Echinacea purpurea polysaccharides regulate the intestinal microbiota and enhance immune function in broilers

Abstract

Echinacea purpurea polysaccharide (EP), one of the main active components extracted from E. purpurea, exhibits immunomodulatory, anti-inflammatory, antioxidant, and intestinal mucosal protective effects. This study investigated the effects of EP on growth performance, antioxidant capacity, serum immune function, nutrient metabolism rate, and the composition of the intestinal microbiota in broilers. A total of 288 one-d-old Arbor Acres broilers (with an average body weight of 41.83 ± 0.32 g) were randomly allocated into four dietary treatments: a control group (CON; without EP) and three EP supplemented groups receiving 0.3%, 0.6%, or 0.9% EP designated as EPL, EPM, and EPH groups, respectively. The experimental feeding period lasted for 42 d. The results showed that, compared with the CON group, the addition of EP to the diet had no significant effect on average daily gain (ADG), the average daily feed intake (ADFI), or feed-to-gain ratio (F/G) of the broilers (P > 0.05), but did significantly reduced mortality, muscle drip loss rate, and shear force (P < 0.05). At 42 d of age, a notable increase in the serum total antioxidant capacity (T-AOC) levels and reduced malondialdehyde (MDA) levels in EPH group, compared with the CON group (P < 0.05). Compared with the CON group, the EPM group had higher high-density lipoprotein cholesterol (HDL-C) levels and lower serum total cholesterol (TC) and triglyceride (TG) levels (P < 0.05). Analysis of the caecal microbiota demonstrated that, compared with the CON group, supplementation with EP led to a marked increase in the proportional representation of beneficial bacterial populations (Alistipes, Ruminococcus_torques_group, and Bacteroides) while concurrently reducing the relative abundance of detrimental bacterial species (Streptococcus). Additionally, compared with the CON group, the addition of 0.6% EP elicited a significant increase in the concentration of short-chain fatty acids (SCFAs) in the caecal contents of the broilers both at 21 and 42 d (P < 0.05). The inclusion of EP in the diet substantially increased the nutrient metabolism rates (ether extract, crude ash, crude fibre, and calcium) of the broilers compared with those in the control group (P < 0.05). This study revealed the mechanisms by which EP enhanced broiler production performance, primarily through modulation of intestinal microbiota composition and promoting SCFA production. These changes, in turn, strengthened serum immune and antioxidant functions while improving nutrient metabolism rates. This ultimately translated into reduced mortality and improved meat quality under practical production conditions.

1. Introduction

With rising economic development and living standards, the global demand for animal products has consistently increased, spurring a steady evolution towards intensive, large-scale poultry farming (Perera and Ravindran, 2025). In recent decades, antibiotics have been extensively utilized as growth-enhancing agents in the poultry industry (Castanon, 2007). Nonetheless, the extensive use of antibiotics in poultry production has triggered public concern regarding antimicrobial resistance and potential risks to human health (Van Boeckel et al., 2015). In response, many countries have recently implemented restrictions or outright prohibitions on incorporating antibiotics into animal feed (Zhou et al., 2025). Notably, since July 1, 2020, China has outlawed the inclusion of antibiotics used solely for growth promotion in feed formulations (Schoenmakers, 2020). Hence, the imperative to diminish or substitute antibiotic utilization has emerged as a critical and time-sensitive challenge for the livestock industry.

Research has indicated that plant-derived bioactive compounds have various health benefits, including enhancing animal immunity, promoting the growth of beneficial bacteria, maintaining intestinal integrity, reducing oxidative stress, and exerting anti-inflammatory effects (Lillehoj et al., 2018). Echinacea purpurea (L.) Moench is an herbaceous perennial belonging to the Asteraceae family, and is native to North America. It is rich in various bioactive compounds, such as polysaccharides, alkylamides, caffeic acid derivatives, and phenolic compounds, and possesses significant medicinal value (Panahirad et al., 2024). According to previous reports, Echinacea has antioxidant, immunomodulatory, and antiviral functions in chickens (Lee et al., 2012b; Wang et al., 2024a). According to previous research, incorporating 1.0% Echinacea powder into the diets of broilers significantly improved growth efficiency and upregulated interleukin-10 (IL-10) expression in the spleen (Rady et al., 2023). Ashour et al. (2025) reported that adding Echinacea solution to broiler drinking water could enhance antioxidant activity and growth performance in broilers. A study conducted by Wang et al. (2024a) revealed that incorporating Echinacea extract into the diet could enhance intestinal mechanical barriers and regulate the Toll-like receptor 4-mitogen-activated protein kinase signaling pathway, thereby enhancing and prolonging broilers' immunity to avian influenza virus.

Polysaccharides are high-molecular-weight polymers composed of numerous monosaccharide molecules linked by glycosidic bonds (Fu et al., 2024). Research has indicated that polysaccharides not only enhance growth performance in broilers and modulate immune responses to reduce inflammation and stress hormone levels but also improve gut health, increase antioxidant capacity, and exert bidirectional immunomodulatory effects during stress and recovery periods. This comprehensive approach significantly increases the overall immunity and health status of broilers (Xing et al., 2023). Studies have demonstrated that incorporating Glycyrrhiza polysaccharides into the diet could enhance broiler growth performance and liver anti-inflammatory and antioxidant capacity (Ji et al., 2024). Previous research has shown that dietary supplementation with Lycium barbarum polysaccharides improve broiler immune function, antioxidant capacity, digestive enzyme activity, and growth performance (Long et al., 2020).

E. purpurea polysaccharides (EPs) are the primary natural biomolecules in E. purpurea and possess anti-inflammatory, immune-modulating, antioxidant, and gut mucosa-protective properties (Jiang et al., 2022; Jing et al., 2024). The EP can inhibit apoptosis and oxidative stress in both in vitro and in vivo oxidative damage models (Hou et al., 2020). In summary, dietary EP supplementation may improve health outcomes in broilers by enhancing immune function and antioxidant capacity through regulation of the caecal microbiota, thereby influencing production performance. Therefore, this study systematically investigated the effects of dietary EP supplementation on growth performance, immune and antioxidant functions, nutrient metabolism rates, and the gut microbiota in broilers, providing a theoretical basis for the application of EP in broiler production.

2. Materials and methods

2.1. Animal ethics statement

All experimental protocols received approval from the Animal Ethics Committee of Jilin Agricultural University (No. 20240327), and ethical and moral guidelines were rigorously adhered to throughout.

2.2. Test materials

The EP used in this study was provided by Shanxi Baichuan Kangze Biotechnology Co., Ltd. (Xianyang, Shanxi, China). The main components of EP were polysaccharides (80.12 ± 0.98)%, soluble proteins (1.92 ± 0.20)%, glucuronic acid (3.12 ± 0.02)%, and flavonoids (10.35 ± 0.07)%.

2.3. Animals and experimental design

The animal trials were carried out at the educational and research facility of Jilin Agricultural University (Changchun, Jilin, China). All the experimental broilers were raised in standard cage-type broiler houses. A total of 288 one-d-old Arbor Acres broilers (half male and half female) with an average initial body weight of 41.83 ± 0.32 g was purchased from Chundong Wood Industry Co., Ltd. (Jilin, Jilin, China). The broilers were allocated at random into four distinct groups, each consisting of six replicates, with every replicate containing 12 broilers (stocking density: 12 broilers per 2.04 m²). The groups were as follows: CON group, fed a corn-soybean meal-based diet; EPL group, 0.3% EP addition; EPM group, 0.6% EP addition; and EPH group, 0.9% EP addition. The experiment lasted 42 d and was segmented into two stages: 1 to 21 d and 22 to 42 d.

The experiment was conducted using a tiered cage rearing system, in which broilers had ad libitum access to feed via pan feeders and water via nipple drinkers. Rearing management was conducted in accordance with the GB/T 19664-2005 (China National Standard, 2005). The temperature (maintained at 32–35 °C during the first week and then reduced by 2 °C weekly until reaching 22 °C, which was maintained thereafter), humidity (50%–65%), and lighting (with a minimum of 12 h of light per d) were strictly controlled. On the 7th and 14th d of the experiment, the live Newcastle disease vaccine in chickens (La Sota strain) and the live Newcastle disease and infectious bronchitis vaccine in chickens (La Sota strain + H120 strain) were administered in the drinking water (Harbin Pharmaceutical Group Biological Vaccine Co., Ltd., Harbin, Heilongjiang, China). The feeding management protocols and biosafety guidelines for the broilers were strictly followed. The experimental diets were formulated according to the NY/T 33-2004 (Ministry of Agriculture of the People's Republic of China, 2004), and their detailed composition is presented in Table 1.

Table 1.

Ingredients and nutrient levels of the basal diet (air-dry basis, %).

Items	Groups2 (1–21 d)				Groups2 (22–42 d)
	CON	EPL	EPM	EPH	CON	EPL	EPM	EPH
Ingredients
EP	0.00	0.30	0.60	0.90	0.00	0.30	0.60	0.90
Corn	56.60	56.20	55.90	55.45	63.70	63.30	63.00	62.60
Soybean meal	35.45	35.55	35.55	35.70	28.60	28.70	28.70	28.80
Premix1	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00
Soybean oil	2.95	2.95	2.95	2.95	2.70	2.70	2.70	2.70
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Nutrients
Metabolizable energy, MJ/kg	12.46	12.47	12.48	12.48	12.61	12.62	12.63	12.63
Crude protein	21.52	21.71	21.12	21.22	19.10	19.07	19.24	19.01
Crude fiber	2.86	3.48	3.49	3.27	2.20	2.25	2.19	2.07
Calcium	0.92	0.92	0.92	0.92	0.86	0.86	0.86	0.86
Total phosphorus	0.52	0.52	0.52	0.52	0.47	0.47	0.47	0.47
Met	0.44	0.44	0.44	0.44	0.41	0.41	0.41	0.41
Lys	1.27	1.27	1.27	1.28	1.08	1.09	1.08	1.09
Thr	0.83	0.83	0.83	0.83	0.72	0.72	0.72	0.72
Try	0.27	0.27	0.27	0.27	0.23	0.23	0.23	0.23

EP = Echinacea purpurea polysaccharide; CON = control.

¹Premix provided per kg of diet (1–21 d): vitamin A, 6250 IU; vitamin D₃, 2700 IU; vitamin E, 22.5 IU; vitamin K₃, 2.2 mg; vitamin B₁, 4.25 mg; vitamin B₂, 6.25 mg; vitamin B₆, 2.55 mg; vitamin B₁₂, 1.38 mg; folic acid, 0.8 mg; calcium pantothenate, 32.5 mg; niacin, 27.5 mg; copper, 16.25 mg; iron, 265 mg; zinc, 111.25 mg; manganese, 87.5 mg; iodine, 2.5 mg; selenium, 0.33 mg; Met, 0.1%; Lys, 0.075%; calcium, 0.79%; phosphorus, 0.14%; salt, 0.28%; moisture, ≤0.5%. Premix provided per kg of diet (22-42 d): vitamin A, 6250 IU, vitamin D₃, 2700 IU; vitamin E, 28 IU; vitamin K₃, 2.2 mg; vitamin B₁, 4.25 mg; vitamin B₂, 5.5 mg; vitamin B₆, 2.55 mg; vitamin B₁₂, 1.25 mg; folic acid, 1.05 mg; calcium pantothenate, 32.5 mg; niacin, 27.5 mg; copper, 1 mg; iron, 262.5 mg; zinc, 75 mg; manganese, 132.5 mg; iodine, 2.55 mg; selenium, 0.325 mg; Met, 0.1%; Lys, 0.075%; calcium, 0.76%; phosphorus, 0.11%; Salt: 0.28%; Moisture, ≤0.5%.

²CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

2.4. Chemical analysis

The nutrient levels of the diet were determined according to the methods of the AOAC (2006). Crude protein (CP; method 984.13) was analyzed by a Kjeldahl apparatus (K9840, Hanon Keystone Technology Co., Ltd., Jinan, Shandong, China). Dry matter (DM; method 934.01) was determined by an electric blast drying oven (GZX-9140MBE, Boxun Medical Bioinstrument Co., Ltd., Shanghai, China). Crude ash (method 942.05) was determined by an intelligent integrated muffle furnace (SX2-5-12Z, Boxun Medical Bioinstrument Co., Ltd., Shanghai, China). Ether extract (EE; method 920.39) was determined using a standard Soxhlet extraction apparatus (glass components from DWK Life Sciences Co., Ltd.‌, Millville, NJ, USA). Crude fibre (CF; method 978.10) was analyzed by an automatic fiber analyzer (ANKOM DELTAi, ANKOM Technology, Madison, NY, USA). P (method 965.17) was determined by a visible spectrophotometer (721N, Shanghai Jingqi Instrument Co., Ltd., Shanghai, China) and Ca (method 968.31) was determined by ethylene diamine tetraacetic acid (EDTA) titration using a standard titration apparatus (Luda Instrument and Building Materials Co., Ltd., Hefei, Anhui, China). The metabolizable energy (ME) in the feed was analyzed by a ZDHW-5 fully automatic calorimeter (Hengfeng Coal Quality Analysis Equipment Co., Ltd., Hebi, Henan, China) according to the GB/T 45104-2024 (China National Standard, 2024).

2.5. Sample collection

Sampling was conducted at 22 and 43 d of age. One broiler was randomly selected from each replicate group. At the time of euthanasia, the selected broilers underwent venipuncture. Blood samples were collected into 2-mL sterile centrifuge tubes (Shenggong Biotechnology Co., Ltd., Shanghai, China) immediately after euthanasia. The samples were then centrifuged at 6000 × g for 15 min at 4 °C. Serum was separated and transferred to new centrifuge tubes for measurement of immunoglobulin (Ig) G, IgA, IgM, interleukin (IL)-10, IL-6, glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), malondialdehyde (MDA), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. The middle parts of the duodenum, jejunum, ileum, and liver were collected. Each tissue sample (1 cm) was promptly placed into a 5-mL cryogenic tube, rapidly frozen in liquid nitrogen, and stored at −80 °C. Separately, a 2 cm tissue sample was immersed in tissue fixative to maintain structural integrity. At the same time, 1 g fresh caecal content was collected. The samples were rapidly transferred to 5-mL sterile cryogenic tubes, immediately frozen in liquid nitrogen, and stored at −80 °C for short-chain fatty acid (SCFA) and intestinal microbiota analyses. Finally, the breast and thigh meat samples were collected for subsequent analyses.

2.6. Growth performance

All the broilers were weighed at the start of the experiment. Throughout the experimental period, body weights were measured weekly, and feed intake and mortality were recorded concurrently. Based on the experimental data, the average daily feed intake (ADFI), average daily gain (ADG), feed-to-gain ratio (F/G), and mortality of each experimental group were calculated.

2.7. Meat quality

Five indicators were used to evaluate the effect of EP on meat quality: pH value, drip loss rate, meat color, shear force, and cooking loss rate. Twenty-four hours post–slaughter, the pH values of the breast and thigh muscle tissues were determined using an FE28 pH meter (Mettler-Toledo Instruments Co., Ltd., Shanghai, China) following standard calibration procedures. At 24 h post–slaughter, samples of uniformly colored breast and thigh meat (of sufficient thickness to be opaque) were cut and then measured using a HunterLab ColorFlex color difference metre (HunterLab, Reston, VI, USA) for brightness (L∗), redness (a∗), and yellowness (b∗). The cooking loss rate, drip loss rate, and shear force at 24 h were measured using the method described by Wang et al. (2024b).

2.8. Serum immunological and antioxidant function measurements

The levels of IgG (YX-063466C), IgA (YX-0562147C), IgM (YX-0622387C), IL-10 (YX-167934C), and IL-6 (YX-126804C) in broiler serum samples at 21 and 42 d were quantified using an enzyme-linked immunosorbent assay (ELISA) with a microplate reader for absorbance detection (ST-360, Shanghai Kehua Experimental System Co., Ltd., Shanghai, China). All ELISA kits used in this study were procured from Shanghai Youxuan Biotechnology Co., Ltd. (Shanghai, China) and were employed strictly according to the manufacturer's instructions. In addition, GSH-Px (A005-1-2) activity, and T-AOC (A015-2-1) and MDA (A003-1-2) levels in the serum of the broilers at 21 and 42 d were assayed using kits provided by Nanjing Jiancheng Bioengineering Co., Ltd. (Nanjing, Jiangsu, China).

2.9. Determination of serum lipid metabolism indicators

The concentrations of TG (A110-1-1), TC (A111-1-1), LDL-C (A113-1-1), and HDL-C (A112-1-1) in serum samples at 21 and 42 d were determined using a Kehua ST-360 Microplate Reader. The kits used were provided by Nanjing Jiancheng Bioengineering Co., Ltd. All steps were performed in strict accordance with the operational guidelines provided by the reagent manufacturer.

2.10. Determination of nutrient metabolic rate

Before sampling on d 22 and 43, the excreta of the broilers were collected for three consecutive days and cleaned to remove impurities including feathers and feed. The excreta collected over three consecutive days were dried at 65 °C, crushed and mixed evenly. The contents of CP, DM, ash, EE, CF, P, and Ca in the excreta were determined according to the methods of the AOAC (2006). The determination of ME followed the same procedure as described in Section 2.4. The metabolic rates of each nutrient were computed according to the following formula:

Nutrient metabolic rate (%) = [1 – (Weight of excrement × Content of nutrient in excrement)/(Feed intake × Content of nutrient in feed)] × 100.

2.11. Determination of caecal pH and SCFA content

On d 21 and 42 of the feeding trial, the caecal contents were collected to determine the caecal pH and SCFA concentration. Caecal contents (0.1 g) were diluted with distilled water at a ratio of 1:10. After homogenization, the pH was measured with a FE28 pH meter (Mettler-Toledo Instrument Co., Ltd., Shanghai, China). The SCFA content in the caecum was determined by gas chromatography in accordance with the methods of Cao et al. (2023).

2.12. Analysis of intestinal microbiota

On d 21 and 42, after a 12-h fast, one broiler was randomly selected from each replicate. The broilers were euthanized by venous exsanguination. The caecal content samples were promptly collected in sterile cryotubes under aseptic conditions. The samples were then snap-frozen in liquid nitrogen and stored at −80 °C. Bacterial DNA was extracted from each caecal content sample using the Soil and Faecal Genomic DNA Extraction Kit (TianGen Biotech Co., Ltd., Beijing, China). The DNA concentration and A_260/280 ratio were measured using a Qubit 4.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), respectively. Barcoded fusion primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) targeting the V3–V4 region were designed for PCR amplification. After magnetic bead-based purification, the library was constructed. The molar concentration of effective library fragments was determined using a KAPA qPCR quantification kit (KAPA Biosystems Co., Ltd., Wilmington, MA, USA). Library quality was assessed via a Qubit fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the fragment size was verified using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The library mixing ratio was adjusted based on the quantitative real-time PCR results. The libraries were pooled and subjected to paired-end 250 bp (PE250) sequencing on the NovaSeq 6000 platform (Illumina, Inc., San Diego, CA, USA). The raw data were subjected to quality filtering with Fastp (version 0.23.1) to remove low-quality reads (Q < 20) and adapter contamination (Bokulich et al., 2012). Amplicon sequence variants (ASVs) were generated through denoising with DADA2 (version QIIME2-202202) (Wang et al., 2021). Species annotation was performed using QIIME2 with the Silva 138.1 database. Data analysis subsequently included species taxonomy analysis, community diversity analysis, and species differential analysis.

2.13. Statistical analysis

Statistical analysis was conducted using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). All the data were first assessed for normality using the Shapiro–Wilk test. If the P values for each treatment group were > 0.05 and the absolute skewness was < 1 and the absolute kurtosis was < 3, the data were deemed to follow a normal distribution. One-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) test was employed to determine significant differences among all treatment groups. A general linear model analysis of variance was applied to test the significance of interactions between dietary EP and age on immune function, antioxidant capacity, lipid metabolism, short-chain fatty acid levels, and nutrient metabolism rates. The following model was used:

$$Y_{ij}=\mu +a_i+{\epsilon }_{ij}\text{,}$$

where Yᵢⱼ is the dependent variable; μ is the overall mean; aᵢ is the fixed treatment effect of EP supplementation; and εᵢⱼ is the random error.

The linear and quadratic effects of different dietary EP levels were determined by using orthogonal polynomial comparison. The results are reported as the mean values and standard errors of the mean (SEM), with statistical significance set at P < 0.05 for all comparisons.

3. Results

3.1. Growth performance

The effects of the experimental treatments on growth performance are presented in Table 2. Dietary supplementation with EP did not affect ADFI, ADG, or F/G in broilers (P > 0.05). Dietary supplementation with EP linearly and quadratically reduced the mortality of the broilers (P < 0.001). Compared with those in the CON group, mortality in the EPM and EPH treatment groups was significantly lower (P < 0.001).

Table 2.

Effects of dietary supplementation of Echinacea purpurea polysaccharides (EP) on growth performance of broilers.

Items	Groups1				SEM	P-value
	CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic
1–21 d
ADG, g	29.22	29.33	29.63	30.25	0.562	0.934	0.509	0.800
ADFI, g	43.19	43.84	44.02	44.26	0.758	0.972	0.645	0.893
F/G	1.48	1.49	1.49	1.47	0.016	0.953	0.752	0.839
22–42 d
ADG, g	73.83	69.19	69.39	67.66	1.682	0.645	0.260	0.492
ADFI, g	140.31	138.17	138.76	133.26	2.662	0.839	0.402	0.684
F/G	1.91	2.00	2.00	1.97	0.033	0.746	0.616	0.551
1–42 d
ADG, g	52.07	49.75	49.99	49.42	0.911	0.769	0.392	0.632
ADFI, g	92.93	92.15	92.55	89.84	1.339	0.876	0.470	0.735
F/G	1.79	1.86	1.86	1.82	0.023	0.715	0.714	0.515
Total mortality, %	4.44a	4.17a	2.50b	2.50b	0.142	<0.001	<0.001	<0.001

ADG = average daily gain; ADFI = average daily feed intake; F/G = feed-to-weight ratio; CON = control; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.2. Meat quality

As presented in Table 3, dietary supplementation with EP quadratically reduced the drip loss rate of broiler breast meat (P = 0.002). The drip loss rate of thigh meat decreased linearly (P = 0.003) and quadratically (P = 0.004). The dietary inclusion of EP also reduced the shear force of the broiler thigh meat in a quadratic manner (P = 0.025).

Table 3.

Effects of Echinacea purpurea polysaccharides (EP) supplementation on the meat quality of broilers in 42 d.

Items	Groups1				SEM	P-value
	CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic
Breast meat
pH 24 h	6.24	6.14	6.27	6.27	0.025	0.263	0.276	0.499
Drip loss rate, %	4.73a	2.91b	3.01b	3.53b	0.220	0.001	0.133	0.002
Cooking loss rate, %	0.20	0.19	0.22	0.20	0.007	0.679	0.978	0.926
Shear force, N	13.53	13.17	12.56	16.24	1.745	0.902	0.620	0.762
Brightness	47.80	50.08	46.99	46.32	0.730	0.299	0.209	0.355
Redness	6.23	6.15	5.82	6.44	0.287	0.906	0.887	0.804
Yellowness	15.16	16.22	16.00	15.33	0.676	0.943	0.989	0.841
Thigh meat
pH 24 h	6.59	6.61	6.58	6.49	0.024	0.328	0.125	0.171
Drip loss rate, %	4.58a	3.35ab	2.88b	2.67b	0.249	0.011	0.003	0.004
Cooking loss rate, %	0.20	0.21	0.19	0.20	0.011	0.931	0.699	0.884
Shear force, N	11.87a	11.04ab	7.25b	13.02a	0.720	0.009	0.953	0.025
Brightness	48.41	48.52	47.08	48.75	0.990	0.942	0.974	0.903
Redness	8.87	7.35	8029	7.20	0.413	0.458	0.313	0.602
Yellowness	14.94	13.92	14.62	12.59	0.662	0.630	0.293	0.538

SEM = standard error of mean.

Within a row, means without a common superscript letter differ at P < 0.05.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.3. Serum immune and antioxidant functions

As presented in Table 4, dietary EP supplementation elicited significant linear (P = 0.001) and quadratic (P = 0.004) increases in serum IgA concentrations in the broilers at d 21. Compared with those in the CON group, the serum IgA levels in the EPM and EPH groups were significantly elevated (P = 0.015). Compared with those in the CON group, the IL-6 levels in the other three groups were significantly lower (P < 0.001). At 42 d, IgM levels were significantly elevated in the EPH group (linear and quadratic, P < 0.001). Dietary supplementation with EP linearly (P = 0.006) and quadratically (P = 0.014) increased serum IL-10 levels in broilers, with significant differences observed between the EPH groups (P = 0.041). As the broilers grew, the serum IgM and IL-10 levels in the EPH group increased significantly, and an interaction effect was observed between EP supplementation level and age (P < 0.05).

Table 4.

Effects of dietary addition of Echinacea purpurea polysaccharides (EP) on serum immune function of broilers.

Items	Age, d	Groups1				SEM	P-value
		CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic	EPs × age
IgA, μg/mL	21	223.4b	248.5ab	276.4aA	285.3aA	7.804	0.015	0.001	0.004	0.410
	42	191.1	241.3	231.0B	233.1B	7.688	0.092	0.147	0.109
IgG, μg/mL	21	172.1A	165.2A	170.9A	163.8	3.358	0.042	0.025	0.036	0.004
	42	131.1abB	123.6bB	148.6aB	147.3a	3.487	0.013	0.010	0.041
IgM, μg/mL	21	265.7	226.0	256.8	255.2B	10.477	0.595	0.918	0.774	0.001
	42	232.3b	201.9b	227.1b	351.6aA	13.094	<0.001	<0.001	<0.001
IL-6, pg/mL	21	15.4aA	12.9bA	13.1bA	10.1c	0.513	<0.001	<0.001	<0.001	<0.001
	42	11.2B	10.6B	11.3B	10.8	0.164	0.414	0.768	0.929
IL-10, pg/mL	21	20.5	20.0	21.6	19.5B	0.330	0.135	0.584	0.315	0.049
	42	20.5b	21.8ab	23.0ab	23.2aA	0.395	0.041	0.006	0.014

Ig = immunoglobulin; IL = interleukin; CON = control; SEM = standard error of the mean.

Different lowercase letter superscripts in the same row indicate significant differences among EP addition levels (0.0, 0.3%, 0.6%, and 0.9%) (P < 0.05). Different uppercase letter superscripts within the same column indicate significant differences between age stages (21 and 42 d) (P < 0.05); EPs × age denotes the interaction between EP addition levels and ages.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

As detailed in Table 5, at 21 d, dietary supplementation with EP linearly (P = 0.003) and quadratically (P = 0.001) increased the serum T-AOC levels of the broilers. Serum T-AOC levels were significantly greater in the EPH group than in the control group (P = 0.031). Moreover, the MDA levels in the three EP-treated groups decreased significantly (linear, P = 0.005; quadratic, P = 0.012). On d 42, the addition of EP to the diet linearly (P < 0.001) and quadratically (P = 0.004) increased T-AOC levels in broilers, with the EPH group reaching a significant level (P = 0.013). The MDA levels were significantly lower in the EPM and EPH groups (linear and quadratic, P < 0.001). As the broilers grew, the MDA levels decreased significantly in both the EPM and EPH groups (P = 0.001), with a trend towards interaction between EP supplementation level and age (P = 0.051).

Table 5.

Effects of dietary addition of Echinacea purpurea polysaccharides (EP) on antioxidant capacity of broilers.

Items	Age, d	Groups1				SEM	P-value
		CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic	EPs × age
T-AOC, mmol/mL	21	0.76b.A	0.84abA	1.01ab	1.05a	0.043	0.031	0.003	0.011	0.710
	42	0.59bB	0.62bB	0.72ab	0.79a	0.026	0.013	<0.001	0.004
GSH-Px, U/mL	21	1235B	1441B	1239B	1294B	40.323	0.241	0.462	0.565	0.126
	42	3858abA	3360bA	3949aA	3460abA	89.604	0.026	0.533	0.793
MDA, nmol/mL	21	7.32a	4.78b	5.56bA	4.60bA	0.285	0.001	0.005	0.012	0.051
	42	6.31a	4.71ab	3.19bB	3.60bB	0.317	<0.001	<0.001	<0.001

T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde; CON = control; SEM = standard error of the mean.

Different lowercase letter superscripts in the same row indicate significant differences among EP addition levels (0.0, 0.3%, 0.6%, and 0.9%) (P < 0.05). Different uppercase letter superscripts within the same column indicate significant differences between age stages (21 and 42 d) (P < 0.05); EPs × age denotes the interaction between EP addition levels and ages.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.4. Serum lipid metabolism

As presented in Table 6, serum TC levels at 21 d were quadratically lower in broilers from the EPL and EPM groups than in those from the control group following dietary EP supplementation (P < 0.001). A quadratic reduction in serum TG levels was also observed in the EPM group (P = 0.005). Serum HDL-C levels were elevated in the EPM and EPH groups compared with those in the CON group following dietary EP supplementation (linear and quadratic, P < 0.001). At 42 d, the serum TC (P = 0.001) and TG (P = 0.004) levels were significantly lower in the EPM group than in the CON group, and the HDL-C level was greater (P = 0.038). As the broilers grew, the TG levels in the EPH group decreased significantly (P < 0.05).

Table 6.

Effects of dietary addition of Echinacea purpurea polysaccharides (EP) on serum lipid metabolism indicators of broilers (mmol/L).

Items	Age, d	Groups1				SEM	P-value
		CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic	EPs × age
TC	21	4.48a	3.85b	3.68b	4.28a	0.081	<0.001	0.464	<0.001	0.356
	42	4.43a	4.14ab	3.84b	4.34a	0.066	0.002	0.455	0.001
TG	21	1.60a	1.40ab	1.22b	1.48abA	0.045	0.014	0.271	0.005	0.496
	42	1.42a	1.33a	1.04b	1.20abB	0.043	0.004	0.012	0.004
HDL-C	21	2.47bB	2.58bB	3.36aB	3.29a	0.111	0.001	<0.001	<0.001	0.091
	42	3.42bA	3.56abA	3.93aA	3.66ab	0.067	0.038	0.083	0.032
LDL-C	21	0.94	0.93	0.90	0.92	0.011	0.589	0.347	0.496	0.796
	42	0.96	0.92	0.89	0.92	0.011	0.108	0.157	0.045

TC = total cholesterol; TG = triglyceride; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol; CON = control; SEM = standard error of the mean.

Different lowercase letter superscripts in the same row indicate significant differences among EP addition levels (0.0, 0.3%, 0.6%, and 0.9%) (P < 0.05). Different uppercase letter superscripts within the same column indicate significant differences between age stages (21 and 42 d) (P < 0.05); EPs × age denotes the interaction between EP addition levels and ages.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.5. Microbial composition of the caecum

3.5.1. The ASVs and diversity analysis

As depicted in Fig. 1, a total of 482 common ASVs were shared by the caecal microbiota of broilers at 21 d (Fig. 1A). Among these, 254, 230, 265, and 242 ASVs were unique to the CON, EPL, EPM, and EPH groups, respectively. At 42 d (Fig. 1B), 701 ASVs were shared among the four groups in the caecal microbiota of broilers, among which the number of ASVs specific to the CON, EPL, EPM, and EPH groups was 460, 336, 300, and 349, respectively.

Fig. 1.

The effects of Echinacea purpurea polysaccharides (EP) on the diversity of the intestinal microbiota of broilers. (A and B) The Venn analysis of amplicon sequence variants (ASV) at 21 and 42 d. (C and D) Principal component analysis at 21 and 42 d. CON21, EPL21, EPM21, and EPH21 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 21 d, respectively; CON42, EPL42, EPM42, and EPH42 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 42 d, respectively; n = 6. CON = control.

As shown in Fig. 1C and D, the beta diversity assessment, which was conducted using principal component analysis (PCA), indicated no statistically significant differences in the microbial community compositions across the four groups. Alpha diversity serves as an integrated metric reflecting species richness and community diversity. As presented in Fig. 2, at 21 and 42 d, no significant differences were observed in the Chao1, Shannon, or Simpson indices among the four groups (P > 0.05).

Fig. 2.

The effect of Echinacea purpurea polysaccharides (EP) supplementation on alpha diversity in the cecum of broilers. Alpha diversity analysis at 21 (A) and 42 d (B). CON21, EPL21, EPM21, and EPH21 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 21 d, respectively; CON42, EPL42, EPM42, and EPH42 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 42 d, respectively; n = 6. CON = control.

3.5.2. Relative abundance at the phylum and genus levels

At the phylum level, when the broilers reached 21 d (Fig. 3A), Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteria emerged as the predominant phyla within the caecal microbiota of these broilers. In comparison, a notable reduction in the relative abundance of Proteobacteria was observed in the EPL, EPM, and EPH groups (P = 0.005). At 42 d (Fig. 3B), Firmicutes, Bacteroidota, Proteobacteria, and Campylobacterota emerged as the predominant phyla within the caecal microbiota of the broilers. Compared with the CON group, other groups exhibited a marked decrease in the relative abundance of Actinobacteria (P = 0.015).

Fig. 3.

Relative abundance of cecum microbiota in broilers at the phylum level. Analysis of phylum level at 21 (A) and 42 d (B). CON21, EPL21, EPM21, and EPH21 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 21 d, respectively; CON42, EPL42, EPM42, and EPH42 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 42 d, respectively; n = 6. EP = Echinacea purpurea polysaccharides; CON = control; F:B = Firmicutes: Bacteroidota. Different lowercase letters above columns represent significant differences among treatments at P < 0.05.

On d 21 (Fig. 4A), at the genus level, Alistipes, Faecalibacterium, Streptococcus, and Ruminococcus_torques_group constituted the predominant genera within the caecal microbiota of the broilers (collectively accounting for more than 30% of the total abundance). Compared with those in the CON group, the relative abundances of Alistipes and Ruminococcus_torques_group significantly increased in the EPM and EPH groups, whereas the relative abundance of Streptococcus was lowest in the EPH group (P < 0.05). On d 42 (Fig. 4B), Parabacteroides, Bacteroides, Faecalibacterium, and Alistipes constituted the predominant genera within the caecal microbiota of the broilers (collectively accounting for more than 40% of the total abundance). Compared with that in the control group, the relative abundance of Bacteroides in the EPL and EPM groups was significantly greater (P = 0.041). Additionally, compared with the CON group, the EPH group demonstrated a significantly greater relative abundance of Alistipes (P = 0.044).

Fig. 4.

Relative abundance of cecum microbiota in broilers at the genus level. Analysis of genus level at 21 (A) and 42 d (B). CON21, EPL21, EPM21, and EPH21 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 21 d, respectively; CON42, EPL42, EPM42, and EPH42 represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet at 42 d, respectively; n = 6. EP = Echinacea purpurea polysaccharides; CON = control. Different lowercase letters above columns represent significant differences among treatments at P < 0.05.

3.6. Caecal pH and SCFA levels

At 21 d (Table 7), compared with those in the CON group, the levels of total SCFAs, propionic acid, butyric acid, and acetic acid in the EPM group significantly increased (P < 0.05). However, the EPM did not significantly affect the pH value or other SCFAs (valeric acid, isovaleric acid, or isobutyric acid) (P > 0.05). Compared with those in the CON group, the levels of total SCFAs, propionic acid, butyric acid, and acetic acid also significantly increased in the EPM group at 42 d (P < 0.05). Moreover, the isobutyric acid concentration increased linearly (P = 0.002) and quadratically (P = 0.008) at 42 d. Among these groups, the EPH group presented significantly higher isobutyric acid concentration than the CON group (P = 0.025). As the broilers grew, the propionic acid, isobutyric acid, and total SCFAs contents significantly increased in the EPH group (P < 0.05). At 42 d, the valeric acid content in all EP-supplemented groups was significantly greater than that at 21 d (P < 0.05).

Table 7.

Effects of dietary addition of Echinacea purpurea polysaccharides (EP) on SCFA in the caecum of broilers.

Items	Age, d	Groups1				SEM	P-value
		CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic	EPs × age
pH	21	7.53	7.38	7.32	7.54	0.057	0.464	0.951	0.271	0.507
	42	7.68	7.38	7.38	7.28	0.081	0.346	0.117	0.240
Acetic acid, mmol/L	21	7.53b	11.46ab	13.81a	10.99ab	0.848	0.047	0.143	0.021	0.740
	42	10.24b	10.91ab	16.14a	13.90ab	0.968	0.013	0.062	0.095
Propionic acid, mmol/L	21	0.70b	0.99ab	1.69a	0.89bB	0.106	0.029	0.539	0.096	0.472
	42	1.41b	1.76ab	2.73a	2.57abA	0.225	0.047	0.025	0.055
Butyric acid, mmol/L	21	1.30b	1.22b	3.13a	1.67ab	0.282	0.036	0.254	0.137	0.728
	42	1.70b	1.95ab	3.02a	2.35ab	0.201	0.037	0.106	0.087
Isobutyric acid, mmol/L	21	0.16	0.12	0.18	0.10B	0.018	0.415	0.499	0.622	0.054
	42	0.19b	0.26b	0.36ab	0.43aA	0.032	0.025	0.002	0.008
Valeric acid, mmol/L	21	0.19	0.15B	0.17B	0.12B	0.018	0.613	0.250	0.525	0.195
	42	0.34	0.36A	0.46A	0.45A	0.031	0.187	0.027	0.091
Isovaleric acid, mmol/L	21	0.16B	0.18	0.13B	0.15B	0.014	0.863	0.668	0.914	0.257
	42	0.45A	0.37	0.49A	0.55A	0.048	0.159	0.033	0.098
Total SCFA, mmol/L	21	10.16b	15.04ab	19.74a	13.80abB	1.277	0.045	0.263	0.028	0.725
	42	14.24b	15.62ab	23.29a	20.33abA	1.437	0.044	0.043	0.068

SCFA = short-chain fatty acid; CON = control; SEM = standard error of the mean.

Different lowercase letter superscripts in the same row indicate significant differences among EP addition levels (0.0, 0.3%, 0.6%, and 0.9%) (P < 0.05). Different uppercase letter superscripts within the same column indicate significant differences between age stages (21 and 42 d) (P < 0.05); EPs × age denotes the interaction between EP addition levels and ages.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.7. Nutrient metabolic rate

As presented in Table 8, at 21 d, dietary supplementation with EP led to a reduction in the DM digestibility of broilers in both the EPM and EPH groups (linear and quadratic, P < 0.001). Additionally, the digestibility of EE, ash, and Ca in the three EP-treated groups were notably greater than those of the CON group (P < 0.05). At 42 d, compared with those of the CON group, the digestibility of EE, CF, and P in the EPL and EPM groups increase quadratically (P < 0.05). Furthermore, compared with those in the CON group, the digestibility of CF and Ca in the three EP-treated groups significantly increased (P < 0.05). As the broilers grew, the DM and ash digestibility in the EPL group increased significantly (P < 0.05). At 42 d, the CF digestibility in all EP supplementation groups was significantly greater than that at 21 d, with an interaction effect observed between EP supplementation level and age (P = 0.018).

Table 8.

Effects of dietary addition of Echinacea purpurea polysaccharides (EP) on nutrient metabolic rate of broilers (%).

Items	Age, d	Groups1				SEM	P-value
		CON	EPL	EPM	EPH		ANOVA	Linear	Quadratic	EPs × age
DM	21	92.7b	92.5bB	95.5a	95.3a	0.458	<0.001	<0.001	<0.001	<0.001
	42	95.5b	96.4aA	95.8ab	96.1ab	0.119	0.020	0.491	0.479
CP	21	72.6	77.0	75.2	75.6	0.973	0.481	0.498	0.537	0.722
	42	78.6	80.3	77.6	79.0	0.668	0.599	0.768	0.952
EE	21	75.0bA	82.8aA	83.2aA	80.7aA	0.992	0.001	0.083	<0.001	0.450
	42	68.5cB	77.8aB	76.7abB	71.7bcB	1.052	<0.001	0.571	<0.001
Ash	21	34.5c	41.3bB	47.8a	42.3ab	1.289	<0.001	0.011	<0.001	0.009
	42	35.6b	48.3aA	41.0ab	38.6ab	1.643	0.020	0.903	0.122
CF	21	27.1c	42.8aB	34.8bB	27.3cB	1.771	<0.001	0.439	0.003	0.018
	42	36.4b	51.6aA	52.9aA	50.2aA	2.135	0.005	0.038	0.003
Ca	21	51.8bB	65.0aB	63.1a	63.6a	1.519	<0.001	0.020	0.002	0.187
	42	58.4cA	70.5aA	67.3ab	63.5b	1.271	<0.001	0.467	0.003
P	21	77.0A	79.5A	79.0	76.9A	0.518	0.146	0.716	0.078	0.274
	42	70.6bB	76.8aB	77.3a	71.8abB	0.938	0.007	0.810	0.002
ME	21	80.0B	81.7B	81.9	82.4	0.411	0.190	0.052	0.112	0.077
	42	82.4bA	85.5aA	83.5ab	82.7b	0.387	0.006	0.535	0.059

DM = dry matter; CP = crude protein; EE = ether extract; Ash = crude ash; CF = crude fiber; ME = metabolizable energy; CON = control; SEM = standard error of the mean.

Different lowercase letter superscripts in the same row indicate significant differences among EP addition levels (0.0, 0.3%, 0.6%, and 0.9%) (P < 0.05). Different uppercase letter superscripts within the same column indicate significant differences between age stages (21 and 42 d) (P < 0.05); EPs × age denotes the interaction between EP addition levels and ages.

¹CON, EPL, EPM, and EPH, represent EP addition levels of 0.0, 0.3%, 0.6%, and 0.9% in the diet, respectively (n = 6).

3.8. Spearman correlation analysis

Spearman correlation analysis was employed to evaluate the relationships among the caecal microbiota, serum immune function, serum antioxidant capacity, and serum lipid metabolism (Fig. 5). At 21 d (Fig. 5A), a notable positive correlation was observed between the Faecalibacterium abundance and serum IgG concentration, whereas a significant negative correlation was detected between the Faecalibacterium abundance and serum MDA level (P < 0.01). The abundance of Escherichia-Shigella was strongly positively correlated with the serum IL-6 concentration and strongly negatively correlated with serum GSH-Px activity (P < 0.01). Conversely, UCG-005 levels were significantly inversely correlated with serum IL-6 levels and strongly positively correlated with serum GSH-Px activity (P < 0.01). At 42 d (Fig. 5B), the abundance of Bacteroides was significantly negatively correlated with the LDL-C and TC levels (P < 0.01). The Ruminococcus_torques_group and the NK4A214_group were significantly positively correlated with HDL-C concentrations but strongly negatively correlated with serum MDA and TC levels (P < 0.01). This study revealed a substantial increase in the relative abundance of Bacteroides, suggesting that Bacteroides might be related to host lipid metabolism.

Fig. 5.

Heat map of Spearman correlation analysis. Correlation analysis at 21 (A) and 42 d (B). The color scale ranges from light green to deep red, indicating correlation coefficients from low to high. The darker the color, the stronger the correlation. Ig = immunoglobulin; IL = interleukin; T-AOC = total antioxidant capacity; GSH-Px = glutathione peroxidase; MDA = malondialdehyde; TC = total cholesterol; TG = triglycerides; L-DLC = low-density lipoprotein cholesterol; H-DLC = high-density lipoprotein cholesterol. ∗∗ P < 0.01.

4. Discussion

This study revealed that adding EP to the diet of broilers had no effect on the ADFI, ADG, or F/G. Lee et al. (2012a) used dry E. purpurea powder as a feed additive for broilers and reported that it did not significantly affect their growth performance. In a study conducted by Nosrati et al. (2017), incorporating E. purpurea extract into the diet of broilers did not notably affect indicators associated with their growth performance. Nevertheless, the inclusion of EP in the dietary regimen led to a significant decrease in mortality among the broilers.

These findings may imply that the EP feed additive strategy also contributes to the health and disease resistance of broilers (Sarangi et al., 2016). Natural polysaccharides can intervene in diseases by regulating intestinal microecology, thereby acting as prebiotics (Xu et al., 2023). In the past several decades, improving the quality of raw meat for consumers has always been key (Leroy et al., 2023). In this research, the influence of EP on the meat quality of 42-d-old broilers was investigated. Meat color, pH, water-holding capacity, and tenderness are considered the core nutritional indicators for measuring the quality of chickens (Wang et al., 2020). Polysaccharides have multiple functions in meat and meat products, such as improving texture, increasing moisture retention and extending shelf life (Abubaker et al., 2025). This research revealed that, compared with the control group, dietary supplementation with EP reduced the rate of drip loss in the breast and thigh meat. Additionally, the shear force of the thigh meat decreased quadratically, enhancing the water-holding capacity and tenderness of the muscle. Huang et al. (2021) reported that polysaccharides derived from Morinda officinalis could reduce the rate of drip loss in the muscles of broilers with tibial dyschondroplasia, improve their water-holding capacity, and enhance meat quality. Zhou et al. (2024) revealed that incorporating Pleurotus citrinopileatus polysaccharide into the diet of broilers could reduce drip loss and shear force in muscles. The improvement in water-holding capacity directly affects muscle tenderness (Wang et al., 2023), thereby enhancing meat quality.

Under antigen induction, humoral immunity activates B lymphocytes to secrete specific antibodies, neutralizing antigen activity to establish immune protection (Zhang et al., 2021). Different Ig types perform distinct functions in broilers: IgA protects mucosal surfaces from microbial invasion (Kondo et al., 2025); IgM serves as the primary antibody in adaptive immunity, dominating the early response (Watson et al., 2025); and IgG can be transferred maternally, specifically binding pathogens to enhance immune recognition (Chang et al., 2022). Cytokines (such as proinflammatory IL-6 and anti-inflammatory IL-10) act as biomarkers for evaluating systemic defence responses (Xiao et al., 2024). Plant polysaccharides activate lymphocytes, promoting immunoglobulin and cytokine secretion to regulate immunity (Wang et al., 2025a). For instance, polysaccharides from Yingshan Yunwu tea was found to increase serum IgA and IgG levels in broilers (Xiang et al., 2020). Codonopsis pilosula polysaccharides have been shown to improve the immune status of immunocompromised mice and restore serum IL-6, IgM, and IgG levels to normal (Rong and Shu, 2024). Similarly, EP can suppress the abnormal elevation of inflammatory factors such as serum IL-6, in lung cancer mice, thereby improving the immune environment (Shen et al., 2025). Cytokines increase B-cell activity, and B cells produce immunoglobulins (Banskota et al., 2000). This study revealed that EP supplementation increased serum IgM, IgA, and IL-10 concentrations while decreasing IL-6 levels in broilers. These findings suggest that EP has a beneficial effect on the immunological reactivity of broilers. As broilers grow, serum IgM and IL-10 levels significantly increase, with an interaction observed between EP supplementation levels and age. With increasing age, the immune organs of broilers gradually develop and mature, increasing the number and function of immune cells; consequently enhancing synthesis and secretion of immunoglobulins and cytokines (Lebedev et al., 2024).

Polysaccharides are known to possess intrinsic antioxidant characteristics, attributes that underlie their prospective advantages for health and their applicability in therapeutic contexts (Gao et al., 2024). Oxidative stress arises from an imbalance between the formation of oxidants and their elimination by the cellular antioxidant system, leading to the oxidation of biomolecules (such as DNA, lipids, and proteins) and causing tissue damage (Gessner et al., 2016). Under steady-state physiological conditions, small amounts of reactive oxygen species do not cause damage and work in coordination with the body's antioxidant system to maintain normal function (Lian et al., 2022). However, excessive accumulation of reactive oxygen species stimulates excessive antioxidant defences, causing oxidative stress damage and leading to the onset of various diseases (Jiang et al., 2016). The T-AOC represents the cumulative measure of all antioxidant capacities within the serum, thereby indicating, to a certain degree, the organism's comprehensive ability to scavenge free radicals (Ghiselli et al., 2000). The MDA frequently serves as an indicator of endogenous lipid peroxidation and the harm inflicted by free radicals within biological systems (Pitino et al., 2021). Studies have shown that incorporating Glycyrrhiza polysaccharides into the diet of broilers increases T-AOC level and antioxidant enzyme activity. Concurrently, this dietary supplementation effectively decreases MDA levels in both the serum and liver of broilers, thereby bolstering their overall antioxidant capacity (Ji et al., 2024). According to a study conducted by Lee et al. (2012b), incorporating E. purpurea into the diet of broilers markedly elevated the activity levels of antioxidant enzymes in the serum, liver, and spleen, consequently improving their overall antioxidant capacity.

This study revealed that dietary supplementation with EP led to a notable increase in serum T-AOC levels among broilers but concurrently decreased MDA levels. These findings suggest that EP has the potential to strengthen systemic antioxidant defence mechanisms in broilers. Furthermore, as the broilers grew, serum MDA levels decreased significantly, with a trend towards interaction between EP supplementation levels and age. As broilers mature, their antioxidant systems gradually develop and mature, enabling more efficient clearance of free radicals and oxidative products within the body and thereby reducing MDA levels (Pesti-Asbóth et al., 2023). In vivo experiments have revealed that EP can significantly reduce oxidative damage in the serum and liver of mice and exhibits significant antioxidant activity (Hou et al., 2020). Lipid metabolism, encompassing both biosynthetic and breakdown processes, plays a pivotal role for virtually all aspects of cellular operation. Among the various metabolic routes involved in lipid metabolism, the pathways responsible for fatty acid and cholesterol metabolism stand out as the most critical (Sun et al., 2022). A diverse array of lipids, including cholesterol and TG, participates in lipid biosynthesis, a process of fundamental importance for cellular function (Howie et al., 2018). Dyslipidaemia is characterized by an imbalance in lipid metabolism, with elevated plasma levels of TG, TC, and LDL-C coupled with reduced concentrations of HDL-C (Sun et al., 2022).

Previous studies have shown that plant polysaccharides can promote and regulate lipid metabolism in animals. A study revealed that dietary supplementation with Ganoderma lucidum polysaccharides could significantly increase serum HDL-C concentrations while concurrently lowering serum TG levels in broilers (Gao et al., 2024). Moreover, investigations revealed that Radix rehmanniae praeparata polysaccharides markedly decreased the serum concentrations of TG, TC, and LDL-C in broilers (Yang et al., 2023). In this study, incorporating EP into the diet resulted in an increase in serum HDL-C levels, which was accompanied by a concurrent reduction in the serum levels of TC and TG, and as the broilers grew, the TG levels decreased significantly; this might be because the microbiota ferments polysaccharides to produce SCFAs, which hinder the biosynthesis of cholesterol and facilitate the transformation of primary bile acids into their secondary forms, which is crucial for the emulsification of cholesterol (Silva et al., 2021; Wahlström et al., 2016). These results indicate that EP can effectively regulate serum lipid metabolism and had the greatest effect observed on the EPM group.

The gut microbiota exists in a mutually beneficial symbiosis with its host, regulating serum metabolites and influencing immune responses (Dodd et al., 2017). Microbiome imbalance disrupts the body's homeostasis, triggering various diseases, such as inflammatory bowel disease and irritable bowel syndrome (Singh et al., 2021). The caecum has the greatest microbial density and diversity in broilers (Stanley et al., 2015). Upon entering the animal body, polysaccharides provide energy, exert effects within the gut, and can be metabolized by microorganisms (Yu et al., 2024). Previous studies have shown that polysaccharides can regulate the structure of microbial communities, thereby promoting intestinal health (Lai et al., 2023; Yin et al., 2020). In the current research, at 21 and 42 d, Firmicutes and Bacteroidota emerged as the predominant phyla within the intestinal microbiota, accounting for more than 80% of the intestinal microbiota (Zhao et al., 2024). Notably, Firmicutes play a pivotal role in facilitating polysaccharides breakdown and are linked to enhanced nutrient assimilation (Gu et al., 2024). Bacteroidota are assumed to play a significant role in the breakdown of carbohydrates, with a particular emphasis on the degradation of starch and pectin (He et al., 2024). This study revealed that adding EP to the diet led to a decrease in the proportional representation of Proteobacteria and Actinobacteria within the caecal microbiota. Proteobacteria include many pathogenic bacteria associated with intestinal inflammation, such as Escherichia coli, Salmonella, and Vibrio (Cuesta et al., 2022). Proteobacteria are gram-negative bacilli that use lipopolysaccharides as the main component of their outer membrane (Cani et al., 2008). Lipopolysaccharides induce oxidative stress, trigger inflammatory responses, and lead to dysbiosis of the intestinal microbiota (Sampath, 2018; Zhong et al., 2019). The Actinobacteriota can play a role in modulating the equilibrium of the host's intestinal microbial community during the initial developmental phase, and its relative abundance gradually decreases as gut function stabilizes (Ruan et al., 2024). Research has revealed that the Actinobacteriota is composed of various pathogenic bacteria, and their relative abundance is negatively correlated with intestinal health (Gao et al., 2025).

When analyzed at the genus level, the inclusion of EP in the diet led to a marked increase in the proportional representation of genera such as Alistipes, Bacteroides, and Ruminococcus_torques_group. Bacteroides can increase host utilization of polysaccharides (Bäckhed et al., 2004), increase host immunity and maintain the balance of the intestinal microbiota (Stappenbeck et al., 2002; Hooper et al., 2001). Furthermore, correlation analysis revealed that the abundance of Bacteroides was significantly negatively correlated with LDL-C and TC levels. Bacteroides is involved in the synthesis of SCFAs in the body (Yang et al., 2024a), among which butyric acid regulates lipid metabolism (Han et al., 2018; Zhang et al., 2011). Propionic acid may inhibit the biosynthesis pathways of fatty acids and cholesterol in the body and may also participate in and mediate the role of soluble dietary fibre in lowering blood lipids (Wright et al., 1990). Studies have demonstrated that incorporating polysaccharides derived from Astragalus membranaceus and Glycyrrhiza uralensis into the diet can lead to an increase in the relative abundance of Alistipes and Ruminococcus within the caecal microbiota of broilers (Qiao et al., 2022).

Alistipes is a rod-shaped, gram-negative anaerobic bacteria that is commonly found in the intestines of healthy humans (Parker et al., 2020). A study revealed that adding Polygonatum sibiricum polysaccharides to the daily diet increased the relative abundance of Alistipes in the caecum (Yang et al., 2024b). According to Li et al. (2022), the relative proportion of Alistipes within the caecal contents appears to increase the levels of small intestinal mucosal factors, including mucin 2, while concurrently decreasing the concentrations of inflammatory cytokines. Furthermore, Ruminococcus_torques_group belongs to the Ruminococcus family and can produce SCFAs by fermenting polysaccharides (Rao et al., 2024). The SCFAs represent a category of fatty acid compounds that can be assimilated by the host organism and subsequently utilized as energy sources (Biddle et al., 2013); they can regulate lipid metabolism and enhance immune function in broilers (Morrison and Preston, 2016). In the current research, incorporating EP into the diet led to a decrease in the relative proportion of Streptococcus within the caecal microbiota of broilers. Streptococcus is a common pathogenic bacterium that primarily colonizes the respiratory tract and causes respiratory infections (Brouwer et al., 2016). Additionally, a study revealed that under pathological conditions, certain Streptococcus strains can proliferate abnormally and become dominant in the intestine (Zoetendal et al., 2012). These bacteria can cause various infectious diseases, including local inflammation, systemic sepsis, and secondary complications (Kitamura et al., 2023). Overall, these findings suggested that the addition of EP to the diet promotes the colonization of beneficial bacteria (Alistipes, Ruminococcus_torques_group, and Bacteroides) while inhibiting pathogenic bacteria (Streptococcus), with the best results observed in the EPM and EPH groups.

The SCFAs are metabolites produced by the gut microbiota, with propionic acid, acetic acid, and butyric acid being the most important SCFAs in the intestine (Burananat et al., 2025). The SCFAs serve as key mediators in the communication between the gut microbiota and immune system, improving gut health by maintaining intestinal barrier integrity, suppressing proinflammatory factor expression, and regulating the state of innate immune cells (Botticelli et al., 2020; Ratajczak et al., 2021; Wasiewska et al., 2025). Research has indicated that polysaccharides enhance the metabolic activities of the intestinal microbiota, promote probiotic proliferation, and increase SCFA levels (Guo et al., 2025). For instance, Crataegus pinnatifida polysaccharides increase acetate production (Guo et al., 2021), whereas fucoidan enhances butyrate metabolism (Liu et al., 2022). In the present study, at the ages of 21 and 42 d, the concentrations of propionic acid, acetic acid, and butyric acid within the caecal contents of the broilers in the EPM group were markedly greater than those in the CON group. The levels of propionic acid, isobutyric acid, valeric acid, and total acid in 42-d-old broilers were significantly greater than those in 21-d-old broilers. These findings may be closely related to the increase in certain bacterial populations. As a pivotal genus belonging to Firmicutes, Ruminococcus is capable of generating SCFAs and plays a vital role in preserving intestinal well-being (Gaffney et al., 2021).

Systematic classification revealed that Bacteroides and Alistipes belong to Bacteroidota, which can produce propionic acid, acetic acid, and butyric acid (Macfarlane and Macfarlane, 2007). These findings further indicated that EP can maintain intestinal health, regulate immune function, and modulate lipid metabolism through increasing the relative proportion of bacteria capable of producing SCFAs. Moreover, SCFAs significantly promote the growth and proliferation of intestinal epithelial cells, improving the morphology of the small intestine in broilers and maintaining overall intestinal integrity (Tan and Coussens, 2007). By enhancing intestinal barrier function and reducing the leakage of undigested nutrients, the metabolic rate of nutrients is improved (Wang et al., 2025b). This study revealed that EP can increase the metabolic rates of DM, ash, EE, CF, Ca, and P in broilers; promote nutrient absorption; enhance immune function, antioxidant capacity, and lipid metabolism; reduce mortality; and promote overall health. As the broilers grew, the metabolic rates of DM and ash significantly increased, with an interaction observed between EP supplementation levels. By regulating the Toll-like receptor 4/nuclear factor κ-B (TLR4/NF-κB) pathway, EP markedly promotes immune cell proliferation and antibody production during the later growth stages. This reduces energy expenditure and immune responses, thereby redirecting more nutrients towards metabolic processes (Zhang et al., 2025).

The addition of EP to feed might maintain the stability of the intestinal microbiota by promoting the growth of beneficial bacteria (such as Alistipes, Ruminococcus_torques_group, and Bacteroides) and inhibiting the proliferation of harmful bacteria (such as Streptococcus). A stable gut microbiota can ferment EP to produce SCFAs, thereby promoting gut health. A healthy gut is the foundation for efficient nutrient metabolism and robust immune function (Wells et al., 2017). Enhanced immune function and improved antioxidant capacity can effectively eliminate pathogens, protect immune cells from free radical damage, and reduce the incidence of infection, thereby lowering the mortality of broilers and ultimately promoting their overall health.

5. Conclusions

In conclusion, incorporating EP into the diet significantly reduced the shear force and drip loss rate of the breast and thigh muscles of broilers, thereby effectively improving the meat quality. Moreover, the addition of EP optimized the gut microbiota structure and increased SCFA concentrations, thereby enhancing immune function, improving antioxidant capacity, optimizing lipid metabolism, and reducing mortality. A healthy intestinal environment also increased nutrient metabolism rates. The results revealed that the addition of EP at 0.6% and 0.9% had significant effects. However, further studies are needed to elucidate the underlying mechanisms and to determine the optimal EP inclusion levels for maximizing benefits in broiler production.

Credit Author Statement

Wenting Gou: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. Ze Wang: Validation, Investigation. Yuqian Liu: Validation, Investigation. Jia Fu: Validation, Investigation. Mengxue Liu: Validation, Investigation. Qiang Si: Writing – review & editing. Nanyi Zhang: Writing – review & editing. Hongmei Shang: Writing – review & editing, Project administration, Formal analysis.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.