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Effects of Three Artemisia Extracts and Their Combinations on the Gut Barrier Function and Transcriptomic Regulation of the Jejunum in Laying Hens
2026 年 63 巻 論文ID: 2026023
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2026 年 63 巻 論文ID: 2026023
Maintaining intestinal health during the late-laying period is important for sustaining feed efficiency and bird health. Plant-derived feed additives have been explored as alternatives to antibiotic growth promoters. This study evaluated the effects of dietary supplementation with the extracts of three Artemisia species, individually and in combination, on laying performance, antioxidant status, and intestinal barrier function in laying hens. A total of 720 62-week-old Jingfen No. 6 laying hens were assigned to eight dietary treatments, with six replicate cages per treatment and 15 hens per cage. The treatments included a basal diet and diets supplemented with single or combined aqueous extracts of Artemisia argyi, Artemisia ordosica, or Artemisia annua for eight weeks. Compared to the control group, hens receiving combined Artemisia extracts showed a reduced feed-to-egg ratio (P < 0.05). The combined treatments also improved jejunal morphology, as indicated by increased villus height and villus height-to-crypt depth ratio (P < 0.05). In addition, the supplemented groups exhibited enhanced antioxidant capacity and modulated intestinal immune-related indices, as reflected by higher total superoxide dismutase, Glutathione peroxidase, Total antioxidant capacity, and Secretory immunoglobulin A levels (P < 0.05). The combined extract supplementation reduced serum IL-1β and IL-6 levels and Diamine oxidase activity while increasing Claudin-1 expression compared with the control group (P < 0.05). Jejunal transcriptomic analysis suggested that lipid metabolism, retinol metabolism, and peroxisome proliferator-activated receptor (PPAR)-related pathways might be involved in the response to combined Artemisia supplementation. Overall, the combination of Artemisia argyi, Artemisia ordosica, and Artemisia annua extracts may improve antioxidant status and intestinal barrier function, thereby contributing to improved feed utilization under the present experimental conditions.
The late-laying period is a critical stage in commercial egg production during which advancing age is often accompanied by a decline in laying performance, redox balance, immune responsiveness, and intestinal physiological functions[1]. In aged laying hens, oxidative stress and impaired intestinal barrier function may reduce nutrient utilization efficiency and increase susceptibility to intestinal dysfunction[2]. Therefore, maintaining intestinal health during the late-laying period is important for sustaining feed utilization, physiological stability, and overall bird health.
Jingfen No. 6 is a representative Chinese pink-shell laying hen strain that has been used in commercial laying-hen research and studies related to egg quality and aging-associated physiological changes[3,4]. Similar to other high-producing laying hens, maintaining intestinal function and physiological homeostasis during the late-laying stage remains a practical challenge for Jingfen No. 6 hens. In this context, nutritional strategies that support the intestinal antioxidant capacity, mucosal immune balance, and epithelial barrier integrity may be particularly relevant for late-laying Jingfen No. 6 hens.
The intestine is the main site of nutrient digestion and absorption and serves as an essential physical and immunological barrier. The integrity of the intestinal mucosal structure and tight junction proteins collectively upholds the selective permeability of the intestinal tract. This system effectively blocks the entry of pathogenic microorganisms, endotoxins, and other detrimental substances into the systemic circulation[5,6]. Poultry are continuously exposed to a multitude of stressors such as high stocking density, environmental fluctuations, and routine management procedures in intensive production systems[7]. Therefore, the development of safe and effective feed additive strategies to support intestinal health has become increasingly important in poultry production, particularly in the context of reducing or replacing antibiotic growth promoters[8]. Plant extracts are increasingly recognized as promising feed additives owing to their antimicrobial effects, potential to shape intestinal microbial communities, disease-preventive effects, and growth-promoting potential, along with their inherent safety, efficacy, and natural origin[9].
As a prominent genus in the Asteraceae, Artemisia consists of diverse widely distributed species. Their aqueous extracts contain abundant secondary metabolites applied in traditional medicine and animal husbandry. Artemisia argyi exhibits antibacterial, anti-inflammatory, immunomodulatory and antioxidant activities[10,11]. Artemisia ordosica has multiple bioactive functions and relieves murine inflammatory bowel disease[12,13]. As a natural and ecofriendly botanical feed additive, Artemisia annua exhibits immunomodulatory, anti-inflammatory, and antioxidant properties[14].
The chemical compositions of Artemisia extracts vary among the species. Phenolic acids and caffeoylquinic acids such as chlorogenic acid and dicaffeoylquinic acid derivatives are common phenolic constituents of Artemisia species, whereas flavonoids such as apigenin, luteolin, rutin, isoquercitrin, and related glycosides have also been reported in different Artemisia plants[11]. Artemisia argyi is characterized by phenolic acids, flavonoids, polysaccharides, volatile oils, and sesquiterpenes; Artemisia ordosica contains flavonoids, phenylpropanoids, terpenoids, and polysaccharide-related components; whereas Artemisia annua is known for artemisinin-related sesquiterpenes, flavonoids, phenolic compounds, and polysaccharides[15]. Inner Mongolia is rich in wild germplasm resources of the genus Artemisia, such as Artemisia argyi, Artemisia annua, and Artemisia ordosica[16,17,18], providing a basis for screening representative Artemisia species for the development of plant-derived feed additives. Because these three Artemisia species differ in their phytochemical compositions, they may exert partially distinct biological activities. Previous studies have mainly focused on individual Artemisia species. However, limited information is available regarding the comparative effects of different Artemisia extracts and their combinations on the intestinal function of laying hens. We hypothesized that combined Artemisia extracts, owing to their complementary phytochemical profiles, would provide broader support for intestinal antioxidant status, mucosal immune regulation, and barrier function than single extracts alone. Accordingly, this study evaluated the effects of three Artemisia aqueous extracts, individually and in combination, on the production performance, intestinal morphology, immune-related indices, antioxidant capacity, barrier-related parameters, and jejunal transcriptomic profiles of late-laying Jingfen No. 6 hens. This study is expected to provide useful information for the development of Artemisia-based phytogenic feed additives aimed at supporting intestinal function in laying hens.
The Artemisia argyi, Artemisia annua, and Artemisia ordosica used in the present study were harvested from Hohhot, Inner Mongolia, in 2023. The aqueous extracts were prepared according to previously established methods[16,17,18]. A brief description of the extraction procedure is provided below: The three Artemisia species were harvested separately, shade-dried at room temperature, and cut into small segments. Each sample was extracted with distilled water at a ratio of 1:25 (w/v) in a constant-temperature water bath at 80 °C for 6 h. The extracts were then concentrated using a rotary evaporator and freeze-dried to obtain powdered extracts for feeding trials. The extraction yields were 18.04% for Artemisia argyi, 23.56% for Artemisia annua, and 14.85% for Artemisia ordosica. All experimental dosages were calculated based on freeze-dried extract powder.
The phytochemical characteristics of the three Artemisia aqueous extracts have been reported in our previously published laboratory analyses. The aqueous extract of Artemisia ordosica contained relatively high levels of total flavonoids (60.20 ± 0.13 mg RE/g extract) and polysaccharides (29.43 ± 1.23 mg GE/g extract), with a DPPH radical scavenging rate of 85.52%[16]. The aqueous extract of Artemisia annua contained relatively high levels of total polyphenols (39.58 ± 6.01 mg GAE/g extract) and total flavonoids (77.04 ± 0.55 mg RE/g extract)[17]. The aqueous extract of Artemisia argyi contained phenylpropanoid- and sesquiterpene-related constituents, as well as active polysaccharides (14.91%)[18]. These compositional differences provided the basis for comparing the effects of the three extracts, individually and in combination.
Animals, Experimental Design, and DietsIn total, 720 healthy 62-week-old Jingfen No. 6 laying hens with similar initial laying rates and egg weights were used in this study. Birds were randomly assigned to eight treatment groups in a single-factor, completely randomized design, with six replicates per treatment and 15 hens per replicate. All hens were allowed a 7-day adaptation period before the start of the experiment, followed by an 8-week feeding trial. The supplementation level was selected based on preliminary dose-screening trials conducted on laying hens in our laboratory. In particular, a previous study on Artemisia argyi aqueous extract evaluated its effects on laying performance, antioxidant status, and immune function in laying hens, and provided useful information for the selected inclusion level[19]. Based on previous and preliminary dose-screening results, 1,500 mg/kg was selected as the dietary inclusion level for each Artemisia aqueous extract powder in the formal trial. Accordingly, the control group was fed a basal diet, whereas the experimental groups received diets supplemented with the following: 1,500 mg/kg Artemisia argyi aqueous extract (AA), 1,500 mg/kg Artemisia ordosica aqueous extract (AO), 1,500 mg/kg Artemisia annua aqueous extract (AL), 750 mg/kg Artemisia argyi + 750 mg/kg Artemisia ordosica aqueous extracts (AA+AO), 750 mg/kg Artemisia argyi + 750 mg/kg Artemisia annua aqueous extracts (AA+AL), 750 mg/kg Artemisia ordosica + 750 mg/kg Artemisia annua aqueous extracts (AO+AL), and 500 mg/kg Artemisia argyi + 500 mg/kg Artemisia ordosica + 500 mg/kg Artemisia annua aqueous extracts (AA+AO+AL).
The experimental hens were raised under standardized management at Inner Mongolia Lushui Qingshan Agriculture and Animal Husbandry Co., Ltd. (Chifeng, China). A closed stacked-cage housing system was used, accommodating 15 hens per replicate. Illumination was maintained for 16 h, and the feed was manually administered five times daily. Feed was provided ad libitum, and the hens had free access to water via nipple drinkers. The crude protein (CP), calcium (Ca), and phosphorus (P) in the feed were determined in accordance with the Chinese national standards GB/T 6432-2018, GB/T 6436-2018, and GB/T 6437-2018, respectively. The metabolizable energy and amino acid content were calculated based on the agricultural industry standard NY/T 3645-2020. Supplementary Table S1 presents the composition and nutritional status of the basal diet.
Sample collectionDuring the experimental period, egg weight and number were recorded daily on a per-replicate basis, and feed intake was measured on day 56 to calculate production performance variables. At the end of the experiment (day 56), one hen with body weight close to the replicate average was selected for sample collection. Six birds per treatment (one bird per replicate) were used for serum biochemical analysis, intestinal morphology analysis, determination of immune and antioxidant parameters, and tight junction gene expression analysis. The replicates served as experimental units for the statistical analysis. For the transcriptomic analysis, four jejunal samples per treatment group were randomly selected from the same sampling pool and subjected to RNA sequencing. Blood samples were collected prior to euthanasia and centrifuged at 3,000 × g for 10 min at 4 °C to obtain serum, which was used for the determination of diamine oxidase (DAO) activity and d-lactic acid content. A 2-cm segment of the middle jejunum was excised, fixed in 4% paraformaldehyde for 48 h, and processed for histological analysis. Jejunal tissue was rinsed with physiological saline, snap-frozen in liquid nitrogen, and stored at −80 °C for immune and antioxidant assays. Jejunal mucosa was carefully scraped and stored at −80 °C for tight junction gene expression analysis.
Production PerformanceDuring the experiment, the laying rate and average egg weight were calculated. At the end of the 56 day trial, total feed consumption was measured, and the average daily feed intake and feed-to-egg ratio (FCR) were calculated. The calculation formulas were as follows: laying rate (%) = 100 × number of eggs per day/number of laying hens; average egg weight (g) = total egg weight/total egg number; average daily feed intake (g) = total feed intake/number of laying hens/test days; and feed-to-egg ratio = total feed consumption/total egg weight.
Tissue Morphology of JejunumIntestinal samples were processed using standard paraffin embedding techniques, including graded ethanol dehydration, xylene clearing, and paraffin infiltration. Sections (measuring 5 μm) were stained with hematoxylin and eosin (H&E) and mounted with neutral gum. Intestinal morphology was examined using a microscope (OLYMPUS SZX10, Olympus, Tokyo, Japan), and images were analyzed using Image-Pro Plus 6.0 software. For each sample, ten intact villi and their associated crypts were randomly selected for measurement. Villus height (VH), crypt depth (CD), and villus-to-crypt ratio (VH/CD) were determined.
Jejunal Immune and Antioxidant ParametersJejunal tissues were homogenized in saline at a ratio of 1:9 (w/v) using a high-speed homogenizer and then centrifuged at 3,000 × g for 10 min at 4 °C. The resulting 10% jejunal supernatants were stored at −20 °C until analysis. Levels of IgG, IgM, secretory IgA (sIgA), IL-1β, IL-4, and IL-6 were measured using commercial ELISA kits (Wuhan Gene Med Technology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions.
For antioxidant assessment, the total protein content of jejunal tissue was determined using a commercial kit. Subsequently, total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, total superoxide dismutase (T-SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) content were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Intestinal PermeabilitySerum samples were analyzed for diamine oxidase (DAO) activity and d-lactic acid (D-LA) content using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), following the manufacturer’s instructions.
RNA Extraction and SequencingJejunal tissue samples collected at the end of the 56-day formal feeding trial were used for transcriptomic RNA-seq analysis. Four biological replicates per treatment group were randomly selected from the independent replicate cages, with one bird selected from each replicate cage to ensure biological independence. Total RNA was extracted from jejunal tissue samples using TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, followed by DNase I treatment (Takara Bio Inc., Kusatsu, Japan) to remove genomic DNA contamination. The RNA quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific), respectively. Only high-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RNA integrity number ≥ 6.5, and RNA amount > 10 μg) were used for library construction. RNA-seq libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Poly(A)+ mRNA was enriched using oligo(dT) magnetic beads, fragmented, and used for first- and second-strand cDNA synthesis. After end repair, A-tailing, and adapter ligation, cDNA fragments (200–300 bp) were selected and amplified using PCR. The libraries were sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
Raw reads were processed using fastp (v0.23.2) for quality control. The clean reads were aligned to the Gallus gallus reference genome (GRCg7b) using HISAT2 (v2.2.1). Gene annotation was performed using the National Centre for Biotechnology Information (NCBI) RefSeq database. Alignment statistics were evaluated using SAMtools (v1.15.1). Gene expression levels were quantified using FeatureCounts (v2.0.3). Differentially expressed genes (DEGs) were identified using DESeq2 (v1.38.0) based on four biological replicates per group, with thresholds of |log2FoldChange| > 1 and P < 0.05. Gene Ontology and KEGG enrichment analyses were performed using the clusterProfiler R package (v3.8.1), and enriched GO terms and KEGG pathways were considered significant at P-value < 0.05.
RNA Extraction, cDNA Synthesis, and RT-qPCR AnalysisJejunal tissue samples were collected to determine the mRNA expression of tight junction-related genes and validate the selected RNA-seq results. Total RNA was extracted from jejunal tissue using TRIzol reagent (Thermo Fisher Scientific), and its purity and concentration were assessed using a Nucleic acid analyzer (Pultton, Shanghai, China). Reverse transcription was conducted using the Hifair® II 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology Co., Ltd., Shanghai, China) on a Labcycler PCR system (SensoQuest GmbH, Göttingen, Germany) under the following conditions: 85 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min.
Quantitative real-time PCR (qPCR) was conducted using Hieff® qPCR SYBR® Green Master Mix (No Rox) (Yeasen Biotechnology Co., Ltd.) on a LightCycler® 96 system (Roche Diagnostics GmbH, Mannheim, Germany). The PCR amplification program was as follows: initial denaturation at 95 °C for 30 s; 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, and extension at 72 °C for 20 s; followed by melt curve analysis at 95°C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. β-actin was used as the internal reference gene, and relative gene expression levels were calculated using the 2−ΔΔCt method.
For tight junction-related gene expression, the expression of Claudin-1, Occludin, and ZO-1 was analyzed. To validate selected RNA-seq results, five differentially expressed genes related to immune regulation and oxidative-stress pathways, including TLR-4, PPARG, IL-6, Nrf-2, and NF-κB, were selected for RT-qPCR analysis. Primers were designed using the NCBI Primer-BLAST. The primer sequences for all genes used in the RT-qPCR analysis are listed in Table S2.
Statistical AnalysisData were analyzed by one-way analysis of variance (ANOVA) using SAS software (version 9.4). When significant effects were detected, mean comparisons among treatment groups were performed using Duncan’s multiple-range test. Differences were considered statistically significant at P < 0.05.
The effects of dietary supplementation with Artemisia extracts and their combinations on production performance are shown in Figure 1. There were no significant differences in egg production (Figure 1A) or ADFI (Figure 1D) between the groups (P > 0.05). The average egg weights (Figure 1B) in the AA+AO, AA+AL, AO+AL, and AA+AO+AL groups were significantly higher than those in the AO and AL groups (P < 0.05); however, the Artemisia extracts groups were not significantly different from the control group. In addition, the FCR (Figure 1C) in the AA+AL and AA+AO+AL groups was significantly lower than that in the control group (P < 0.05).
Effects of Artemisia extract supplementation on laying performance. (A) Egg production rate (%). (B) Average egg weight (g/egg). (C) Feed conversion ratio (F/E). (D) Average daily feed intake (ADFI) (g/d). Bars labeled with different lowercase letters indicate significant differences among treatment groups (P < 0.05), whereas bars with the same letters indicate no significant difference. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
The effects of dietary supplementation with Artemisia extracts and their combinations on jejunal morphology are shown in Table 1 and Figure 2. Representative H&E-stained images of jejunal sections from each treatment group are shown in Figure 2. VH was the highest in the AA+AO+AL group, and the combined treatment groups showed significantly greater VH than the control group (P < 0.05). No significant differences in CD were observed among the groups (P > 0.05), although all treatment groups showed lower values than the control group. The VH/CD ratios in the AL, AA+AO, AA+AL, AO+AL, and AA+AO+AL groups were significantly higher than those in the control group (P < 0.05).
| Combination of extracts from Artemisia plants | SEM | P-Value | ||||||||
| CON | AA | AO | AL | AA+AO | AA+AL | AO+AL | AA+AO+AL | |||
| Villus height/μm | 1044.09c | 1064.73abc | 1053.11bc | 1147.17abc | 1167.27ab | 1083.47b | 1169.08ab | 1175.18a | 13.67 | 0.027 |
| Crypt depth/μm | 116.48 | 106.66 | 105.63 | 110.08 | 111.76 | 102.82 | 113.33 | 109.92 | 1.24 | 0.127 |
| VH/CD | 9.01b | 10.06ab | 10.00ab | 10.62a | 10.46a | 10.55a | 10.31a | 10.77a | 0.14 | 0.050 |
Note: Values within a row with different lowercase letters indicate significant differences among the treatment groups (P < 0.05); values sharing the same letter indicate no significant differences. CON, basal diet; AA, basal diet supplemented with Artemisia argyi; AO, basal diet supplemented with Artemisia ordosica; AL, basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, and AA+AO+AL), basal diet supplemented with the corresponding combinations of Artemisia extracts.
Representative H&E-stained images of jejunal morphology in laying hens fed diets supplemented with different Artemisia aqueous extracts. (A) CON; (B) AA; (C) AO; (D) AL; (E) AA+AO; (F) AA+AL; (G) AO+AL; (H) AA+AO+AL. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts. Scale bar = 500 μm.
The effects of dietary supplementation with Artemisia extracts and their combinations on jejunal antioxidant capacity are shown in Table 2. No significant differences were observed in the CAT activity or MDA content among the groups (P > 0.05). In contrast, significant differences were detected in the T-SOD, GSH-Px, and T-AOC activities among the treatment groups (P < 0.05). T-SOD activity in the AA+AO and AA+AO+AL groups was significantly higher than that in the control, single-extract, and AA+AL groups (P < 0.05). The GSH-Px activity in all treatment groups was significantly higher than that in the control group (P < 0.05). Additionally, T-AOC activity in the AA+AO and AA+AO+AL groups was significantly higher than that in the control and AL groups (P < 0.05).
| Items | Combination of extracts from Artemisia plants | SEM | P -value | |||||||
| CON | AA | AO | AL | AA+AO | AA+AL | AO+AL | AA+AO+AL | |||
| CAT (U/mg prot) | 30.31 | 37.95 | 33.69 | 32.13 | 31.05 | 34.57 | 34.39 | 40.37 | 0.940 | 0.101 |
| T-SOD (U/mg prot) | 46.9b | 49.43b | 47.75b | 47.54b | 68.11a | 51.21b | 56.08ab | 65.77a | 1.886 | 0.006 |
| GSHPx (U/mg prot) | 29.08c | 43.93b | 63.44a | 63.57a | 48.39b | 50.19b | 60.64a | 45.19b | 1.995 | <0.001 |
| T-AOC (μmol/g prot) | 51.74b | 56.83ab | 58.50ab | 52.46b | 64.86a | 62.42ab | 56.92ab | 68.69a | 1.471 | 0.034 |
| MDA (nmol/mg prot) | 0.27 | 0.23 | 0.21 | 0.25 | 0.18 | 0.19 | 0.23 | 0.23 | 0.018 | 0.951 |
Note: Values within a row with different lowercase letters indicate significant differences among the treatment groups (P < 0.05); values sharing the same letter indicate no significant differences. CON, basal diet; AA, basal diet supplemented with Artemisia argyi; AO, basal diet supplemented with Artemisia ordosica; AL, basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, and AA+AO+AL), basal diet supplemented with the corresponding combinations of Artemisia extracts.
The effects of dietary supplementation with Artemisia extracts and their combinations on jejunal immune function are shown in Table 3. No significant differences were observed in the IgG, IgM, or IL-4 levels among the groups (P > 0.05). The concentrations of sIgA, IL-1β, and IL-6 were significantly affected by the treatments (P < 0.05). sIgA levels in the AA+AO, AA+AL, and AO+AL groups were significantly higher than those in the control group (P < 0.05). The IL-1β level in the AA+AO+AL group was significantly lower than that in the control and AO groups (P < 0.05). In addition, the IL-6 levels in the AA+AL and AA+AO+AL groups were significantly lower than those in the control and AA+AO groups (P < 0.05).
| Items | Combination of extracts from Artemisia plants | SEM | P-value | |||||||
| CON | AA | AO | AL | AA+AO | AA+AL | AO+AL | AA+AO+AL | |||
| SIgA (ug/mL.prot) | 7.58b | 7.64b | 9.07ab | 9.24ab | 9.79a | 10.70a | 9.86a | 9.54ab | 0.240 | 0.014 |
| IgG (ug/mg.prot) | 0.27 | 0.29 | 0.25 | 0.34 | 0.46 | 0.34 | 0.38 | 0.39 | 0.018 | 0.091 |
| IgM (ng/mg.prot) | 34.37 | 33.94 | 32.38 | 36.15 | 42.36 | 37.98 | 38.41 | 37.66 | 1.012 | 0.295 |
| IL-1β (pg/mL.prot) | 8.73ab | 8.19abc | 9.20a | 7.43bc | 7.00bc | 7.02bc | 7.37bc | 6.64c | 0.223 | 0.029 |
| IL-6 (pg/mL.prot) | 5.38a | 3.71ab | 3.76ab | 3.78ab | 5.08a | 3.37b | 4.04ab | 2.74b | 0.206 | 0.018 |
| IL-4 (pg/mL.prot) | 7.39 | 5.83 | 4.74 | 5.15 | 7.67 | 6.32 | 6.75 | 6.69 | 0.273 | 0.071 |
Note: Values within a row with different lowercase letters indicate significant differences among the treatment groups (P < 0.05); values sharing the same letter indicate no significant differences. CON, basal diet; AA, basal diet supplemented with Artemisia argyi; AO, basal diet supplemented with Artemisia ordosica; AL, basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, and AA+AO+AL), basal diet supplemented with the corresponding combinations of Artemisia extract.
The effects of dietary supplementation with Artemisia extracts and their combinations on intestinal permeability and tight junction gene expression are shown in Figure 3. No significant differences were observed in the serum D-LA levels (Figure 3B) among the groups (P > 0.05), although all treatment groups showed numerically lower values than the control group. The DAO activity (Figure 3A) was significantly affected by the treatments (P < 0.05), with significantly lower levels observed in the AA+AO and AA+AO+AL groups than in the control group (P < 0.05). No significant differences were detected in Occludin (Figure 3D) or ZO-1 (Figure 3E) expression between the groups (P > 0.05). However, Claudin-1 (Figure 3C) expression in the AO + AL and AA + AO + AL groups was significantly higher than that in the control group (P < 0.05).
Assessment of intestinal permeability by serum biomarkers and of tight junction gene expression by RT-qPCR. (A) Serum diamine oxidase (DAO) activity (U/L); (B) Serum D-lactate (D-LA) concentration (nmol/mL); (C) Relative mRNA expression of Claudin-1; (D) Occludin; and (E) ZO-1. Bars with different lowercase letters indicate significant differences among groups (P < 0.05); bars sharing the same letter indicate no significant difference. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
A total of 1.55 billion raw reads and 232.694 Gb of raw bases were obtained from 32 samples. After quality filtering, an average of 48.44 million clean reads were generated per sample (Table S3). The mapping rate to the reference genome ranged from 90.99% to 95.29%, with an average unique mapping rate of 92.76%. These sequencing metrics supported the use of the data for downstream analyses. Pearson’s correlation analysis was performed to assess the consistency of gene expression among the samples (Figure 4A). The correlation heatmap showed high intragroup consistency with correlation coefficients exceeding 0.90 among biological replicates. Principal component analysis (PCA) was performed to provide an overview of the global transcriptomic variation among jejunal samples. The first two principal components (PC1, 21.7%; PC2, 12.6%) explained 34.3% of the total variance (Figure 4B). The PCA plot showed a partial clustering of samples within some treatment groups, whereas an overlap was observed among several groups. These results indicated that dietary supplementation with Artemisia extracts did not induce complete separation of the overall jejunal transcriptome profiles. Therefore, differential expression and functional enrichment analyses were performed to evaluate the treatment-related transcriptomic responses. Volcano plots were used to visualize the differentially expressed genes (DEGs) in each comparison (Figure 5). The numbers of DEGs were as follows: AA vs CON, 1,367 (469 upregulated and 898 downregulated) (Figure 5A); AO vs CON, 1,381 (440 upregulated and 941 downregulated) (Figure 5B); AL vs CON, 1,136 (574 upregulated and 562 downregulated) (Figure 5C); AA+AO vs CON, 487 (212 upregulated and 275 downregulated) (Figure 5D); AA+AL vs CON, 755 (355 upregulated and 400 downregulated) (Figure 5E); AO+AL vs CON, 1,280 (463 upregulated and 817 downregulated) (Figure 5F); and AA+AO+AL vs CON, 759 (380 upregulated and 379 downregulated) (Figure 5G).
Transcriptomic consistency and variance among jejunal samples. (A) Pearson correlation heatmap of gene expression among samples. (B) Principal component analysis (PCA) of the jejunal transcriptome based on global gene expression profiles. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
Volcano plots of differentially expressed genes between the Artemisia extract-treated groups and the control group in the jejunal tissue of laying hens. DEGs were identified using thresholds of |log2FoldChange| > 1 and P-value < 0.05. Orange dots indicate upregulated genes, blue dots indicate downregulated genes, and grey dots represent genes without significant differences. (A) AA vs. CON; (B) AO vs. CON; (C) AL vs. CON; (D) AA+AO vs. CON; (E) AA+AL vs. CON; (F) AO+AL vs. CON; (G) AA+AO+AL vs. CON. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
The results of GO enrichment analysis are shown in Figure 6 and Table S4. Distinct gene expression patterns were observed among the treatment groups. Compared to the control group, the AA group showed significant upregulation of genes related to morphogenesis in the BP category and vascular endothelial growth factor receptor activity in the MF category, whereas actin cytoskeleton-related genes in the CC category were significantly downregulated (Figure 6A). The AO group exhibited significant enrichment in lipid metabolic processes (BP), and antioxidant enzyme activities, including monooxygenase activity, were markedly upregulated in the MF category. Conversely, the genes associated with cell junctions in the CC were significantly downregulated (Figure 6B). The AL group exhibited enrichment in growth-related processes (BP), with significantly increased oxidoreductase and glutathione transferase activities (MF), but decreased epithelial cell–cell adhesion (BP) (Figure 6C). In the AA+AO group, the terms related to skeletal and organ development (BP) were predominantly enriched. However, detoxification-related processes (BP) and peroxidase activity (MF) were significantly downregulated (Figure 6D). The AA+AL group exhibited significant upregulation of genes involved in the positive regulation of myeloid cell differentiation (BP), indicating a pronounced immune activation profile (Figure 6E). The AO+AL group showed increased oxidoreductase activity (MF), but decreased expression of genes related to adhesion junctions and the extracellular matrix (CC) (Figure 6F). The AA+AO+AL group showed significant enrichment and upregulation of small-molecule metabolic processes, particularly lipid metabolism (BP). In addition, oxidoreductase activity (MF) was significantly enhanced, indicating a marked improvement in the antioxidant capacity (Figure 6G).
Bar plot of Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs). BP, biological process; CC, cellular component; MF, molecular function. (A) AA vs. CON; (B) AO vs. CON; (C) AL vs. CON; (D) AA+AO vs. CON; (E) AA+AL vs. CON; (F) AO+AL vs. CON; (G) AA+AO+AL vs. CON. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
The results of the KEGG pathway enrichment analysis are shown in Figure 7 and Table S5, with the top 15 significantly enriched pathways presented in a bubble plot. Distinct metabolic and immunoregulatory patterns were observed in the treatment groups. Among the single treatment groups, the AA group showed significant upregulation of the MAPK signaling pathway and downregulation of cytokine-cytokine receptor interaction (Figure 7A). The AO group exhibited significant enrichment of the PPAR signaling pathway (Figure 7B), whereas the AL group showed significant upregulation of both the PPAR signaling pathway and retinol metabolism (Figure 7C).The AA+AO group showed significant upregulation of neuroactive ligand-receptor interactions, but downregulation of the PPAR signaling pathway (Figure 7D). Among the combined treatment groups, the AA+AL group showed upregulation of the MAPK signaling pathway and downregulation of cytokine-cytokine receptor interaction (Figure 7E). The AO+AL group exhibited a significant upregulation of retinol metabolism and the PPAR signaling pathway (Figure 7F). Notably, the AA+AO+AL group showed significant enrichment in the PPAR signaling pathway, retinol metabolism, and glycerophospholipid metabolism (Figure 7G). Genes involved in the PPAR signaling pathway in the relevant treatment comparisons are listed in Supplementary Table S6, together with their log2FoldChange values, nominal P values, and FDR-adjusted values. These enriched pathways were related to lipid metabolism, retinol metabolism, inflammatory regulation, and epithelial barrier-associated functions, suggesting that Artemisia extract supplementation may influence jejunal function through metabolic- and immune-related molecular responses.
Bubble plot of top 15 KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in the jejunal tissue of laying hens. (A) AA vs. CON; (B) AO vs. CON; (C) AL vs. CON; (D) AA+AO vs. CON; (E) AA+AL vs. CON; (F) AO+AL vs. CON; (G) AA+AO+AL vs. CON. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
To validate the reliability of the RNA-seq results, five genes were selected for RT-qPCR analysis. The effects of dietary supplementation with Artemisia extracts and their combinations on the mRNA expression of PPARG, Nrf-2, NF-κB, IL-6, and TLR-4 in the jejunum of laying hens are shown in Figure 8. Compared to the CON group, PPARG expression was significantly upregulated in the AO, AL, AA+AO, AO+AL, and AA+AO+AL groups (P < 0.05) (Figure 8A). The expression of Nrf-2 was significantly increased in all treatment groups, except AA (P < 0.05) (Figure 8B). In contrast, the expression of proinflammatory genes was significantly downregulated in several treatment groups. Specifically, NF-κB expression was significantly reduced in the AA, AA+AO, AA+AL, and AA+AO+AL groups (P < 0.05) (Figure 8C). IL-6 expression significantly decreased in the AO, AA+AL, and AA+AO+AL groups (P < 0.05) (Figure 8D). TLR-4 expression was significantly downregulated in the AA, AO, AA+AO, AA+AL, and AA+AO+AL groups (P < 0.05) compared with that in the CON group (Figure 8E). Overall, the RT-qPCR results were highly consistent with the RNA-seq data, confirming the reliability of the transcriptomic analysis.
Effects of dietary supplementation with AA, AO, AL, and their combinations on the relative mRNA expression of genes in the jejunal tissue of laying hens, as determined by RT-qPCR. (A) PPARG; (B) Nrf-2; (C) NF-κB; (D) IL-6; (E) TLR-4. Bars labeled with different lowercase letters indicate significant differences among treatment groups (P < 0.05), whereas bars sharing the same letter indicate no significant difference. CON: basal diet; AA: basal diet supplemented with Artemisia argyi; AO: basal diet supplemented with Artemisia ordosica; AL: basal diet supplemented with Artemisia annua; combined groups (AA+AO, AA+AL, AO+AL, AA+AO+AL): basal diet supplemented with the corresponding combinations of Artemisia extracts.
The three Artemisia aqueous extracts used in this study differed in their phytochemical characteristics, as described in the Introduction and Materials and Methods. These compositional differences may partly explain the distinct responses observed among the single- and combined-extract treatments and provide a rationale for comparing different supplementation strategies. As the extracts were provided as whole aqueous preparations, the observed effects should be interpreted as treatment-level responses rather than as the effects of individual purified compounds.
In the present study, dietary supplementation with Artemisia extract did not result in broad improvements in laying performance (Figure 1). However, the feed-to-egg ratio was reduced in the AA+AL and AA+AO+AL groups, suggesting that some combined Artemisia extract treatments may improve feed utilization efficiency rather than broadly enhancing laying performance. Previous studies have reported that Artemisia extracts and their metabolites may improve the nutrient utilization and growth performance of monogastric animals[16,20]. Production performance is closely associated with intestinal nutrient digestion and absorption; the beneficial effects observed in this study may be related to improvements in the intestinal physiological status[21]. Therefore, the intestinal antioxidant status, immune response, morphology, and barrier function were evaluated. Overall, these results suggest that Artemisia extract supplementation may support intestinal function and feed utilization in laying hens, with different treatments showing distinct response patterns across the measured parameters. However, as the present study was conducted using 62-week-old Jingfen No. 6 laying hens under specific feeding and management conditions, whether similar responses occur in other laying hen strains, ages, production stages, or production systems requires further investigation. Body weight and weight gain were not recorded during the feeding trial. Therefore, production-related responses can only be interpreted based on laying performance and feed-to-egg ratio.
Previous studies have demonstrated that phenolic compounds, flavonoids, and polysaccharide-related components in Artemisia plants may contribute to antioxidant responses in animals[22,23,24,25,26]. In poultry, Artemisia argyi supplementation has been reported to increase SOD, GSH-Px, and T-AOC activities and reduce MDA levels[24]. Similarly, Artemisia annua aqueous extracts have been associated with increased intestinal antioxidant capacity and Nrf2/HO-1-related responses in broilers[25]. In addition, Artemisia ordosica polysaccharides improve antioxidant capacity by modulating the Nrf2/Keap1 pathway[26]. These findings suggest that different phytochemical classes in Artemisia plants may participate in antioxidant regulation through both direct antioxidant activity and the activation of endogenous antioxidant defenses. Consistent with these studies, the present study showed that dietary supplementation with Artemisia aqueous extract affected the jejunal antioxidant status, as reflected by changes in SOD, GSH-Px, and T-AOC activities (Table 2). Mechanistically, flavonoid-related components may contribute to antioxidant regulation by reducing oxidative stress and lipid peroxidation, thereby decreasing the oxidative pressure on intestinal epithelial cells[27]. Polysaccharide-related components may further contribute to antioxidant defense by activating Nrf2/Keap1-related signaling, which is closely associated with the regulation of antioxidant enzymes[26]. As the present study used whole aqueous extract preparations, the observed antioxidant responses are likely to reflect the combined contribution of multiple phytochemical classes rather than the action of a single compound. The transcriptomic findings provide a further molecular context for these potential local antioxidant actions via redox- and metabolism-related pathways. In the AA+AO+AL group, KEGG enrichment analysis indicated enrichment of PPAR signaling (Figure 7) and glycerophospholipid metabolism, accompanied by GO terms related to lipid metabolism, retinol metabolism, monooxygenase activity, arachidonic acid epoxygenase activity, acyltransferase activity, and heme or iron ion binding (Figure 6). These pathways and functional terms may be associated with lipid handling, membrane lipid remodeling, epithelial metabolic homeostasis, and oxidative metabolism. PPAR signaling is involved in lipid metabolism and oxidative stress responses, retinol metabolism has been linked to epithelial antioxidant defense and barrier-related functions, and glycerophospholipid metabolism is closely related to membrane lipid remodeling and membrane oxidative stability[28,29]. In addition, RT-qPCR showed increased PPARG and Nrf2 expression in the AA+AO+AL group, further suggesting that PPAR- and Nrf2-related regulation may participate in the jejunal antioxidant response[30]. Therefore, Artemisia aqueous extract may support jejunal redox status through local phytochemical antioxidant activity in the intestinal environment and regulation of selected redox- and metabolism-related pathways. Further studies using purified compounds, fractionated extracts, and appropriate interaction models are needed to identify the key active constituents and evaluate potential interactions among the extracts.
Gut epithelial cells are continuously and directly exposed to oxidative stress and pathogenic challenges, and maintenance of local immune homeostasis is essential for intestinal health[7]. Existing research has reported that Artemisia species may attenuate inflammatory responses through TLR-4/NF-κB-related pathways in poultry or other animal models[24,25,31]. These effects may be associated with the phytochemical classes present in Artemisia extracts. Flavonoid- and phenolic-related components may influence mucosal immune status partly through their antioxidant activity and regulation of redox-sensitive inflammatory signaling, whereas polysaccharide-related components may participate in cytokine regulation and mucosal antibody-related responses[24,25,26,31]. In the present study, Artemisia extract supplementation affected selected jejunal immune-related indices, mainly involving mucosal sIgA and pro-inflammatory cytokines IL-1β and IL-6, whereas IgG, IgM, and IL-4 were not significantly changed (Table 3). This pattern suggests a selective immunomodulatory response rather than a broad alteration in all measured immune parameters. Notably, the hens used in the present study were healthy and were not subjected to inflammatory or pathogenic challenges. IL-1β and IL-6 are not only inflammatory mediators but also participate in normal mucosal immune regulation and epithelial homeostasis[32,33]. Under normal physiological conditions, the lower IL-1β and IL-6 concentrations observed in some treatment groups should be interpreted as changes in basal immune status or mucosal immune tone rather than direct evidence of improved disease resistance or a purely beneficial anti-inflammatory effect[34]. Meanwhile, the increased sIgA concentrations observed in several combined treatment groups suggested that Artemisia extract supplementation may also support mucosal immune activity[35]. The enrichment of PPAR signaling and retinol metabolism may provide a molecular context for these immune-related responses, as both pathways have been implicated in intestinal immune regulation and epithelial differentiation[28,29]. Therefore, Artemisia extract supplementation may help modulate jejunal immune homeostasis through interactions between mucosal immune activity, cytokine regulation, and metabolism-related pathways.
Changes in antioxidant and immune statuses may also be related to intestinal morphology. Epithelial renewal and villus development are influenced by oxidative and inflammatory status[7,36]. Villus height, crypt depth, and villus-to-crypt ratio are commonly used indicators of intestinal development and absorptive capacity[37]. Previous studies have reported that Artemisia-derived preparations can improve intestinal morphology in broilers under stress or challenge conditions[38,39,40]. In the present study, several combined extract treatments increased villus height and the villus height-to-crypt depth ratio, indicating favorable changes in jejunal morphology (Table 1). Transcriptomic results indicated that several pathways related to lipid metabolism, retinol metabolism, and redox processes were altered in the AA+AO+AL group. These pathways may be associated with membrane remodeling, metabolic homeostasis, epithelial differentiation, and antioxidant status[41,42]. Therefore, the observed changes in jejunal morphology may be linked to the combined effects of changes in the antioxidant status, mucosal immune regulation, and metabolic pathways. However, these relationships remain associative, and the present data did not establish a direct causal link between transcriptomic pathway enrichment and villus development.
Intestinal morphology contributes to absorptive surface area and may be related to epithelial barrier integrity[43,44]. Excessive reactive oxygen species and inflammatory cytokines have been reported to impair tight junction integrity by inducing lipid peroxidation and promoting tight junction protein internalization and degradation[45,46]. The maintenance of selective intestinal permeability is critical for preventing the translocation of pathogens and harmful metabolites across the epithelium[43]. This function largely depends on tight junction complexes composed of Claudin-1, Occludin, and ZO-1[44]. Disruption of these structures leads to the release of intracellular DAO into circulation, resulting in elevated serum DAO activity[47]. In this study, Artemisia extract supplementation reduced serum DAO activity and increased Claudin-1 expression in some treatment groups, indicating favorable changes in barrier-related parameters (Figure 3). Consistent with these findings, previous studies have reported that individual Artemisia species can improve intestinal barrier function in animals[48,49]. GO and KEGG enrichment analyses provided molecular context for these barrier-related responses, mainly involving epithelial structure-, adhesion-, redox-, and lipid metabolism-related processes. Since intestinal barrier integrity depends on the coordinated regulation of the cytoskeleton, adhesion complexes, extracellular matrix, tight junction proteins, and membrane lipid composition, these transcriptomic changes may reflect epithelial remodeling or adaptive barrier-related regulation rather than direct evidence of barrier impairment[43,44,50,51]. In the AA+AO+AL group, the enrichment of lipid metabolism, redox processes, and glycerophospholipid metabolism may be related to membrane lipid remodeling and epithelial metabolic homeostasis[52,53]. Together with the observed reduction in serum DAO activity and increased Claudin-1 expression, these transcriptomic patterns suggest that Artemisia aqueous extracts may influence jejunal barrier-related responses through interactions among redox status, tight junction regulation, and membrane/metabolism-related pathways.
Overall, both single and combined Artemisia extract treatments showed favorable effects on the selected intestinal functional indices. Among these treatments, the AA+AO+AL group exhibited a relatively integrated response profile, as reflected by changes in the feed-to-egg ratio, jejunal antioxidant and immune indices, intestinal morphology, barrier-related parameters, and enrichment of pathways related to PPAR signaling, retinol metabolism, glycerophospholipid metabolism, redox processes, and lipid metabolism. However, at the global transcriptomic level, the treatment groups did not show a marked separation from the control group in the PCA plot (Figure 4), indicating that these favorable responses were not accompanied by extensive transcriptomic remodeling. Instead, the effects of AA+AO+AL treatment may be more closely related to the selected genes and pathways involved in metabolic regulation, antioxidant responses, and epithelial barrier-associated functions. Thus, transcriptomic results should be interpreted together with biochemical, morphological, and barrier-related measurements. It should also be noted that the transcriptomic screening and enrichment analyses were based on nominal p-values. As shown in Supplementary Table S6, the expression of many PPAR signaling-related genes remained unchanged after FDR correction. Therefore, the PPAR-related transcriptomic results should be regarded as exploratory molecular evidence that provides supportive context for the observed physiological responses, rather than definitive gene-level confirmation of this pathway.
A limitation of the present study is that the gut microbiota composition was not analyzed; therefore, whether gut microbial changes contributed to the observed effects of Artemisia extract supplementation on jejunal antioxidant status, immune-related indices, morphology, and barrier-related parameters requires further investigation.In conclusion, aqueous extracts from three Artemisia species show great potential as natural feed additives for laying hens. This study offers preliminary molecular insights into herb-regulated intestinal responses. Further studies incorporating body weight monitoring, long-term production evaluation, and comprehensive egg quality assessment are required to confirm their practical application potential.
The raw sequence data reported in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1451457, which will be released on 2027-05-01.
The experimental animal care protocol used in this study was approved by the Animal Ethics and Welfare Committee of the Inner Mongolia Agricultural University (approval no. NND2022119, approved on 29 December 2022).
The authors declare that there are no acknowledgements.
This work was supported by the Key Research and Achievement Transformation Project of the Inner Mongolia Autonomous Region (project number 2023YFDZ0014).
Yuechen Liu: Writing – original draft, Formal analysis, and Data curation. Xiao Jin: Supervision, Resources, Project administration, Writing - review and editing, and Conceptualization. Yuanyuan Xing: Visualization, Validation, Methodology, and Investigation. Hanqiong Zhou: Data curation and Investigation. Dengsheng Sun: Software and Formal analysis. Binlin Shi: Writing – review, editing, and validation.
The authors declare that they have no conflicts of interest related to this study.
The authors have not used AI and AI-assisted technologies in the production of this study.
The online version contains supplementary material available
at https://doi.org/10.2141/jpsa.*******
