The effects of degraded polysaccharides from Acanthopanax senticosus on growth, antioxidant and immune effects in broiler chicks based on intestinal flora

Abstract

The aim of this study was to evaluate the effect of degraded polysaccharide of Acanthopanax senticosus (ASPS-1) on the immunological effects and appropriate dosage of broiler chicks with a view to developing a new feed additive. For the experimental design, 180 broiler chicks were randomly divided into six groups, ASPS-1 low, medium and high dose groups, undegraded Acanthopanax senticosus polysaccharide (ASPS) low and medium dose groups and blank control group. The drug was administered for 21 consecutive days, and the growth and data of immune organ index and immune factors were recorded on the 7th, 14th and 21st d. Finally, the effect of ASPS-1 on the intestinal flora of broiler chicks was investigated by high-throughput sequencing of the 16S rRNA gene and the correlation between the main flora and intestinal indexes was analyzed, and the function of microbial community was predicted by using PICRUSt2. The results showed that the addition of high dose of ASPS-1 could promote the body weight growth of broiler chicks, had no significant effect on immune organs, significantly promoted the increase of intestinal villi and crypt ratio, and effectively regulated the levels of serum antioxidant factors and immune indexes. Analysis of the intestinal flora showed that ASPS-1H promoted the proliferation of Lactobacillus, Faecalibacterium, Negativibacillus, and Eubacterium and inhibited the colonization of Desulfovibrio and Turicibacter, and that proliferation of Faecalibacterium, Negativibacillus and Eubacterium was associated with the development of intestinal villi. Predictive analysis of PICRUSt2 function indicates that proliferation of Lactobacillus, Faecalibacterium, Negativibacillus and Eubacterium functions through amino acid metabolism, global and overview maps, replication and repair pathways function. In summary, the addition of high doses of ASPS-1 can improve the immunity of broilers and has the potential to be used as a feed additive.

Introduction

As one of the most important poultry species globally, broilers' growth performance significantly influences the economic efficiency and sustainable development of the farming industry. While traditional growth promoters, such as antibiotics, can enhance the growth rate and feed conversion rate of broilers, their prolonged and excessive use may lead to antibiotic resistance, environmental pollution and food safety concerns (Cissé, et al., 2024; Mehdi, et al., 2018; Mensah, et al., 2014). Therefore, the search for alternatives to antibiotics has become one of the hot topics of current research. Immune function serves as the innate defense mechanism against exogenous infections against exogenous infections, and chicks are particularly vulnerable to bacterial and other diseases due to their underdeveloped immune systems. Thus, enhancing chick immunity and reducing disease incidence have become critical research priorities (Zheng, et al., 2023). Recent studies have highlighted the benefits of plant-derived bioactive compounds, including polysaccharides, in poultry production to improve growth performance, immunity and gut health (Cheng, et al., 2024; He, et al., 2024; Zhou, et al., 2024a). Acanthopanax senticosus polysaccharide (ASPS), a bioactive compound demonstrated to enhance immunity and growth performance in poultry, is emerging as a promising feed additive for broiler chickens (Long, et al., 2021; Yang, et al., 2021).

It is one of the important methods to solve the poultry diseases by adding the active substance of traditional Chinese medicine polysaccharide (Du, et al., 2023; Zhu, et al., 2024). Some studies have confirmed that many traditional Chinese medicine polysaccharides, such as Astragalus polysaccharide (Hao, et al., 2024; Wei, et al., 2022), Atractylodes macrocephala polysaccharide (Li, et al., 2021), Glycyrrhiza uralensis polysaccharide (Zhou, et al., 2022), etc., can increase the index of the immune organs of the animal organism and improve the damage of the immune organs.

The bioactivity of polysaccharides is significantly influenced by their molecular weight (Yuan, et al., 2024). Polysaccharides with larger molecular weights exhibit lower bioavailability, so researchers have focused on degrading polysaccharides to improve their bioactivity and utilization (Fan, et al., 2024; Liu, et al., 2024b; Yao, et al., 2024b). The degradation of Flammulina velutipes residue polysaccharides (FVRP) using an ultrasound-assisted H₂O₂-VC (ascorbic acid) technique yielded FVRPV. Compared with FVRP, the in vitro antioxidant capacity of FVRPV was significantly enhanced. Additionally, FVRPV was shown to enrich probiotics and reduce the abundance of pathogenic bacteria through intestinal microbiota-mediated metabolic pathways, thereby exerting its potential probiotic effects (Que, et al., 2024). Yao et al. (2024a) obtained and verified the antioxidant capacity of two degraded polysaccharides from Gleditsia sinensis seeds under ultrasonic power treatments of 300 W and 450 W. Their results indicated that ultrasonic treatments significantly improved the polysaccharides' scavenging and reducing capacities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals (Yao, et al., 2024a). Huo et al. purified the degraded polysaccharides (PHP) from the Porphyra haitanensis to obtain three fractions (P1, P2, and P3). Cellular experiments revealed that P2 and P3, which had smaller molecular weights, exhibited superior immunomodulatory activities compared to P1 and PHP (Huo, et al., 2021). These findings align with numerous studies demonstrating that polysaccharide bioactivity is enhanced following degradation. Although previous studies have obtained and validated the in vitro probiotic effects of degraded Acanthopanax senticosus polysaccharides, in vivo validation experiments remain lacking (Wang, et al., 2024c).

The effects of degraded Acanthopanax senticosus polysaccharides on the immune organs of chicks, their mechanisms of action in regulating intestinal flora to improve immune function, and their optimal dosage require further exploration. Therefore, this study aims to investigate the effects of varying additive amounts of Acanthopanax senticosus degradation polysaccharides on the immune organ index, serum immunity and antioxidant indices, and intestinal flora of broiler chicks. By elucidating the underlying mechanisms, this research seeks to provide a scientific basis for the application of Acanthopanax senticosus degradation polysaccharides in broiler chicken production.

Materials and methods

Materials

ReagentAcanthopanax senticosus was acquired from Limin Pharmacy in Liaocheng, China. 30% H₂O₂ (GR, Sinopharm Chemical Reagent Co., Ltd. Shanghai, China), L-Ascorbic acid (Bomei Biotechnology Co., Ltd. Hefei, China), Tissue fixative (4% paraformaldehyde, Servicebio Technology Ltd. Wuhan, China), Newcastle disease vaccine (NDV) was purchased from Sinopharm Animal Health Co. Nobleryder, China. Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, USA), Cetyltrimethylammonium bromide (CTAB, Nobleryder, China), 2% agaros gels (biowest, Spain), Universal DNA Purification Kit (TianGen, China).

Instrument Electronic scale (LE203E/02, METTLER TOLEDO, Shanghai, China), Three-hole electric thermostatic water tank (DK-8D, Shanghai Yiheng Scientific Instrument Co., Ltd.), Centrifuge (H1065-W, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.) Inverted microscope (IX73, OLYMPUS, Tokyo, Japan), Full wavelength microplate reader (MULTISKAN Sky, Thermo Fisher Scientific, USA), High-throughput sequencing instrument Miseq (Illumina, USA), Nucleic acid Electrophoresis System (DYCP-32C, Beijing Liuyi Instrument Factory, China), Polymerase Chain Reaction Machine (T100PCR, Bio-Rad, USA), Bioanalyzers (Agilent 5400 Agilent Technologies CoLtd. USA)

Preparation of polysaccharide and degraded polysaccharide

Polysaccharides from Acanthopanax senticosus were isolated using a composite enzyme extraction technique. The composite enzyme consisted of cellulase: pectinase: hemicellulase = 2: 1: 2, was added at a concentration of 1,200 U/g. The enzymatic digestion was performed at pH 7 and 30°C for 60 min, with a solid-to-liquid ratio of 1:50 (g/mL) (Wang, et al., 2023). The extracted Acanthopanax senticosus polysaccharides (ASPS) were freeze-dried and prepared into a 50 mg/mL polysaccharide solution for further use.

Referring to the previous studies, the degradation of polysaccharides of Acanthopanax was carried out by H₂0₂-VC technique. Accurately weighed 0.25 g of enzyme extracted polysaccharide of Acanthopanax senticosus and add 2.5 ml of 1% H₂O₂, then add 2.5 ml of 0.5 mol/L VC, and put into a 50 °C constant temperature water bath for degradation. After the degradation was completed, appropriate amount of NaHSO₃ was added to remove the H₂O₂ in the solution, which produced bubbles, and then NaOH was added to adjust the pH to 7. The polysaccharide solution was loaded into a dialysis bag of 500 Da, and dialysed under running water for 12 h and then prepared for use. After degradation, degraded polysaccharides (ASPS-1) were obtained by freeze-drying and formulated into a 50 mg/mL degraded polysaccharide solution for spare use.

Animals and experimental design

The animal experiments were approved by the Ethics Committee of Liaocheng University and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC). A total of 180 healthy 1-day-old 817 crossbred chicks with similar body weights were randomly divided into 6 groups, with 3 replicates of 10 chicks per group. Adaptive rearing under the same feed, water, and light conditions for 7 days prior to the start of the trial and vaccination against NDV by eye-dotting at 3 days of age ensured that the trial was not interfered with by other diseases. The experimental groups included: (1) ASPS-1 low-dose (ASPS-1L, 250 mg/kg·bw), medium-dose (ASPS-1M, 500 mg/kg·bw), and high-dose (ASPS-1H, 1000 mg/kg·bw) groups; (2) ASPS medium-dose (ASPSM, 500 mg/kg·bw) and high-dose (ASPSH, 1000 mg/kg·bw) groups; and (3) a blank control group receiving normal drinking water without any drug. The trial lasted 21 days, with daily dosages adjusted based on initial body weight. To ensure complete consumption, ASPS and ASPS-1 were administered via drinking water after a 3-hour water withdrawal period, followed by unrestricted access to water. Body weights were recorded on days 7, 14, and 21, and 10 chicks per group were randomly selected for cardiac blood collection and dissection. Serum, immune organs, and small intestines were collected for analysis. Additionally, cecal contents from 6 chicks per group at 21 days were analyzed for intestinal flora composition.

Indicators and methods of measurement

Growth performance

Chicks were weighed every 7 days during the experimental period (fasted for two hours before weighing) and average daily gain (ADG) was calculated.

$$\text{ADG}=\frac{\text{Average}\text{weight}\text{at}\text{the}\text{end}\text{of}\text{the}\text{trial}-\text{Average}\text{weight}\text{at}\text{the}\text{beginning}\text{of}\text{the}\text{trial}}{\text{Test}\text{days}}$$

Determination of immune organ index

On days 7, 14, and 21 of the experiment, 10 chicks (one replicate) were randomly selected from each group for fasting body weight measurement. After cardiac blood collection, the chicks were euthanized by cervical dislocation. The spleen, thymus, and bursa of Fabricius were then dissected, weighed, and used to calculate the immune organ index.

$$\text{Immune}\text{organ}\text{index}(\%)=\frac{\text{Fresh}\text{weight}\text{of}\text{immune}\text{organs}\left(\mathrm{g}\right)}{\text{Live}\text{body}\text{weight}\left(\mathrm{g}\right)}\times 100$$

Immune organ and intestinal histomorphological observations

After dissection at 7, 14, and 21 days, the spleen, thymus, and bursa of Fabricius from three randomly selected chicks per group were collected. A section of the small intestine from the same region was fixed in tissue fixative for 24 h. Following routine dehydration, clearing, paraffin embedding, and sectioning, 5 μm-thick paraffin sections were prepared. The sections were dewaxed, stained with hematoxylin for 3–5 min, dehydrated through a graded alcohol series, counterstained with eosin for 5 min, and mounted with neutral gum. Images were captured using an inverted microscope (OLYMPUS, IX73) for histological analysis. Using ImageJ software, five intact villi and crypts per tissue section were selected, and the villus height (VH) to crypt depth (CD) ratio was calculated.

Serum indicator measurements

Three chicks were randomly selected from each group at 7 d, 14 d and 21 d. Two milliliters of blood were drawn, and the serum was allowed to sit at room temperature for three hours. After centrifuging, the serum was kept in reserve at -20°C. CAT, MDA, GSH-Px, FRAP, and SOD were assayed according to the kit manufacturer's instructions. Catalase (CAT), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and total antioxidant capacity (FRAP) assay kits were purchased from Isejyu Biotech Co. Superoxide dismutase (SOD) was purchased from Beijing Solebao Technology Co., Ltd. Determination of immune factors was carried out using chicken ELISA test kits for IFN-γ, IL-2, and IgG levels according to the manufacturer's instructions. Chicken gamma interferon (chicken-gamma) ELISA kit, chicken interleukin 2 (IL-2) ELISA kit and chicken immunoglobulin G (IgG) ELISA kit were purchased from Shanghai Yuanquan Biotechnology Center.

16S rRNA gene sequencing

Following standard fecal sampling procedures and adhering to aseptic techniques, cecal contents from six chicks per group were collected in sterile cryotubes at 21 days and stored at −80°C for subsequent 16S rDNA sequencing of intestinal microbiota. DNA extraction and PCR amplification were performed according to previously published methods (Su, et al., 2024a).

Bioinformatic analysis of 16S rRNA sequencing

Briefly, raw data FASTQ files were imported into the format which could be operated by QIIME2 system using qiime tools import program. Demultiplexed sequences from each sample were quality filtered and trimmed, de-noised, merged, and then the chimeric sequences were identified and removed using the QIIME2 dada2 plugin to obtain the feature table of amplicon sequence variant (ASV) (Callahan, et al., 2016). The QIIME2 feature-classifier plugin was then used to align ASV sequences to a pre-trained GREENGENES 13.8 99% database (trimmed to the V3V4 region bound by the 341F (5’-CCTAYGGGRBGCASCAG-3’) and 806R (5’-GGACTACNNGGGTATCTAAT-3’) primer pair) to generate the taxonomy table (Bokulich, et al., 2018). Any contaminating mitochondrial and chloroplast sequences were filtered using the QIIME2 feature-table plugin. Appropriate methods include ANCOM, ANOVA, Kruskal Wallis, LEfSe and DEseq2 were employed to identify the bacteria with different abundance among samples and groups (Love, et al., 2014; Mandal, et al., 2015; Segata, et al., 2011). Diversity metrics were calculated using the core-diversity plugin within QIIME2. Feature level alpha diversity indices, such as observed OTUs, Chao1 richness estimator, Shannon diversity index, and Faith's phylogenetics diversity index were calculated to estimate the microbial diversity within an individual sample. Beta diversity distance measurements, including Bray Curtis, unweighted UniFrac and weighted UniFrac were performed to investigate the structural variation of microbial communities across samples and then visualized via principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) (Vázquez-Baeza, et al., 2013). Co-occurrence analysis was performed by calculating Spearman's rank correlations between predominant taxa and the network plot was used to display the associations among taxa. In addition, the potential KEGG Ortholog (KO) functional profiles of microbial communities was predicted with PICRUSt (Langille, et al., 2013). Table 1

Table 1.

Ingredient composition and calculated nutrient levels of the basal diet.

Ingredients	Contents (%)	Nutrient components2	Contents (%)
Corn	53.90	Metabolic energy (MJ/Kg)	12.56
Soybean meal	34.90	Crude protein	20.61
Bran	2.00	Calcium	1.00
Fish meal	2.50	Total phosphorus	0.66
Soybean oil	3.00	Lysine	1.20
Calcium hydrophosphate	1.80	Methionine	0.50
Limestone	1.20
Salt	0.30
Vitamin-mineral premix1	0.40

¹Vitamin-mineral premix provided per kg diet: vitamin A 10, 000 IU, vitamin D₃ 4, 500 IU; vitamin E 40 mg, vitamin K 3.5 mg, vitamin B₁ 3 mg, vitamin B₂ 7 mg, vitamin B₆ 4.5 mg, vitamin B₁₂ 0.04 mg, nicotinic acid 40 mg, pantothenic acid 13 mg, biotin 0.25 mg, folic acid 2.1 mg, Cu 10 mg, Fe 60 mg, Zn 80 mg, Mn 100mg, I 1.4 mg, Se 0.4 mg.

²Nutrient levels were measured values.

Data analysis

Data were analyzed using SPSS 21 software for multiple comparisons and significance testing. Results are expressed as mean ± SEM and were analyzed by one-way ANOVA. Statistically significant differences were identified using the chi-square LSD and Duncan methods (P ≤ 0.05). Graphs were generated using OriginPro 2021 software. Gut microbiota diversity data were analyzed using the Micromeritics Alliance Bioscience Cloud platform (https://www.bioincloud.tech).

Results

Effects of different doses of ASPS-1 and ASPS on the growth performance of broiler chicks

On the first day of the experiment, 10 chicks from each group were randomly selected for weight measurement and recorded as the initial weight. The weight measured before euthanasia on the 21st day was considered the final weight. As shown in Table 2, there were no significant differences in the initial body weights among the groups (P > 0.05). The final body weight of the other four groups except the ASPSM group was significantly higher than that of the blank control group, Specifically, the final body weight and ADG of the ASPS-1H group were significantly higher than those of the ASPS high-dose group, and the final body weight of the ASPS-1L and ASPS-1M groups were significantly higher than that of the ASPSM group (P ≤ 0.05).

Table 2.

Effects of different doses of ASPS and ASPS-1 on the growth performance of broiler chicks. (n = 10).

Groups	Initial weight (g)	Final weight (g)	ADG
ASPS-1L	73.385 ± 1.133a	213.555 ± 4.339b	6.675 ± 0.216b
ASPS-1M	73.480 ± 0.662a	219.232 ± 4.596a	6.941 ± 0.214a
ASPS-1H	73.549 ± 0.675a	222.900 ± 4.820a	7.112 ± 0.227a
ASPSM	73.050 ± 0.959a	205.973 ± 4.203c	6.330 ± 0.221a
ASPSH	73.387 ± 1.177a	210.986 ± 4.981b	6.552 ± 0.263b
Blank	73.526 ± 0.819a	205.696 ± 3.757c	6.294 ± 0.196a

Note: Results are expressed as mean ± SD. Different superscript letters indicate significant differences (P ≤ 0.05); same superscript letters indicate non-significant differences (P > 0.05).

Effects of different doses of ASPS-1 and ASPS on immune organ index in broiler chicks

As shown in Table 3, the thymus index increased over time in all groups, comparing the thymus index at 7 d, 14 d and 21 d, the thymus index increased over time in all groups. At the same dose, the thymus index of the ASPS-1 group was higher than that of the ASPS group, but there was no statistical difference (P > 0.05).

Table 3.

Determination results of thymus index of broilers in each group. (n = 10).

Groups	7 d	14 d	21 d
ASPS-1 L	0.223 ± 0.022b	0.248 ± 0.029b	0.263 ± 0.013b
ASPS-1 M	0.240 ± 0.016ab	0.276 ± 0.040ab	0.284 ± 0.042ab
ASPS-1 H	0.257 ± 0.030a	0.306 ± 0.064a	0.315 ± 0.020a
ASPS M	0.211 ± 0.026b	0.264 ± 0.061b	0.280 ± 0.016b
ASPS H	0.231 ± 0.039b	0.276 ± 0.043ab	0.299 ± 0.028ab
Blank	0.227 ± 0.020b	0.242 ± 0.018b	0.264 ± 0.015b

Note: Results are expressed as mean ± SD. Comparisons were made between groups of the same number of days, with different superscript letters indicating significant differences (P ≤ 0.05); the same superscript letters indicating non-significant differences (P > 0.05).

As can be seen from Table 4 as the trial progressed, comparing the spleen indices at 7 d, 14 d, and 21 d, there was a certain increase in the spleen indices of all groups. Among them, at 14 d and 21 d, the splenic index was significantly higher in the ASPS-1M and ASPS-1H groups than in the ASPSM and ASPSH groups (P ≤ 0.05). Compared with the blank group, there were no significant changes in the ASPS-1L group at any of the three measurement stages (P > 0.05).

Table 4.

Determination results of spleen index of broilers in each group. (n = 10).

Groups	7 d	14 d	21 d
ASPS-1L	0.054 ± 0.007c	0.098 ± 0.020bc	0.117 ± 0.015ab
ASPS-1M	0.059 ± 0.007c	0.103 ± 0.022b	0.131 ± 0.026a
ASPS-1H	0.071 ± 0.011a	0.126 ± 0.017a	0.138 ± 0.026a
ASPSM	0.055 ± 0.006c	0.083 ± 0.020c	0.105 ± 0.018b
ASPSH	0.062 ± 0.012bc	0.110 ± 0.004b	0.106 ± 0.018b
Blank	0.054 ± 0.007c	0.085 ± 0.015c	0.103 ± 0.022b

Note: Results are expressed as mean ± SD. Comparisons were made between groups of the same number of days, with different superscript letters indicating significant differences (P ≤ 0.05); the same superscript letters indicating non-significant differences (P > 0.05).

As shown in Table 5, the bursa of Fabricius index increased in all groups over the course of the experiment, comparing the fabricius index at 7 d, 14 d, and 21 d, there was a certain increase in the bursa of fabricius index in all groups. The bursa of fabricius index was significantly higher in the ASPS-1H group than in the ASPSH group at all three stages (P ≤ 0.05), and the index was also higher in the ASPS-1M group than in the ASPSM group, but there was no statistical difference (P > 0.05).

Table 5.

Determination results of the bursa of Fabricius index of broilers in each group. (n = 10).

Groups	7 d	14 d	21 d
ASPS-1 L	0.229 ± 0.025b	0.218 ± 0.040c	0.233 ± 0.015c
ASPS-1 M	0.241 ± 0.021ab	0.294 ± 0.023b	0.290 ± 0.016b
ASPS-1 H	0.266 ± 0.035a	0.351 ± 0.025a	0.317 ± 0.021a
ASPS M	0.207 ± 0.015b	0.263 ± 0.056b	0.271 ± 0.030bc
ASPS H	0.220 ± 0.048b	0.286 ± 0.032b	0.274 ± 0.019bc
Blank	0.215 ± 0.025b	0.208 ± 0.045c	0.262 ± 0.024c

Note: Results are expressed as mean ± SD. Comparisons were made between groups of the same number of days, with different superscript letters indicating significant differences (P ≤ 0.05); the same superscript letters indicating non-significant differences (P > 0.05).

Effects of different doses of ASPS-1 and ASPS on the histomorphology of immune organs in broiler chicks

The histomorphology of the thymus, spleen, and bursa of Fabricius in broilers from each group at 21 d is shown in Fig. 1, Fig. 2-3. All organs exhibited well-developed structures with no obvious pathological changes. In the Blank group, the thymus cortex was thin, the medulla was sparsely populated, and the staining intensity was light after HE staining. Additionally, the number of splenic nodules was low, the red pulp was abundant, and the follicular spaces in the bursa of Fabricius were enlarged. In contrast, the five medicated groups showed a thickened thymus cortex with deeper staining, a denser medulla, and narrower lymphoid follicles in the bursa of Fabricius. Specifically, in the ASPS-1M and ASPS-1H groups, the thymus lobule edges were clearly defined, the number of thymic corpuscles increased, and the splenic nodules were more numerous and larger in size. Furthermore, the lymphocytes were densely packed, and the staining intensity was enhanced. With increasing concentrations of ASPS-1, the lymphoid follicles in the bursa of Fabricius became elongated and larger, the interfollicular arrangement became denser, and the proportion of the medulla increased.

Fig. 1.

Histomorphology sections of the thymus of broiler chicks from each group.

(HE, 40×)

Fig. 2.

Histomorphology sections of spleen from broiler chicks of each group. (HE, 40×)

Fig. 3.

Histomorphology sections of the bursa of fasciola of broiler chicks from each group. (HE, 40×)

Effects of different doses of ASPS-1 and ASPS on intestinal morphology of broiler chicks

The structure of the small intestine at 21 d in each group is shown in Fig. 4. The small intestine was morphologically intact and well developed in all groups. The intestinal villus in the ASPS-1H group was more intact and regular in morphology compared to the other groups, while the differences among the remaining groups were minimal.

Fig. 4.

Histomorphology sections of the small intestine of broiler chicks from each group. (HE, 40×)

Effects of different doses of ASPS-1 and ASPS on intestinal villus height and crypt depth in broiler chicks

The height of five intact villi (VH) and the depth of crypts (CD) were measured for each group, and the results are presented in Table 6. The study revealed that the VH in the ASPS-1L group was significantly higher than that in the blank group (P ≤ 0.05), while the CD was significantly lower, resulting in a significantly elevated VH/CD ratio compared to the Blank group (P ≤ 0.05). No significant differences were observed in VH, CD, or VH/CD between the ASPS-1M and ASPSM groups, although the VH/CD ratio in the ASPS-1M group was slightly higher (P > 0.05). Notably, the VH in the ASPSH group was significantly higher than that in the ASPS-1H group (P ≤ 0.05), but the CD in the ASPS-1H group was significantly lower than that in the ASPSH group (P ≤ 0.01). Consequently, the VH/CD ratio in the ASPS-1H group was significantly higher than that in the ASPSH group (P ≤ 0.01).

Table 6.

Measurements of villus height and crypt depth in broiler chicks. (n = 5).

Groups	villus height (VH)/μm	Crypt depth (CD)/μm	VH/CD
ASPS-1L	504.524 ± 11.038a	119.046 ± 12.018bc	4.280 ± 0.536ab
ASPS-1M	502.094 ± 24.927a	109.074 ± 13.202b	4.645 ± 0.472b
ASPS-1H	498.146 ± 12.365a	86.884 ± 8.920a	5.776 ± 0.545c
ASPSM	522.300 ± 24.250ab	117.898 ± 9.774bc	4.447 ± 0.315ab
ASPSH	553.618 ± 30.243b	118.444 ± 17.573bc	4.753 ± 0.688b
Blank	520.740 ± 46.733bc	135.446 ± 21.785c	3.892 ± 0.455c

Note: Results are expressed as mean ± SD. Different superscript letters indicate significant differences (P ≤ 0.05); same superscript letters indicate non-significant differences (P > 0.05).

Effects of different doses of ASPS-1 and ASPS on serum antioxidant indexes in broiler chicks

As shown in Table 7, the CAT content in the ASPS-1H group was significantly higher than that in the ASPSH group at 7 d and 21 d (P ≤ 0.05). At 14 d, the CAT content in the ASPS-1H group was significantly higher than that in the ASPS-1L group (P ≤ 0.05). Although the CAT content in the ASPS-1M group was consistently higher than that in the ASPSM group at all three time points, the differences were not statistically significant (P > 0.05).

Table 7.

Effects of different doses of ASPS-1 and ASPS on serum antioxidant indexes in broiler chicks.

	Days	ASPS-1L	ASPS-1M	ASPS-1H	ASPSM	ASPSH	Blank
	7 d	6.426 ± 1.917cd	8.599 ± 0.748ab	10.435 ± 0.701a	8.660 ± 0.781ab	8.201 ± 1.095bc	4.866 ± 0.954d
CAT (nmol/min/mL)	14 d	9.945 ± 1.222c	14.994 ± 1.939ab	18.329 ± 1.015a	13.342 ± 1.402bc	14.045 ± 1.147abc	10.649 ± 5.334bc
	21 d	16.585 ± 0.781c	21.940 ± 0.795b	26.836 ± 2.128a	23.378 ± 1.162b	21.298 ± 0.841b	14.933 ± 2.223c
	7 d	1.029 ± 0.110bc	1.037 ± 0.081bc	0.807 ± 0.181c	1.142 ± 0.219ab	0.984 ± 0.041bc	1.364 ± 0.080a
MDA (nmol/mL)	14 d	0.862 ± 0.039b	0.853 ± 0.243b	0.827 ± 0.065b	1.066 ± 0.119ab	0.918 ± 0.262b	1.254 ± 0.063a
	21 d	0.896 ± 0.218bc	0.791 ± 0.083bc	0.604 ± 0.052c	0.872 ± 0.081bc	0.987 ± 0.285b	1.379 ± 0.124a
	7 d	188.815 ± 3.748e	253.742 ± 4.074b	294.621 ± 1.455a	221.118 ± 2.648d	234.303 ± 6.085c	169.126 ± 4.174 f
GSH-Px (nmol/min/mL)	14 d	153.225 ± 4.347d	225.692 ± 3.955b	281.329 ± 5.753a	217.080 ± 4.867c	230.158 ± 3.816b	121.493 ± 4.000e
	21 d	219.331 ± 4.737c	232.981 ± 4.586b	258.959 ± 1.310a	219.081± 5.170c	225.013 ± 1.608c	218.402 ± 1.845c
	7 d	0.488 ± 0.041bc	0.517 ± 0.007b	0.580 ± 0.011a	0.495 ± 0.008bc	0.460 ± 0.032c	0.474 ± 0.024bc
FRAP (umol/mL)	14 d	0.546 ± 0.008b	0.555 ± 0.007b	0.597 ± 0.006a	0.542 ± 0.012b	0.570 ± 0.030ab	0.496 ± 0.022c
	21 d	0.587 ± 0.022bc	0.611 ± 0.006ab	0.625 ± 0.019a	0.589 ± 0.009bc	0.599 ± 0.012b	0.571 ± 0.007c
	7 d	7.070 ± 0.595bc	7.829 ± 0.733b	9.235 ± 0.104a	6.663 ± 0.387c	6.945 ± 0.590bc	5.220 ± 0.229d
SOD (U/mL)	14 d	9.289 ± 0.651cd	10.650 ± 0.438b	12.952 ± 0.716a	9.142 ± 0.566cd	10.081 ± 0.894bc	8.881 ± 0.330d
	21 d	12.597 ± 0.232d	14.829 ± 0.578b	20.461 ± 0.200a	12.766 ± 0.382c	15.587 ± 0.876b	11.852 ± 0.266d

Note: Results were obtained from three replicate experiments and are expressed as mean ± SD. Comparisons were made between groups of the same number of days, with different superscript letters indicating significant differences (P ≤ 0.05); the same superscript letters indicating non-significant differences (P > 0.05).

In the MDA content assay, the ASPS-1M and ASPS-1H groups exhibited lower MDA levels than the ASPSM and ASPSH groups at 7 d and 14 d, but these differences were not significant (P > 0.05). However, at 21 d, the MDA content in the ASPS-1H group was significantly lower than that in the ASPSH group (P ≤ 0.05).

In the GSH-Px content assay, the GSH-Px levels in the ASPS-1M and ASPS-1H groups were significantly higher than those in the ASPSM and ASPSH groups at 7 d, 14 d, and 21 d (P ≤ 0.05). At 21 d, the GSH-Px content in the ASPS-1L group (219.331 ± 4.737 nmol/min/mL) was similar to that in the ASPSM group (219.081 ± 5.170 nmol/min/mL).

In the FRAP content assay, the FRAP levels in the ASPS-1H group were significantly higher than those in the ASPSH group at 7 d and 21 d (P ≤ 0.05). At 14 d, no significant differences were observed between the ASPS-1M and ASPS-1H groups compared to the ASPSM and ASPSH groups (P > 0.05). However, the FRAP content in all treated groups was significantly higher than that in the Blank group at all time points (P ≤ 0.05).

In the SOD content assay, the IgG content in the ASPS-1M and ASPS-1H groups was significantly higher than that in the ASPSM and ASPSH groups at 7 d (P ≤ 0.05). At 14 d and 21 d, the IgG concentration in the ASPS-1H group was significantly higher than that in the ASPSH group (P ≤ 0.05)

Effects of different doses of ASPS-1 and ASPS on serum immunity indexes of broiler chicks

As shown in Table 8, at 7 d, the IgG content of both the ASPS-1M and ASPS-1H groups was significantly higher than that of the ASPSM and ASPSH groups (P ≤ 0.05), and at 14 d and 21 d, the IgG concentration in the ASPS-1H group exceeded that of the ASPSH group, and the disparity was statistically significant (P ≤ 0.05). The IFN-γ content assay showed that the IFN-γ levels in all five treated groups were significantly lower than those in the Blank group at 7 d, 14 d, and 21 d (P ≤ 0.05). At 14 d and 21 d, the content of IL-2 in the ASPS-1M group was higher than that in the ASPSM group but statistically insignificant (P > 0.05), and the content of IL-2 in the ASPS-1H group was significantly higher than that in the ASPSH group (P ≤ 0.05). The results of the data of IFN-γ content assay showed that the IFN-γ levels of the five administered groups were significantly lower than those of the Blank group at 7 d, 14 d, and 21 d (P ≤ 0.05). The IFN-γ level in the ASPS-1M group was considerably lower than that in the ASPSM group at 7 days (P ≤ 0.05), and the difference in the level of IFN-γ between the ASPS-1H group and the ASPSH group compared with the ASPS-1H group was not significant (P > 0.05); At 14 d, IFN-γ levels were lower in the ASPS-1M and ASPS-1H groups than in the ASPSM and ASPSH groups, respectively, but the difference was not significant (P > 0.05); The difference between the results of the ASPS-1M group and the ASPSM group at 21 d was not significant (P > 0.05), whereas the IFN-γ level in the ASPS-1H group was significantly lower than that in the ASPSH group (P ≤ 0.05).

Table 8.

Effects of different doses of ASPS-1 and ASPS on serum immune indices.

	Days	ASPS-1L	ASPS-1M	ASPS-1H	ASPSM	ASPSH	Blank
	7 d	20.798 ± 0.227c	21.530 ± 0.097b	25.213 ± 0.419a	19.541 ± 0.381d	21.995 ± 0.221b	19.308 ± 0.385d
IgG (g/L)	14 d	22.813 ± 0.768c	23.978 ± 0.559b	25.569 ± 0.483a	22.966 ± 0.837bc	23.503 ± 0.153bc	21.386 ± 0.223d
	21 d	23.732 ± 0.541b	24.072 ± 0.652b	25.266 ± 0.325a	23.395 ± 0.142b	23.775 ± 0.834b	20.390 ± 0.149c
	7 d	304.325 ± 0.754d	314.306 ± 1.449b	327.421 ± 1.239a	304.226 ± 1.463d	310.496 ± 1.625c	303.373 ± 1.455d
IL-2 (pg/mL)	14 d	305.615 ± 2.533c	315.397 ± 4.623b	329.544 ± 3.282a	312.302 ± 1.475bc	307.401 ± 2.560cd	291.329 ± 2.447e
	21 d	306.905 ± 1.871b	311.746 ± 1.685b	327.937 ± 2.927a	308.115 ± 4.527b	310.952 ± 1.189b	295.218 ± 4.915c
	7 d	128.004 ± 2.044c	126.154 ± 1.402c	122.971 ± 3.229c	133.489 ± 3.954b	124.297 ± 1.337c	139.732 ± 4.478a
IFN-γ (pg/mL)	14 d	126.834 ± 2.733b	124.443 ± 1.045bc	123.055 ± 1.384c	126.310 ± 0.483b	125.412 ± 1.128bc	139.615 ± 0.377a
	21 d	129.794 ± 2.225b	127.609 ± 0.655b	121.327 ± 1.068c	130.435 ± 1.375b	127.965 ± 2.981b	141.923 ± 1.874a

Note: Results were obtained from three replicate experiments and are expressed as mean ± SD. Comparisons were made between groups of the same number of days, with different superscript letters indicating significant differences (P ≤ 0.05); the same superscript letters indicating non-significant differences (P > 0.05).

Structure of the fecal microbiota

To evaluate the effects of different doses of ASPS-1 and ASPS on the intestinal health of chicks, cecal contents from six chicks per group were collected at 21 d for 16S rRNA sequencing.

Fig. 5A illustrates the distribution of operational taxonomic units (OTUs) at the genus level across the six groups, revealing a total of 537 OTUs. This indicates that the core microbiota in the cecum of broilers remained unchanged despite variations in polysaccharide concentrations. As shown in Fig. 5B, the ASPS-1H group exhibited the highest number of OTUs (686).

Fig. 5.

Flower diagram of OTUs (A); total number of OTUs (B). β-Diversity analysis for the different gut microbiota in each group, PCA (C) and PC1 (D). Alpha diversity indexes data of the gut microbiota in each group. Coverage (E), ACE (F), Chao1 (G), Shannon (H), and Simpson index (I). Note: *P < 0.05, **P < 0.01, ***P < 0.001.

α-Diversity analysis The coverage index, reflecting sequencing depth, exceeded 0.999 for all six groups at the OTU level (Fig. 5A). Figs 5E-I present the alpha diversity analysis using five indices. Fig. 5F displays the ACE index, showing highly significant differences between the ASPS-1H and ASPS-1L groups (P ≤ 0.01) and significant differences between the ASPS-1H and ASPSM groups (P ≤ 0.05). Fig. 5G depicts the Chao1 index, with significant differences between the ASPS-1H group and the ASPSM group (P ≤ 0.05) and highly significant differences compared to the Blank group (P ≤ 0.01). Fig. 5H presents the Shannon index, revealing significant differences between the ASPS-1H and ASPSH groups (P ≤ 0.05) and highly significant differences compared to the Blank group. Fig. 5I shows the Simpson's index, with highly significant differences between the ASPS-1H group and the ASPSH group (P ≤ 0.01) and significant differences compared to the Blank group (P ≤ 0.05).

β-Diversity analysis Principal component analysis (PCA) was employed to examine variations in microbial composition among samples. Fig. 5C shows the PCA results, with PC1 and PC2 axes explaining 44.017% and 13.578% of the variance, respectively. The distribution of the six groups was relatively concentrated, suggesting that polysaccharide supplementation did not significantly alter the core components of the intestinal microbiota. Fig. 5D represents the discrete distribution of samples on the PC1 axis for different grouping conditions. The results showed that the ASPS-1H group had the highest percentage on the PC1 principal component analysis, indicating that the differences in bacterial flora in the ASPS-1H group were more different from the other groups.

Effects of ASPSs on key phylotypes of fecal microbiota

Phylum level analysis To further investigate changes in microbial structure, the composition of the intestinal microbiota was analyzed at different taxonomic levels. Fig. 6A shows the distribution of the top 10 phyla in the cecal bacterial community. Firmicutes and Bacteroidetes dominated, accounting for over 90% of the total microbiota, while other phyla showed minimal variation in the cluster analysis.

Fig. 6.

The relative abundance of the gut microbiota at the phylum level (A); Phylum level cluster analysis heat map (B); The relative abundance of the gut microbiota at the genus level (C); Heat diagram of cluster analysis at the genus level (D); Correlation analysis of intestinal villus height (VH), crypt depth (CD), and their ratios (VH/CD) with major flora at the genus level (E); PICRUSt2 Functional Prediction Level 2 Categorical Composite Bar Chart (F).

Fig. 6B presents a clustered heatmap at the phylum level, highlighting bacteria from the Archaea and Bacteria kingdoms. Red regions indicate higher relative abundance, with distinct patterns observed across groups, suggesting differences in microbial composition and relative abundance under varying polysaccharide treatments.

Genus level analysisFig. 6C displays the top 15 bacterial genera by relative abundance in each group. Lactobacillus was significantly more abundant in the ASPS-1M and ASPS-1H groups compared to the ASPSM and ASPSH groups. Lawsonibacter predominated in the ASPSM, ASPSH, and Blank groups but was significantly reduced in the ASPS-1H group. Negativibacillus abundance was significantly higher in the ASPS-1M and ASPS-1H groups than in the ASPSM and ASPSH groups, with abundance increasing with ASPS-1 dosage. Turicibacter was more prevalent in the ASPSM group but decreased significantly in the ASPS-1M and ASPS-1H groups. Genera such as Romboutsia, Faecalibacterium, Mediterraneibacter, and Eubacterium (Firmicutes) and Bacteroides, Parabacteroides, and Phocaeicola (Bacteroidetes) were evenly distributed across ASPS-1 dose groups.

Fig. 6D shows a clustered heatmap at the genus level, with bacteria primarily from Bacteroidetes and Firmicutes. The relative abundances of genera in Fig. 6D correspond to those in Fig. 6C.

Correlation analysis

Fig. 6E illustrates correlations between gut microbiota and villus height (VH), crypt depth (CD), and their ratios at the genus level. Faecalibacterium showed a highly significant positive correlation with VH/CD (P ≤ 0.01) but a significant negative correlation with CD (P ≤ 0.05). Negativibacillus exhibited a highly significant positive correlation with VH (P ≤ 0.001), while Eubacterium showed a significant positive correlation with VH (P ≤ 0.05). Parabacteroides and Phocaeicola were significantly negatively correlated with VH/CD and VH, respectively (P ≤ 0.05).

Functional prediction based on PICRUSt2

Following the functional prediction using PICRUSt2, secondary functional classifications with an average relative abundance greater than 1% were selected for analysis. These functions were categorized into three primary classifications: metabolism, genetic information processing, and environmental information processing. Among these, metabolism was the most dominant functional category, followed by genetic information processing and environmental information processing.

We further analyzed the secondary functional classifications of the predicted genes, which included 17 categories such as carbohydrate metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and glycan biosynthesis and metabolism. Carbohydrate metabolism exhibited the highest average abundance (12.69%), followed by amino acid metabolism (12.21%). The average abundances of energy metabolism, lipid metabolism, global and overview maps, biosynthesis of other secondary metabolites, metabolism of other amino acids, glycan biosynthesis and metabolism, and replication and repair ranged from 5.02% to 8.87%. In contrast, nucleotide metabolism, xenobiotic biodegradation and metabolism, terpenoid and polyketide metabolism, signal transduction, membrane transport, translation, and folding, sorting, and degradation showed average abundances below 5% (Fig. 6F).

The ASPS-1H group exhibited significant differences from other groups in several metabolic functions, including secondary functional metabolism, genetic information processing, and environmental information processing. Notably, these differences were prominent in amino acid metabolism, global and overview maps, replication and repair, translation, folding, sorting and degradation, and membrane transport. These variations in metabolic pathways may influence the metabolic processing of degraded polysaccharides from Acanthopanax senticosus in chicks.

Discussion

The dried root, rhizome, and stem of Acanthopanax senticosus, a member of the Wujiaceae family, which is mainly produced in the northeast of China and is known for its effects of benefiting qi and strengthening the spleen, tonifying the kidneys and tranquilizing the mind. Among its bioactive components, Acanthopanax senticosus polysaccharides (ASPS), which has been shown to possess a variety of pharmacological activities such as immunomodulatory (Zhang, et al., 2019), antioxidant (Zhou, et al., 2024d), and antimicrobial (Liu, et al., 2022). Degraded ASPS exhibit enhanced anti-digestive and probiotic properties, as demonstrated in previous in vitro studies. However, in vivo investigations of degraded ASPS remain unexplored. Consequently, the study of Acanthopanax senticosus degraded polysaccharides holds considerable potential for enhancing the growth performance and immunity of poultry. This study aimed to evaluate the effects of degraded ASPS (ASPS-1) on broiler chicks, with the aim of providing a comprehensive demonstration of the effects of ASPS-1 on growth performance, immune organ index, gut index, serum factors and gut microbiota. Our findings revealed that ASPS-1 significantly improved body weight gain, immune organ indices, and intestinal health, regulated the levels of immune factors and antioxidant factors and affected functional metabolic pathways such as amino acid metabolism by regulating the intestinal flora in broiler chicks, with the most significant effect of ASPS-1H in the high-dose group.

Immune organs, including the spleen, thymus, and bursa of Fabricius, play a pivotal role in avian defense mechanisms (Zhao, et al., 2024b). Spleen, thymus, and bursa of Fabricius, as important immune organs of avian species, are essential for preserving the body's immunological system, resisting the invasion of pathogens, and disease prevention and control (Zhou, et al., 2024b). The immune organ index can evaluate the immune status of chickens (Sun, et al., 2023). In this study, we recorded the changes in the indices of the spleen, thymus, and bursa at 7 d, 14 d, and 21 d. The addition of the ASPS-1H group was effective in increasing the indices of the spleen, thymus, and bursa of Fabricius, which was in agreement with the results of existing studies (Su, et al., 2024b). Observation of histomorphological sections showed that ASPS-1M and ASPS-1H promoted the development of immune organs with clear margins of thymic lobules, increased numbers of thymic vesicles, splenic nodes and lymphocyte counts, and had a lesser effect on the bursa of Fabricius. Numerous scholars have also related results on polysaccharides to promote the development of immune organs in poultry (Dong, et al., 2024; Wang, et al., 2024a; Xu, et al., 2024a). Maintaining the health of these immune organs is therefore important to ensure avian health and performance.

The intestine serves as both a primary immune organ and the main site for nutrient digestion and absorption (Chen, et al., 2019; Sha, et al., 2021). The ratio of villus height to crypt depth is an important index for assessing the absorptive capacity of the small intestine (Calik and Ergün, 2015). Qiao et al (Qiao, et al., 2022) showed that dietary supplementation with 300 mg/kg of astragalus polysaccharide and 150 mg/kg of licorice polysaccharide significantly increased villus height (VH) and the ratio of villus height to crypt depth (VH/CD), indicating that polysaccharides can help to improve the intestinal morphology of broilers. The intervention of Taishan Pinus massoniana pollen polysaccharides (TPPPS) at different doses ranging from 10 to 40 mg/ml was found to be effective in promoting healthy development and growth of chicken intestinal villus. Longer villus indicate increased surface area for intestinal nutrient digestion and absorption (Zhou, et al., 2021). In the present study, it was observed that the ASPS-1H group significantly promoted a reduction in small intestinal crypt depth, increased the VH/CD ratio, and facilitated the absorption of nutrients in the small intestine in comparison with the ASPS group. These findings suggest that the promotion of weight gain by ASPS-1H may be attributable to these effects. In contrast, ASPS-1 had only a minor effect on the morphological development of the small intestine. Histological analysis revealed that the small intestine was intact and well developed.

The intestinal flora plays an important role in the organism, polysaccharides, after entering the animal organism, not only provide energy but more often play a role in the intestines and are utilized by intestinal microorganisms (Qu, et al., 2024; Yu, et al., 2024). The gut microbiota plays a crucial role in host metabolism, immunity, and overall health. Previous studies have shown that Acanthopanax senticosus polysaccharides have a large molecular weight and are easily degraded when digested by gastrointestinal fluids, while the small molecular weight Acanthopanax senticosus degraded polysaccharides obtained by degradation are not readily digested in vitro in simulated gastrointestinal digestion, and in turn, are able to be utilized by the intestinal flora for the production of short-chain fatty acids (Wang, et al., 2024b). Serum biochemical and immunological markers reflect the immune and oxidative status of animals, such as interleukins and immunoglobulins, which can reflect the immune performance of the animal (Xu, et al., 2024b), and antioxidant indicators can reflect the oxidative stress status of the animal body (Kim, et al., 2010). It is evident that the health of poultry is closely related to serum biochemical indicators and intestinal health.

Plant polysaccharides are known to exert immunomodulatory effects by activating lymphocytes to secrete immunoglobulins and cytokines (Liao, et al., 2015). IgG, IL-2, and IFN-γ play important regulatory roles in the immune system of animal organisms. IgG is essential for immunological regulation. IL-2 is an important anti-inflammatory factor in the body, and IFN-γ is a pro-inflammatory factor mainly secreted by Th1 cells, which is crucial in immune response (Sönmez, et al., 2022; Taha-Abdelaziz, et al., 2018). Probiotic powder supplemented with Wolfiporia cocos polysaccharide (PWP) enhanced immunomodulation and increased IgA, IgM, and IgG levels in mice with antibiotic-associated diarrhea (AAD) compared to non-supplemented controls (Tang, et al., 2024). When evaluating the effects of selenium-enriched mulberry rhubarb polysaccharide on antioxidant capacity, immunity, serum biochemistry and production performance of laying hens, Liu et al. found that the mulberry rhubarb polysaccharide group also significantly increased the serum levels of IL-2, IgM, IgA, sIgA, IgG, and IFN-γ of laying hens (Liu, et al., 2023). In their study of Codonopsis polysaccharides, Rong et al. found that oral administration of Codonopsis polysaccharides to immunocompromised mice improved their immunodeficiency and regulated serum immunoglobulins IgG and IgM, cytokines IL-2, IL-6, and TNF-α (Rong and Shu, 2024). In this study, ASPS-1M and ASPS-1H increased IgG and IL-2 levels while reducing IFN-γ, indicating enhanced humoral and cellular immunity in chicks. It was shown that ASPS-1M and ASPS-1H could effectively activate the immune cells to secrete immunoglobulins and cytokines, thus improving the humoral and cellular immune functions of chicks.

Serum antioxidant factors, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), play a critical role in scavenging reactive oxygen species (ROS), maintaining ROS balance, and mitigating oxidative stress and cellular damage (Jiang, et al., 2016). MDA is frequently utilized as a biomarker for endogenous lipid peroxidation and damage caused by free radicals (Pitino, et al., 2021), and may reflect the extent to which the organism is under free radical attack. The FRAP method allows the determination of the total antioxidant capacity of serum. Zhao et al. found that Apocynum venetum polysaccharide (AVFP) significantly increased GSH-Px and SOD activity and decreased MDA content in immunosuppressed mice (Zhao, et al., 2024a); Lee et al. demonstrated that low molecular weight Tremella fuciformis polysaccharides (TFLP) modulated cyclophosphamide-induced immunosuppression in mice more effectively than high molecular weight Tremella fuciformis polysaccharides (TFP), and that TFLP effectively increased serum levels of T-SOD, GSH-Px, and CAT in immunosuppressed mice, and decreased MDA level (Lee, et al., 2024). In this study, ASPS-1M and ASPS-1H significantly increased CAT, GSH-Px, FRAP, and SOD levels while reducing MDA compared to control groups, with ASPS-1H showing the most pronounced effects. The findings of the present study demonstrated that the incorporation of ASPS-1M into broiler chicks resulted in a more pronounced enhancement of antioxidant function when compared to the ASPSM group. This finding suggests that the incorporation of a suitable amount of ASPS-1 has the potential to augment the antioxidant function of broiler chicks.

The cecum, the most microbially dense region in chickens, exhibits higher microbial diversity than the upper gastrointestinal tract (Stanley, et al., 2015). Existing studies have shown that plant polysaccharides can regulate the composition of the body's intestinal flora to a certain extent, promote the growth of beneficial bacteria and inhibit the reproduction of harmful bacteria (Liu, et al., 2024a; Wan, et al., 2024; Zhang, et al., 2024). In this study, ASPS-1H significantly increased α-diversity indices (Chao1, Ace, Shannon, Simpson), indicating richer microbial diversity. The experiment's results indicated that the α-diversity of chicken intestine bacteria was more abundant in the ASPS-1H group, with all four indices surpassing those of the other groups. In beta diversity analysis, principal component analysis (PCA) was used to determine the principal components using the similarity coefficient matrix of the samples. It uses variance decomposition to represent the difference between multiple data sets, and the axes of this coordinate plot are determined by two eigenvalues (Wang, et al., 2012). Principal component analysis (PCA) revealed a centralized distribution of microbial communities across groups, with no major shifts in core flora.

Firmicutes and Bacteroidetes are the dominant phylum in the microbiota (Liu, et al., 2021). Lactobacillus plays a crucial role in maintaining microecological balance in the gastrointestinal tracts of humans and animals (Cesare, et al., 2020). In this study, Lactobacillus abundance was significantly higher in the ASPS-1M and ASPS-1H groups compared to other groups (Yang, et al., 2024). Similarly, supplementation with Pleurotus citrinopileatus polysaccharide (PCP) has been shown to increase Lactobacillus abundance and short-chain fatty acid (SCFA) production in the chicken cecum (Zhou, et al., 2024c). Desulfovibrio a sulfate-reducing, H₂S-producing anaerobe, can damage intestinal epithelial cells and contribute to intestinal sensitization, and several studies have shown that an increase in the number of Desulfovibrio is an important feature of ulcerative colitis disease (Blachier, et al., 2010). In this study, Desulfovibrio abundance was higher in the ASPSM, ASPSH, and Blank groups but significantly reduced in the ASPS-1H group. Negativibacillus of the thick-walled phylum Negativibacillus is a bacillus with a Gram-negative cell wall structure, and there is little literature on the genus (2024), but in previously published studies Negativibacillus has been found to be enriched in increasingly rich microecological environments (Zhao, et al., 2018). Negativibacillus was found to increase in abundance with the addition of ASPS-1. Turicibacter, a pro-inflammatory bacterium associated with colitis development (Peng, et al., 2020; Wan, et al., 2021). In this study, Turicibacter abundance was significantly lower in the ASPS-1M and ASPS-1H groups compared to the ASPSM group. Collectively, these findings demonstrate that ASPS-1 supplementation promotes the colonization of beneficial bacteria (Lactobacillus and Negativibacillus) while inhibiting pathogenic genera (Desulfovibrio and Turicibacter), with the most pronounced effects observed in the high-dose group (ASPS-1H).

The gut microbiota interacts with the host through metabolic processes (Deng, et al., 2024). In chicks, the digestive tract is the primary site for nutrient digestion and absorption, whereas intestinal villi enhance the surface area of the intestinal tract; longer villi enhance nutrient absorption, and shallower crypts promote epithelial cell migration and differentiation, and the rise in maturity, ultimately contributing to intestinal maturity and function. Thus, the villus height-to-crypt depth (VH/CD) ratio is a key indicator of intestinal digestive and absorptive capacity. Faecalibacterium, known for its anti-inflammatory properties, is often reduced in inflammatory conditions (Eppinga, et al., 2016). Eubacterium synthesizes short-chain fatty acids (SCFAs), such as butyric acid, which protect the gastrointestinal mucosal barrier and mitigate inflammation (Karcher, et al., 2020; Vital, et al., 2017).

PICRUSt2, a functional prediction tool, infers the metabolic potential of microbial communities by comparing 16S rRNA data with known microbial genomes (Zhang, et al., 2023). Consistent with previous studies, metabolism was identified as the primary enriched biological function (Du, et al., 2020). Lactobacillus lactis degrades indigestible polysaccharides, enhances butyric acid metabolism, and maintains intestinal homeostasis (Li, et al., 2019). ASPS-1H exhibited distinct metabolic functions compared to other groups, particularly in amino acid metabolism, global and overview maps, and replication and repair pathways. Comprehensive analysis revealed that ASPS-1H promoted the proliferation of beneficial genera, including Lactobacillus, Faecalibacterium, Negativibacillus, and Eubacterium, through these metabolic pathways.

Conclusion

This study demonstrated that the addition of ASPS-1 significantly enhanced body weight growth and intestinal development in broiler chicks compared to ASPS, as evidenced by an increased ratio of intestinal villi to crypts. While ASPS-1 had no significant impact on immune organs, it effectively regulated serum antioxidant levels and immune indices, thereby improving overall immunity in broiler chicks. Analysis of the intestinal microbiota revealed that ASPS-1H promoted the proliferation of beneficial bacteria, such as Faecalibacterium, Negativibacillus, and Eubacterium, which were associated with intestinal villi development. Conversely, ASPS-1H inhibited the growth of Desulfovibrio and Turicibacter. These beneficial effects may be mediated through functional pathways, including amino acid metabolism, global and overview maps, and replication and repair. In conclusion, ASPS-1 exhibits significant potential as a feed additive in poultry production, with higher doses demonstrating greater efficacy.

Declaration of Competing Interest

All authors disclosed no relevant relationships.