phases: starter (1–21 d) and grower (22–42 d). Basal diets met the nutrient recommendations of NRC (1994) and the Chinese feeding standard NY/T 33-2004. Dietary amino-acid balance was achieved using an ideal digestible amino-acid profile. The ingredient composition and nutrient levels of the experimental diets are presented in Table 1.

Ingredients and nutrient composition of diets.

Component 1 ~ 21 d (Level/%) 22 ~ 42 d (Level/%)
Ingredient component
Corn 48.8834 53.08
Soybean meal 38.7448 33.4638
Soybean oil 3.6825 4.704
Wheat middling 5 5
Limestone 1.05 1.05
NaCl 0.1385 0.1326
Sodium bicarbonate 0.2395 0.2502
Choline chloride 0.1 0.1
Dicalcium P 1.6214 1.6954
L-lysine 0.0869 0.1084
DL-methionine 0.2767 0.2424
Threonine 0.0562 0.0533
Vitamin Premix1 0.02 0.02
Mineral Premix2 0.1 0.1
Total 100 100
Nutrient component3
CP% 22 20
Energy 3,000 3,100
Lys% 1.12 1.02
Methionine cystine 0.76 0.76
Threonine 0.67 0.67
Tryptophan 0.2036 0.1993
Valine 0.8201 0.8168
Leucine 1.4454 1.4712
Isoleucine 0.7324 0.7255
Arginine 1.2502 1.2253
Met% 0.5048 0.5002
Histidine 0.4737 0.4748
Glycine Serine 1.7128 1.6931
Tryptophan 0.2036 0.1993
Lys + Met 1.5048 1.5002
Potassium 0.9745 0.8789
Sodium 0.175 0.175
Chlorine 0.175 0.175
Calcium 0.89 0.89

Vitamin premix provided per kg of diet: VA 12500 IU; VB₁ 2.0 mg; VB₂ 6.0 mg; VB₆ 3.0 mg; VB₁₂ 0.025 mg; VD₃ 2,500 IU; VK₃ 2.65 mg; VE 50 mg; pantothenic acid 10.5 mg; niacin 50 mg; biotin 0.0325 mg; folic acid 0.25 mg.

Mineral premix provided per kg of diet: Zn 75 mg; Mn 100 mg; Cu 8 mg; Fe 80 mg; Se 0.15 mg; I 0.35 mg.

All nutrient levels were calculated values.

The EO inclusion levels were selected with reference to the dose response patterns reported by Yang et al. (13) in broilers receiving thymol–carvacrol cocrystal and by Hong et al. (14), who demonstrated clear graded effects of encapsulated essential oils in meat ducks. Based on this evidence, EO was included at 200, 600, and 1,200 g/t to cover a biologically relevant response range. Florfenicol was included at 0.15 g/kg, a dosage commonly applied in poultry practice and widely used as a reference level in comparative studies on antibiotic alternatives. To further assess potential EO antibiotic interactions, two combination treatments were added: low-dose EO + florfenicol and high-dose EO + florfenicol.

At the start of the trial, AA broiler chicks were randomly assigned to one control group and six treatment groups. Each treatment included eight replicates of 12 birds per replicate, resulting in 672 birds after day-one sampling and culling. Grouping details are shown in Table 2.

Experimental design.

Group Dosage level Number of days of breeding Distribution of samples
CK, A (Control group, Feed) Basic diet 0–42 days (all) 96 (8 replicates, 12 each replicate)
EO1, B (Low-dose essential oil group, Feed) EO1 200 g/t 0–42 days (all)
EO2, C (Medium-dose essential oil group, Feed) EO2 600 g/t 0–42 days (all)
EO3, D (High-dose essential oil group, Feed) EO3 1,200 g/t 0–42 days (all)
EO1+AG, E(EO1+Florfenicol) EO1 200 g/t + AG 0.15 g/kg Antibiotics (days 7–21), Feed B for remaining days
EO3+AG, F(EO3+Florfenicol) EO3 1,200 g/t + AG 0.15 g/kg Antibiotics (days 7–21), Feed D for remaining days
AG, G (Florfenicol) AG (0.15 g/kg) Antibiotics (days 7–21), Feed A for remaining days
Determination of indicators and methods Measurement of growth performance of AA broiler chicks

Growth performance was monitored throughout the experiment. Birds were fasted for 12 h prior to weighing. Fasted body weights (BW) were recorded at 1, 21 and 42 d of age, and the weights of sampled birds were noted. Performance parameters were calculated for each replicate and comprised average body weight (ABW), average daily gain (ADG), average daily feed intake (ADFI), feed: gain ratio (F/G) and mortality rate (MER). Calculations were performed as follows:

ADG ( g ) = G 2 − G 1 + G 3 A 1 × T 1 + B 1 × T 1

In Equation (1), the variables were defined as follows: G1 was the initial pen weight (g). G2 was the final pen weight (g). G3 was the combined weight of birds that died or were culled at removal (g). A1 was the number of birds remaining per cage (birds). T1 was the total experimental duration (days, d). B1 was the number of birds that died or were culled (birds).

ADFI ( g ) = G 4 − G 5 A 1 × T 1 + B 1 × T 1

In Equation (2), the variables were defined as follows: G4 was the total amount of feed supplied (g). G5 was the feed remaining in the trough (g). A1 was the number of birds remaining per cage (birds). T1 was the total experimental duration (days, d). B1 was the number of birds that died or were culled (birds).

F / G = C 1 C 2

In Equation (3), the variables were defined as follows: C1 was the average daily feed intake (g). C2 was the average daily gain (g).

 Determination of serum biochemical parameters in AA broiler chickens

Birds were sampled at 21 and 42 d of age. For each replicate, one bird close to the replicate mean body weight was selected. The wing root (brachial) vein was disinfected with 70% ethanol and blood was collected using a single use needle. Whole blood was drawn into plain vacuum tubes without anticoagulant (red top tubes). At least twice the volume required for planned serum assays was collected. Tubes were allowed to clot and were incubated in a 37 °C water bath for approximately 31 min. Samples were centrifuged at 3,500 rpm for 10 min; the pale-yellow supernatant (serum) was transferred to sterile, labeled microcentrifuge (Eppendorf) tubes and stored at −80 °Cuntil analysis.

Serum biochemical parameters were measured using an automated clinical chemistry analyzer. Assays included γ-glutamyl transferase (GGT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), glucose (GLU), triglycerides (TG), total cholesterol (CHOL) and globulin (GLB). All clinical biochemistry tests were performed at the Diagnostic Center of the Veterinary Teaching Hospital, China Agricultural University.

Serum concentrations of interleukin-6 (IL-6) and interleukin-10 (IL-10) were quantified by ELISA. A multifunctional microplate reader (Tecan) was used for absorbance measurements. Commercial ELISA kits were obtained from Shanghai Jianglai Biotech (Shanghai Jianglai Biological Technology Co., Ltd.).

 Determination of villus height and crypt depth in the small intestine of AA broiler chickens

At 42 d of age, 56 AA broilers were slaughtered. Approximately 2 cm segments of the duodenum, jejunum and ileum were excised, gently flushed with phosphate buffered saline (PBS) to remove luminal contents, and fixed in 4% paraformaldehyde. Tissues were processed for paraffin embedding using standard procedures (trimming, dehydration, clearing, infiltration and embedding), sectioned and stained with hematoxylin and eosin (H&E). Slides were examined with a light microscope, and intact, straight villi and their adjacent crypts were measured using Image-Pro Plus 6.0. For each section, five complete villi (red arrows in Supplementary Figure 1) and five crypts (yellow arrows in Supplementary Figure 1) were measured, and the villus height to crypt depth ratio (V: C) was calculated.

 16S rRNA gene sequencing of the gut microbiota

A total of 112 AA broilers were slaughtered by cervical dislocation at 21 and 42 days of age. Fresh cecal digesta were aseptically collected from each bird and immediately snap frozen in liquid nitrogen. Samples were stored at −80 °C until further analysis.

Microbiota quantification was carried out following the protocol of Sun et al. (46). Purified material was submitted to Biomarker Technologies Co., Ltd. (Beijing, China) for 16S rRNA gene sequencing.

 Statistical analysis

Data were entered into Microsoft Excel and analyzed using IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) was used for comparisons among three or more groups. When ANOVA indicated a significant effect, multiple comparisons were performed using Duncan’s multiple range test or the least significant difference (LSD) post-hoc test, as appropriate. Results were reported as mean ± standard error of the mean (SEM). All tests were two tailed, and p < 0.05 was considered statistically significant.

 Results Effects of dietary compound plant essential oils on broiler growth performance

As shown in Table 3, over days 1–42 the experimental groups EO2, EO1+AG, EO3+AG and AG had significantly higher average body weight (ABW) than CK (p < 0.05). EO2 and EO3+AG also showed increased average daily feed intake (ADFI) (p < 0.05). No significant differences in average daily gain (ADG) were observed among groups (p > 0.05). The AG group exhibited the highest mortality rate. During days 1–21, ADG was higher in AG than in CK (p < 0.05). The EO1+AG group had lower ADFI than CK (p < 0.05). F/G was reduced in AG relative to CK (p < 0.05). No differences in ABW were detected among groups (p > 0.05). The highest mortality in this phase was observed in EO3. From days 22–42, ABW was greater in EO2, EO1+AG, EO3+AG and AG compared with CK (p < 0.05). ADFI was lower in EO3+AG (p < 0.05). No significant differences in ADG or F/G were found among groups (p > 0.05). The AG group again showed the highest mortality.

Effects of dietary compound plant essential oils on growth performance of AA broilers.

Items ABW/g ADG/g ADFI/g F/G MER/%
1 ~ 21 Age
A(CK) 957 ± 0.05 42.47 ± 2.99ab 56.31 ± 3.28b 1.33 ± 0.03b 2.08
B(EO1) 963 ± 0.03 42.40 ± 3.25ab 55.74 ± 2.21ab 1.32 ± 0.07ab 4.17
C(EO2) 959 ± 0.02 43.69 ± 2.09ab 55.84 ± 1.06ab 1.28 ± 0.06ab 5.21
D(EO3) 942 ± 0.03 41.08 ± 2.92a 54.13 ± 3.40ab 1.32 ± 0.03ab 8.33
E(EO1+AG) 943 ± 0.03 42.50 ± 3.43ab 53.60 ± 2.49a 1.27 ± 0.07ab 4.17
F(EO3+AG) 965 ± 0.04 42.57 ± 3.87ab 54.21 ± 3.27ab 1.28 ± 0.14ab 1.04
G(AG) 961 ± 0.03 44.80 ± 3.93b 55.50 ± 2.12ab 1.24 ± 0.08a 2.08
22 ~ 42 Age
A(CK) 2,540 ± 0.19 a 61.21 ± 7.73 116.08 ± 11.56ab 1.91 ± 0.10 9.38
B(EO1) 2,663 ± 0.08 ab 61.80 ± 4.63 113.05 ± 5.87ab 1.83 ± 0.12 11.5
C(EO2) 2,728 ± 0.15 b 65.08 ± 12.81 119.35 ± 11.17ab 1.90 ± 0.39 6.25
D(EO3) 2,606 ± 0.37 ab 60.49 ± 9.96 106.30 ± 19.22a 1.75 ± 0.07 6.25
E(EO1+AG) 2,694 ± 0.11 b 64.66 ± 5.29 118.36 ± 9.03ab 1.84 ± 0.11 8.33
F(EO3+AG) 2,705 ± 0.19 b 69.40 ± 9.93 122.45 ± 11.32b 1.78 ± 0.14 9.38
G(AG) 2,706 ± 0.19 b 65.54 ± 14.11 116.27 ± 20.16ab 1.79 ± 0.17 16.7
1 ~ 42 Age
A(CK) 60.85 ± 4.61 a 51.62 ± 4.42 85.46 ± 6.46ab 1.66 ± 0.05b 11.5
B(EO1) 63.82 ± 1.79 ab 51.86 ± 3.25 83.69 ± 3.43ab 1.62 ± 0.08ab 15.6
C(EO2) 65.41 ± 3.49 b 54.12 ± 6.71 86.82 ± 5.75b 1.62 ± 0.15ab 11.5
D(EO3) 62.44 ± 2.48 ab 50.55 ± 6.12 79.58 ± 10.50a 1.57 ± 0.03a 14.6
E(EO1+AG) 64.58 ± 2.51 b 53.31 ± 2.80 85.19 ± 4.95ab 1.60 ± 0.07ab 12.5
F(EO3+AG) 64.86 ± 4.40 b 55.66 ± 4.22 87.50 ± 6.34b 1.57 ± 0.07a 10.4
G(AG) 64.88 ± 4.37 b 54.91 ± 6.62 85.15 ± 9.61ab 1.55 ± 0.08a 18.8

In the same column, values with the same or no letter superscripts mean no significant difference (p > 0.05), while with different small letter superscripts mean significant difference (p < 0.05).

Treatments: (A) Control (CK), basal diet; (B) EO1, essential oils 200 g/t; (C) EO2, essential oils 600 g/t; (D) EO3, essential oils 1,200 g/t; (E) EO1+AG, EO 200 g/t + florfenicol (0.15 g/kg, days 7–21); (F) EO3+AG, EO 1200 g/t + florfenicol (0.15 g/kg, days 7–21); (G) AG, florfenicol (0.15 g/kg, days 7–21).

 Effects of dietary composite plant essential oils on serum biochemical parameters

Figure 1 and Supplementary Table 1 summarize the serum biochemical and cytokine results. At 21d, dietary supplementation with the composite plant essential oil did not affect total protein (TP) or globulin (GLB) (p > 0.05). Albumin (ALB) was higher in EO1+AG than in CK (p < 0.05). Aspartate aminotransferase (AST) activity was increased in EO1, EO1+AG, EO3+AG and AG compared with CK (p < 0.05). Total cholesterol (CHOL) and interleukin-10 (IL-10) were elevated in EO1+AG versus CK (p < 0.05). By contrast, glucose (GLU) in EO1, gamma glutamyl transferase (GGT) in EO3+AG and AG, triglycerides (TG) in EO2, and interleukin-6 (IL-6) in EO2, EO3, EO1+AG, EO3+AG and AG were all lower than CK (p < 0.05).

Effects of dietary compound plant essential oils on serum biochemical. Serum biochemical and immune indices of AA broilers. (1) Total protein; (2) albumin; (3) globulin; (4) glucose; (5) aspartate aminotransferase; (6) γ-glutamyl transferase; (7) total cholesterol; (8) triglycerides; (9) interleukin-6; (10) interleukin-10. Dietary treatments: CK, control (basal diet without antibiotics or essential oils); EO1, low EO (essential oils, 200 g/t); EO2, medium EO (essential oils, 600 g/t); EO3, high EO (essential oils, 1,200 g/t); EO1+AG, low EO + antibiotic (essential oils, 200 g/t + florfenicol, 0.15 g/kg, days 7–21); EO3+AG, high EO + antibiotic (essential oils, 1,200 g/t + florfenicol, 0.15 g/kg, days 7–21); AG, antibiotic (florfenicol, 0.15 g/kg, days 7–21). Different letters indicate significant differences among treatments (p < 0.05).

Ten bar charts display biochemical measurements over two time points labeled in each chart. Each chart is numbered 1 to 10,representing different metrics such as TP, ALB, GLB, GLU, AST, GGT, CHOL,TG, IL-6 and IL-10 levels across various groups labeled CK, EO1, EO2, EO3, EO1+AG,EO3+AG, and AG. Each chart uses green and red bars to indicate the values at different days, with significance levels annotated by letters above the bars.

At 42 d, no treatment effects were detected for GLU, AST or IL-10 (p > 0.05). TP, ALB and GLB were reduced in EO1+AG and EO3+AG compared with CK (p < 0.05). CHOL was lower in EO1+AG, EO3+AG and AG than in CK (p < 0.05), and CHOL in the antibiotic supplemented groups (EO1+AG, EO3+AG, AG) was lower than in the essential oil only groups (EO1, EO2) (p < 0.05). GGT was higher in AG than in CK (p < 0.05). IL-6 was increased in EO1+AG relative to CK (p < 0.05).

 Effects of dietary composite plant essential oils on small-intestinal morphology

As shown in Supplementary Table 2 and Figure 2, ileal villus height (V) and villus to crypt ratio (V/C) did not differ significantly among EO1, EO2, EO3, EO1+AG, EO3+AG and AG versus CK (p > 0.05). Crypt depth (C) was increased in EO3+AG compared with CK (p < 0.05); other groups showed no significant change (p > 0.05). In the duodenum, villus height and crypt depth did not differ among groups (p > 0.05). The duodenal V/C ratio was higher in EO3+AG than in CK (p < 0.05), and was also higher in EO3+AG than in EO3, EO1+AG and AG (p < 0.05). In the jejunum, crypt depth and V/C ratio did not differ among treatments (p > 0.05). Jejunal villus height was greater in EO3+AG than in CK (p < 0.05).

Histological morphology of the small intestine in AA broilers. Representative hematoxylin and eosin (H&E) stained sections of the intestine. Scale bar, 500 μm; magnification, 400×. (A) Jejunum; (B) ileum; (C) duodenum. Numbers 1–7 indicate dietary treatments: 1, CK (control, basal diet); 2, EO1 (EO 200 g/t); 3, EO2 (EO 600 g/t); 4, EO3 (EO 1200 g/t); 5, EO1+AG (EO 200 g/t + florfenicol 0.15 g/kg, days 7–21); 6, EO3+AG (EO 1200 g/t + florfenicol 0.15 g/kg, days 7–21); 7, AG (florfenicol 0.15 g/kg, days 7–21).

Microscopic images of intestinal tissue sections labeled A1 to C7, each displaying intricate pink and purple striations. The sections are shown at a magnification of 400× with a scale bar indicating 500 micrometers. The detailed morphology of the intestinal villi and crypts is clearly visible in each sample. Effects of dietary supplementation with a compound plant essential oil on the intestinal microbiota of broilers Microbial community composition in the cecum of 42-day-old AA broilers Phylum level relative abundance

The phylum level composition of cecal microbiota in 42-day-old AA broilers was examined (Supplementary Figure 2). Samples were grouped as A (CK), B (EO1), C (EO2), D (EO3), E (EO1+AG), F (EO3+AG) and G (AG). The relative abundances of the top ten phyla were plotted as bar charts. Across all groups, Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes and Verrucomicrobia were dominant. Relative to the CK group, all treatments reduced the relative abundance of Firmicutes (EO1, p < 0.05; EO2 and EO3, p > 0.05; EO1+AG, EO3+AG, and AG, p < 0.05). The abundance of Bacteroidetes increased in EO1 (p < 0.05), EO3 (p < 0.05), EO1+AG (p < 0.05), EO3+AG (p < 0.05), and AG (p < 0.05), but decreased in EO2. Proteobacteria increased in EO2 (p < 0.05), EO1+AG, EO3+AG, and AG, but decreased in EO1 and EO3. Tenericutes decreased in EO2 and AG, whereas it increased in EO1, EO3, EO1+AG, and EO3+AG. Phyla with a relative abundance <1% included Verrucomicrobia, Cyanobacteria, Actinobacteria, Fusobacteria, and Acidobacteria.

 Family level relative abundance

Family level composition of the cecal microbiota was assessed in 42-day-old AA broilers (Figure 3). Plot the relative abundances of the top 10 families of samples in different groups on the X-axis. The dominant families across all treatments were Ruminococcaceae, Lachnospiraceae, Rikenellaceae, the Clostridiales vadinBB60 group, Lactobacillaceae, Erysipelotrichaceae, Christensenellaceae and Enterobacteriaceae.

The family level abundance of cecal luminal microbiota. Treatments: (A) Control (CK), basal diet; (B) EO1, essential oils 200 g/t; (C) EO2, essential oils 600 g/t; (D) EO3, essential oils 1,200 g/t; (E) EO1+AG, EO 200 g/t + florfenicol (0.15 g/kg, days 7–21); (F) EO3+AG, EO 1200 g/t + florfenicol (0.15 g/kg, days 7–21); (G) AG, florfenicol (0.15 g/kg, days 7–21).

Stacked bar chart showing the relative abundance percentages of various bacterial families in groups labeled A to G. Each bar is divided by different colors representing bacterial families such as Ruminococcaceae, Lachnospiraceae, and others. A legend on the right indicates family names corresponding to specific colors. Genus level relative abundance

Genus level cecal microbiota in 42-day-old AA broilers was examined (Supplementary Figure 3). Samples were grouped as A (CK), B (EO1), C (EO2), D (EO3), E (EO1+AG), F (EO3+AG) and G (AG). The ten most abundant genera across all groups were Alistipes, Faecalibacterium, the Clostridiales vadinBB60 group, unclassified members of Lachnospiraceae, Ruminococcaceae UCG-014, Ruminococcus, Lactobacillus, the Ruminococcus torques group, Negativibacillus and the Eubacterium coprostanoligenes group.

Relative to the CK group, Alistipes and Clostridiales vadinBB60 were consistently enriched across all treatments, whereas Faecalibacterium was uniformly depleted (p < 0.05). Unclassified Lachnospiraceae declined in EO1, EO1+AG, EO3+AG and AG, but was unchanged or elevated in EO2 and EO3. Ruminococcaceae UCG-014 increased under essential oil only treatments (EO1, EO2, EO3), but declined under the combined regimens (EO1+AG, p < 0.05; EO3+AG) and the antibiotic treatment (AG, p < 0.05). Lactobacillus showed treatment specific responses: it increased in EO1+AG (p < 0.05) and AG (p < 0.05), but decreased in EO2. Negativibacillus showed treatment dependent changes, being reduced in EO1, EO1+AG and EO3+AG while increasing in some EO treatments. These shifts indicated that the composite essential oil and florfenicol modulated key cecal genera, with distinct effects depending on dose and combination.

 Alpha diversity analysis of cecal microbiota

Alpha diversity metrics were calculated for each group. Coverage values approached 1.00 in all treatments, indicating that sequencing depth adequately reflected cecal microbial diversity. Rarefaction curves (Supplementary Figure 4) showed that observed OTU counts increased with sequencing effort and plateaued at high read depths, confirming that sequencing depth was sufficient.

Statistical comparisons of diversity indices are summarized in Supplementary Table 3 and Figure 4. According to the ACE index, EO3+AG and AG differed significantly from CK (p < 0.05), and the essential oil only groups (EO1, EO2) differed from the combined antibiotic groups (EO3+AG, AG) (p < 0.05). Chao1 analysis showed that AG differed from CK and EO1 (p < 0.05), while EO2 differed from EO1+AG, EO3+AG and AG (p < 0.05). Shannon indices revealed significant differences between EO1 and both EO1+AG and AG (p < 0.05), and between EO3 and AG (p < 0.05). No significant differences were detected in Simpson indices among groups (p > 0.05).

Alpha diversity index of sample. (1) Abundance-based Coverage Estimator; (2) Chao Index; (3) Simpson Diversity Index; (4) Shannon Diversity Index. Treatments: (A) Control (CK), basal diet; (B) EO1, essential oils 200 g/t; (C) EO2, essential oils 600 g/t; (D) EO3, essential oils 1,200 g/t; (E) EO1+AG, EO 200 g/t + florfenicol (0.15 g/kg, days 7–21); (F) EO3+AG, EO 1200 g/t + florfenicol (0.15 g/kg, days 7–21); (G) AG, florfenicol (0.15 g/kg, days 7–21). Different letters indicate significant differences among treatments (p < 0.05).

Four bar charts labeled 1 to 4 show biodiversity indices for groups A to G. Chart 1 (green) represents ACE, chart 2 (blue) represents Chao, chart 3 (red) represents Simpson, and chart 4 (multicolored) represents Shannon. Each bar is labeled with letters indicating statistical significance. Discussion

Numerous recent studies have explored the use of essential oils (EOs) in livestock and poultry production (10, 12, 42, 50–52). Accumulating evidence shows that EOs can promote growth by increasing average daily gain (ADG) and average daily feed intake (ADFI) (15). They also lower the feed to gain ratio (F/G) and reduce mortality (16). For example, dietary supplementation with 400 mg/kg EO improved broiler growth and reduced feed conversion (p < 0.05) (17). Zheng et al. reported that blends of monoglyceride lauric acid and cinnamaldehyde (350 and 500 mg/kg) increased ADG and improved F/G. These blends also enhanced intestinal morphology, improved antioxidant status and downregulated inflammatory markers; the 500 mg/kg dose produced the greatest benefit (18). Alagawany et al. found that lemongrass EO enhanced growth performance, lipid metabolism, immune responses and antioxidant capacity in quail, while reducing intestinal pathogens and overall health risk (19). Various plant EOs have been evaluated as dietary supplements for poultry, and their potential as eco-friendly alternatives to antibiotics in organic production has been reported (8, 20).

In the present trial, supplementation with a composite plant essential oil (carvacrol + thymol), either alone or combined with florfenicol, improved body weight in Arbor Acres broilers. From 22 to 42 d, average body weight (ABW) was higher in EO2, EO1+AG, EO3+AG and AG than in CK (p < 0.05). Average daily feed intake (ADFI) was lower in EO3+AG during this interval (p < 0.05). Over the entire 1–42 d period, ABW increased in EO2, EO1+AG, EO3+AG and AG (p < 0.05), while ADFI was higher in EO2 and EO3+AG (p < 0.05). No differences in average daily gain (ADG) were detected among groups (p > 0.05). These results indicate that EO2 and the combined EO + antibiotic regimens (EO1+AG, EO3+AG, AG) enhanced growth performance under the conditions tested, consistent with earlier reports (21–23). The effects may reflect the aroma and bioactivity of essential oils, which can increase feed palatability, stimulate salivation and promote gut motility, thereby supporting feed intake and physiological function. However, several studies reported no effect of EO supplementation on growth metrics (24–27). Such discrepancies likely arise from differences in EO composition, inclusion level, bird strain or experimental conditions.

Blood maintains internal homeostasis, and hematological indices reflect the interplay between nutrition and disease (28, 29). Total protein (TP) and albumin (ALB) are central to acid base balance, plasma oncotic pressure and tissue protein homeostasis. Aspartate aminotransferase (AST), a major hepatocellular transaminase, participates in amino acid catabolism and synthesis and serves as an indicator of amino acid metabolism (30, 31). Higher protein intake raises amino acid turnover and stimulates transaminase activity; elevated TP and ALB thus indicate a favorable nutritional state (8, 32). Glucose (GLU) reflects energy status and, when increased, is generally associated with improved immunity and stress resistance (33). In the present study, at 42 d, TP was significantly lower in EO1+AG and EO3+AG compared with CK (p < 0.05). ALB and globulin (GLB) were also reduced in EO1+AG and EO3+AG (p < 0.05). Serum cholesterol (CHOL) decreased in EO1+AG, EO3+AG and AG relative to CK (p < 0.05). These findings suggest that essential oils promote lipid catabolism. One plausible mechanism is that linoleic acid in thyme essential oil binds cholesterol and inhibits hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This inhibition may enhance conversion of cholesterol to bile acids and increase excretion, thereby lowering circulating lipids. Consistent with our results, Hong et al. reported that dietary supplementation with 125 ppm essential oil extract (blend of lemon, oregano and anise) and 100 ppm antibiotic (oxytetracycline) significantly reduced serum CHOL in broilers (p < 0.05). They also observed significant differences in very low density lipoprotein among control, essential oil and antibiotic groups, with the essential oil group showing the lowest values (14).

Intestinal structural integrity was evaluated by villus height (V), crypt depth (C) and the villus: crypt ratio (V/C) (8, 27, 34). Taller villi and shallower crypts indicate greater digestive and absorptive capacity. The intestine is both the main digestive organ and the largest immune organ; it is essential for nutrient digestion, absorption and defence against pathogens (35, 36). Modern broiler production exposes birds to multiple stressors for example, elevated ammonia, lighting regimes, stocking density, basal diet, transport, ambient temperature and humidity, noise and pathogens which readily induce intestinal damage, diarrhea and growth retardation (37–39). Dietary cinnamaldehyde increased jejunal villus height and tended to raise the jejunal V/C in heat stressed broilers, suggesting improved intestinal morphology and nutrient digestibility (40). Jiménez et al. reported that cinnamaldehyde increased villus height in piglets and protected villi from free radical damage via antioxidant effects (41). Similar benefits enhanced barrier integrity, increased villus height, reduced crypt depth and enlarged villus surface were reported after essential oil supplementation (42–44). In the present study, jejunal villus height and duodenal V/C were significantly increased in the EO3+AG group compared with CK (p < 0.05). These findings indicate that the tested essential oil preparation, at the studied dose, can support intestinal development in AA broilers.

The gut microbiota of livestock and poultry normally exists in a dynamic equilibrium and has a strong self-repair capacity. Disturbances such as environmental stress, dietary shifts, improper antibiotic use and pathogen overgrowth can damage the mucosa and trigger disease. Dietary inclusion of EOs has been reported to reshape microbial composition, improve nutrient digestibility and support immune function. EOs are proposed to suppress harmful taxa (e.g., Clostridium perfringens, Escherichia coli) while promoting beneficial genera (e.g., Lactobacillus, Bifidobacterium), thus optimizing the microbial environment and preserving mucosal integrity. The phylum Proteobacteria includes many opportunistic pathogens, such as E. coli and Salmonella, which can impair productivity (17, 42, 45). By contrast, Lactobacillus is regarded as beneficial in broiler intestines because it acidifies the lumen, inhibits pathogens and contributes to gut health. Consistent with these observations, Irawan et al. found that a carvacrol–thymol blend reduced pathogenic E. coli in the cecum and ileum and modulated Lactobacillus populations in broilers (32).

The taxonomic composition of the gut microbiota is shaped by several factors, including host age, diet, anatomical site and antibiotic exposure (46, 47). Many studies report that Bacteroidetes, Firmicutes and Proteobacteria are the dominant phyla, with Bacteroidetes and Firmicutes typically being the most abundant (17, 48, 49). These groups play central roles in nutrient absorption and energy metabolism and collectively support host nutrient uptake and energy storage. In broilers, the relative abundances of Verrucomicrobia, Cyanobacteria, Actinobacteria, Fusobacteria and Acidobacteria did not decline markedly, suggesting a stable community structure. Changes in host health therefore often reflect shifts in specific pathogenic taxa, rather than broad reductions in overall microbial diversity.

 Conclusion

Supplementation of the basal diet with the composite essential oil (carvacrol + thymol) at 600 g/t, or a regimen of florfenicol (0.15 g/kg during the starter phase) followed by 1,200 g/t essential oil in the grower phase, significantly increased average body weight (ABW) and average daily feed intake (ADFI). Average daily gain (ADG) and feed: gain ratio (F/G) were not affected. The combined regimen of early antibiotic use followed by later essential oil supplementation (200 g/t or 1,200 g/t) significantly reduced total protein (TP), albumin (ALB), globulin (GLB) and total cholesterol (CHOL). Histological analysis showed that 1,200 g/t essential oil supplementation increased jejunal villus height and duodenal villus: crypt ratio (V/C). Cecal 16S rRNA sequencing revealed an increased relative abundance of Bacteroidetes and a concomitant decrease in Firmicutes, indicating a shift in microbial composition. Taken together, the composite essential oil demonstrated potential as an antibiotic alternative to improve production performance, support intestinal development and modulate the gut microbiota under the tested dosing schedules. Further systematic studies on dose response relationships, long term safety, component interactions (synergy/antagonism) and delivery strategies are warranted to enable reproducible and scalable application in antibiotic free poultry production.

 Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

 Ethics statement

The studies involving humans and animals were approved by Laboratory Animal Ethics Committee, Feed Research Institute, Chinese Academy of Agricultural Sciences. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

 Author contributions

XXL: Formal analysis, Writing – original draft, Project administration, Methodology, Data curation, Conceptualization, Software, Investigation, Funding acquisition. XL: Software, Resources, Writing – original draft, Validation, Visualization, Conceptualization. RC: Writing – review & editing, Formal analysis. JL: Validation, Writing – review & editing. RL: Writing – review & editing, Resources. RZ: Project administration, Writing – review & editing. AL: Data curation, Writing – review & editing. JZ: Writing – review & editing, Methodology. JH: Conceptualization, Writing – review & editing. SY: Supervision, Funding acquisition, Writing – review & editing. AC: Supervision, Funding acquisition, Writing – review & editing.

 Acknowledgments

We would like to thank Shuming Yang and Ailiang Chen for instructing this study.

 Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

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

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 Publisher’s note

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 Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2025.1739666/full#supplementary-material

 References FotouE MoulasiotiV PapadopoulosAG KyriakouD BotiM-E MoussisV . Effect of farming system type on broilers’ antioxidant status, performance, and carcass traits: an industrial-scale production study. Sustainability. (2024) 16:4782. doi: 10.3390/SU16114782 NdukuX StempaT LunguSN NdobeniTN MlamboV. Prospects for antibiotic-free poultry production in South Africa: an analysis of the enablers and stumbling blocks. One Health. (2025) 21:21101144. doi: 10.1016/J.ONEHLT.2025.101144 SelimS MegeidASN AlhotanAR EbrahimA HusseinE. Nutraceuticals vs. antibiotic growth promoters: differential impacts on performance, meat quality, blood lipids, cecal microbiota, and organ histomorphology of broiler chicken. Poult Sci. (2024) 103:103971. doi: 10.1016/J.PSJ.2024.103971 SousaM MachadoI SimõesCL SimõesM. Biocides as drivers of antibiotic resistance: a critical review of environmental implications and public health risks. Environ Sci Ecotechnol. (2025) 25:25100557. doi: 10.1016/J.ESE.2025.100557 CarvalhoFA de MoraesNV CrottiAEM CrevelinEJ SantosAGD. Casearia essential oil: an updated review on the chemistry and pharmacological activities. Chem Biodivers. (2023) 20:e202300492. doi: 10.1002/CBDV.202300492 VlaicuAP UnteaEA PanaiteDT VlaicuP UnteaA PanaiteT . Effect of basil, thyme and sage essential oils as phytogenic feed additives on production performances, meat quality and intestinal microbiota in broiler chickens. Agriculture. (2023) 13:874. doi: 10.3390/AGRICULTURE13040874 WińskaK MączkaW ŁyczkoJ GrabarczykM CzubaszekA SzumnyA. Essential oils as antimicrobial agents—myth or real alternative? Molecules. (2019) 24:2130–11. doi: 10.3390/molecules24112130, 31195752 Abd El-HackME El-SaadonyMT SaadAM SalemHM AshryNM Abo GhanimaMM . Essential oils and their nanoemulsions as green alternatives to antibiotics in poultry nutrition: a comprehensive review. Poult Sci. (2022) 101:101584. doi: 10.1016/J.PSJ.2021.101584, 34942519 HassanAHA YoussefIMI AttyASN Abdel-DaimASA. Effect of thyme, ginger, and their nano-particles on growth performance, carcass characteristics, meat quality and intestinal bacteriology of broiler chickens. BMC Vet Res. (2024) 20:269–9. doi: 10.1186/S12917-024-04101-Z MajidG AsiyeA ShahrzadA BasiratpourA ZokaeiM DerakhshanM. Thymol and carvacrol supplementation in poultry health and performance. Vet Med Sci. (2021) 8:267–88. doi: 10.1002/VMS3.663 ChaviraSJ GarcíaBBH. Essential oils, chemical compounds, and their effects on the gut microorganisms and broiler chicken production: review. Agriculture. (2024) 14:1864-1864. doi: 10.3390/AGRICULTURE14111864 LiXL XiaoCC KeYZ TianG DingX BaiS . Effects of thymol and carvacrol eutectic on growth performance, serum biochemical parameters, and intestinal health in broiler chickens. Animal. (2023) 13:2242. doi: 10.3390/ANI13132242 YangJ LiC JiangS FuY ZhouG GaoY . Dietary thymol–carvacrol cocrystal supplementation improves growth performance, antioxidant status, and intestinal health in broiler chickens. Antioxidants. (2025) 14:1323–3. doi: 10.3390/ANTIOX14111323, 41300480 HongJC SteinerT AufyA LienT-F. Effects of supplemental essential oil on growth performance, lipid metabolites and immunity, intestinal characteristics, microbiota and carcass traits in broilers. Livest Sci. (2012) 144:253–62. doi: 10.1016/j.livsci.2011.12.008 HeCQ LiuY DaiY HeC YaoY LiM . The influence of plant essential oil/palygorskite composite on growth performance, blood parameters and intestinal morphology of broiler chickens. Ital J Anim Sci. (2023) 22:1073–82. doi: 10.1080/1828051X.2023.2271921 ReisJH GebertRR BarretaM BaldisseraMD dos SantosID WagnerR . Effects of phytogenic feed additive based on thymol, carvacrol and cinnamic aldehyde on body weight, blood parameters and environmental bacteria in broilers chickens. Microb Pathog. (2018) 125:168–76. doi: 10.1016/j.micpath.2018.09.015, 30205193 DingY HuY YaoX HeY ChenJ WuJ . Dietary essential oils improves the growth performance, antioxidant properties and intestinal permeability by inhibiting bacterial proliferation, and altering the gut microbiota of yellow-feather broilers. Poult Sci. (2022) 101:102087–7. doi: 10.1016/J.PSJ.2022.102087, 36095866 ZhengCJ XiaoGS YanX ZhengC XiaoG QiuT . Complex of lauric acid monoglyceride and cinnamaldehyde ameliorated subclinical necrotic enteritis in yellow-feathered broilers by regulating gut morphology, barrier, inflammation and serum biochemistry. Animals. (2023) 13:516-516. doi: 10.3390/ANI13030516, 36766404 AlagawanyM El-SaadonyMT ElnesrSS FarahatM AttiaG MadkourM . Use of lemongrass essential oil as a feed additive in quail's nutrition: its effect on growth, carcass, blood biochemistry, antioxidant and immunological indices, digestive enzymes and intestinal microbiota. Poult Sci. (2021) 100:101172-101172. doi: 10.1016/J.PSJ.2021.101172, 33951594 ChowdhuryS MandalPG PatraKA KumarP SamantaI PradhanS . Different essential oils in diets of broiler chickens: 2. Gut microbes and morphology, immune response, and some blood profile and antioxidant enzymes. Anim Feed Sci Technol. (2018) 236:39–47. doi: 10.1016/j.anifeedsci.2017.12.003 AgungI CecepH AnuragaJ RatriyantoA. Essential oils as growth-promoting additives on performance, nutrient digestibility, cecal microbes, and serum metabolites of broiler chickens: a meta-analysis. Asian Australas J Anim Sci. (2020) 34:1499–513. doi: 10.5713/AB.20.0668 ChenY WangJ YuL XuT ZhuN. Microbiota and metabolome responses in the cecum and serum of broiler chickens fed with plant essential oils or virginiamycin. Sci Rep. (2020) 10:5382. doi: 10.1038/s41598-020-60135-x, 32214106 Gholami-AhangaranM Ahmadi-DastgerdiA Karimi-DehkordiM. Thymol and carvacrol; as antibiotic alternative in green healthy poultry production. Plant Biotechnol Persa. (2020) 2:22–5. doi: 10.29252/pbp.2.1.22 AbouelezzK Abou-HadiedM YuanJ ElokilAA WangG WangS . Nutritional impacts of dietary oregano and Enviva essential oils on the performance, gut microbiota and blood biochemicals of growing ducks. Animal. (2019) 13:2216–22. doi: 10.1017/S1751731119000508, 30914073 El-HackM AlagawanyM. Performance, egg quality, blood profile, immune function, and antioxidant enzyme activities in laying hens fed diets with thyme powder. J Anim Feed Sci. (2015) 24:127–33. doi: 10.22358/jafs/65638/2015 FernandezEM KembroMJ BallesterosLM CalivaJM MarinRH LabaqueMC . Dynamics of thymol dietary supplementation in quail (Coturnix japonica): linking bioavailability, effects on egg yolk total fatty acids and performance traits. PLoS One. (2019) 14:e0216623. doi: 10.1371/journal.pone.0216623 RuanD FanQ FouadMA SunY HuangS WuA . Effects of dietary oregano essential oil supplementation on growth performance, intestinal antioxidative capacity, immunity, and intestinal microbiota in yellow-feathered chickens. J Anim Sci. (2021) 99:1–11. doi: 10.1093/JAS/SKAB033 AkramiR GharaeiA MansourRM GaleshiA. Effects of dietary onion (Allium cepa) powder on growth, innate immune response and hemato–biochemical parameters of beluga (Huso huso Linnaeus, 1754) juvenile. Fish Shellfish Immunol. (2015) 45:828–34. doi: 10.1016/j.fsi.2015.06.005 CsillaT EdinaS BohumilB NagyO. Changes of total protein and protein fractions in broiler chickens during the fattening period. Vet World. (2019) 12:598–604. doi: 10.14202/vetworld.2019.598-604 KaiserJC ReiderH PabiloniaKL MooreAR. Establishment of biochemical reference values for backyard chickens in Colorado (Gallus gallus domesticus). Vet Clin Pathol. (2022) 51:577–84. doi: 10.1111/vcp.13136 QaidMM Al GaradiMA. Protein and amino acid metabolism in poultry during and after heat stress: A review. Animals. (2021) 11:1167–7. doi: 10.3390/ANI11041167, 33921616 IrawanA HidayatC JayanegaraA RatriyantoA. Essential oils as growth-promoting additives on performance, nutrient digestibility, cecal microbes, and serum metabolites of broiler chickens: a meta-analysis. Asian Australas J Anim Sci. (2020) 34:1499–513. doi: 10.5713/AB.20.0668 AriyoWO KwakyeJ SoviS AryalB GhareebAFA HartonoE . Glucose supplementation improves performance and alters glucose transporters’ expression in pectoralis majorof heat-stressed chickens. Animals. (2023) 13:2911. doi: 10.3390/ANI13182911 ChamorroS RomeroC BrenesA Sánchez-PatánF BartoloméB ViverosA . Impact of a sustained consumption of grape extract on digestion, gut microbial metabolism and intestinal barrier in broiler chickens. Food Funct. (2019) 10:1444–54. doi: 10.1039/c8fo02465k, 30768097 BalasubramanianB LiuCW. Editorial: gut microbiota: allied with livestock nutrition, health, and welfare. Front Vet Sci. (2024):111413671:11. doi: 10.3389/FVETS.2024.1413671 FranciosiniMP Casagrande-proiettiP ForteC BeghelliD AcutiG ZanichelliD . Effects of oregano (Origanum vulgare L.) and rosemary (Rosmarinus officinalis L.) aqueous extracts on broiler performance, immune function and intestinal microbial population. J Appl Anim Res. (2016) 44:474–9. doi: 10.1080/09712119.2015.1091322 GoelA NchoCM ChoiYH. Regulation of gene expression in chickens by heat stress. J. Anim. Sci. Biotechnol. (2021) 12:11. doi: 10.1186/s40104-020-00523-5, 33431031 TianH GuoY DingM SuA LiW TianY . Identification of genes related to stress affecting thymus immune function in a chicken stress model using transcriptome analysis. Res Vet Sci. (2021) 138:90–9. doi: 10.1016/J.RVSC.2021.06.006, 34126450 WastiS SahN MishraB. Impact of heat stress on poultry health and performances, and potential mitigation strategies. Animals. (2020) 10:1266. doi: 10.3390/ani10081266, 32722335 KangH WangQ YuH GuoQ WeberLI WuW . Validating the use of a newly developed cinnamaldehyde product in commercial broiler production. Poult Sci. (2024) 103:103625–5. doi: 10.1016/J.PSJ.2024.103625, 38507831 JiménezMJ RogerB SabineS BracarenseAPFRL. Ingestion of organic acids and cinnamaldehyde improves tissue homeostasis of piglets exposed to enterotoxic Escherichia coli (ETEC). J Anim Sci. (2020) 98:skaa012. doi: 10.1093/jas/skaa012 LiZ JinX WuQ LongL LiY ZhangQ . Effects of encapsulated thymol and carvacrol mixture on growth performance, antioxidant capacity, immune function and intestinal health of broilers. Ital J Anim Sci. (2022) 21:1651–9. doi: 10.1080/1828051X.2022.2151944 ZhangK YanF KeenC WaldroupPW. Evaluation of microencapsulated essential oils and organic acids in diets for broiler chickens. Int J Poult Sci. (2005) 4:612. doi: 10.3923/ijps.2005.612.619 ZouY XiangQ WangJ PengJ WeiH. Oregano essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig model. Biomed Res Int. (2016) 2016:5436738. doi: 10.1155/2016/5436738, 27314026 PanJF ZhuYL Abdel-SamieMA PanJ ZhuY LiC . Biological properties of essential oil emphasized on the feasibility as antibiotic substitute in feedstuff. Grain Oil Sci. Technol. (2023) 6:10–23. doi: 10.1016/J.GAOST.2022.11.001 SunB HouLY YangY. The development of the gut microbiota and short-chain fatty acids of layer chickens in different growth periods. Front Vet Sci. (2021) 8:8666535–5. doi: 10.3389/FVETS.2021.666535, 34277754 WangX TsaiT DengF WeiX ChaiJ KnappJ . Longitudinal investigation of the swine gut microbiome from birth to market reveals stage and growth performance associated bacteria. Microbiome. (2019) 7:109. doi: 10.1186/s40168-019-0721-7, 31362781 StanleyD GeierSM DenmanES HaringVR CrowleyTM HughesRJ . Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed. Vet Microbiol. (2013) 164:85–92. doi: 10.1016/j.vetmic.2013.01.030 ZhaoCZ HuJL LiQ ZhaoC HuJ FangY . Transfer of nitrogen and phosphorus from cattle manure to soil and oats under simulative cattle manure deposition. Front Microbiol. (2022) 13:916610-916610. doi: 10.3389/FMICB.2022.916610 BaoH XueY ZhangY TuF WangR CaoY . Encapsulated essential oils improve the growth performance of meat ducks by enhancing intestinal morphology, barrier function, antioxidant capacity and the cecal microbiota. Antioxidants. (2023) 12:253–3. doi: 10.3390/ANTIOX12020253, 36829812 LianHZ FeiG JunWG LiH XiaF BaiH . Potential of aromatic plant-derived essential oils for the control of foodborne bacteria and antibiotic resistance in animal production: a review. Antibiotics. (2022) 11:1673–3. doi: 10.3390/ANTIBIOTICS11111673 MeligyAMA MarwaAE-H I YonisAE ElhaddadGY Abdel-RaheemSM El-GhareebWR . Liposomal encapsulated oregano, cinnamon, and clove oils enhanced the performance, bacterial metabolites antioxidant potential, and intestinal microbiota of broiler chickens. Poult Sci. (2023) 102:102683–3. doi: 10.1016/J.PSJ.2023.102683

Edited by: Teodora Popova, Institute of Animal Sciences, Bulgaria

Reviewed by: Abhijeet Champati, Siksha O Anusandhan University, India

Alejandra Paola Magnoli, National University of Río Cuarto, Argentina

Barrios-García Hugo, Universidad Autónoma de Tamaulipas, Mexico