Introduction

In modern animal production systems, transitioning from a state focused on survival to one of creation is essential. This shift aims to minimize the negative effects of chronic inflammation and excessive stress, allowing poultry to allocate energy towards growth rather than defense mechanisms1. Yellow-feathered quails are gaining popularity due to their high protein content in both meat and eggs, as well as their significant economic value. However, the intensive farming of these quails has led to the misuse of antibiotics, resulting in increased drug residues and compromised immune systems, which in turn reduces meat and egg production2,3,4. As a result, the search for green, natural feed additives as alternatives to antibiotics has become a key area of research. Studies indicate that alternative products, such as probiotics, prebiotics, and plant polysaccharides, can promote intestinal microflora balance, enhance metabolism, and improve intestinal integrity. These additives have been shown to possess anti-inflammatory, antioxidant, immune-modulating properties, and contribute to the maintenance of the intestinal barrier1.

The Xinjiang turnip (Brassica rapa L.), commonly referred to as “Qiamagu,” is a tuberous plant belonging to the Brassicaceae family and Brassica genus, specifically the rapa species. It is a widely utilized medicinal and edible plant, primarily cultivated in Kepin County, Xinjiang, a region where it is recognized as a geographically protected product. Both its seeds and roots hold medicinal properties and exhibit a range of pharmacological activities. One of its key bioactive components is the Qiamagu polysaccharide (BRP), with the polysaccharide content in its dried tuber ranging between 9.10% and 16.32%5. Yamamoto et al.6 demonstrated that Qiamagu significantly enhances IFN-γ production in the spleens of mice.while Tanaka et al.7 identified Brassica polysaccharides as potent immunostimulants, exerting their effects through TLR2 and TLR4 pathways. Additionally, Shin et al.8 have shown that Qiamagu markedly reduces serum nitric oxide (NO) levels, inflammatory cytokines, and mortality in lipopolysaccharide-challenged mice, These effects are achieved through modulation of nuclear factor-kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression at the transcriptional level, mediated by the inhibition of IκB kinase β. Currently, most research on BRP focuses on optimizing extraction methods, with limited studies reporting its application in poultry.

To the best of our knowledge, no studies have been published regarding the effects of dietary supplementation with BRP on the production performance of yellow-feathered quails. Therefore, this study aims to evaluate the influence of dietary BRP supplementation on growth performance, immune function, antioxidant capacity, and intestinal microbiota composition in yellow-feathered quails.

Materials and methods

Materials

The BRP were sourced from Hana Biotechnology Co., Ltd., Xi’an, China. The quail variety examined in this study is of the Japanese quail, sourced from the Jincheng Breeding Cooperative in Changji City, Xinjiang. The nutritional composition of the basal diet, based on corn and soybean meal, was aligned with the standards set by the National Research Council9 (Table 1).

Table 1 Composition and nutrient content of experimental diets.
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Methods

The husbandry and management of yellow-feathered quails adhered to the “Guidelines for the Care and Use of Agricultural Animals in Research and Teaching”10. All animals were treated humanely, with efforts made to minimize any suffering endured. A total of 250 one-day-old healthy yellow-feathered quails, evenly divided by sex, with an initial body weight of 7.5 ± 0.2 g, were selected. Utilizing a completely randomized experimental design, the quails were allocated into five treatment groups, with each group comprising 10 replicates and each replicate containing 5 quails. The dietary treatments involved the addition of BRP to the basal diet at concentrations of 0 g/kg (CON), 0.25 g/kg (Low dose BRP, LBRP), 0.5 g/kg (Medium dose BRP, MBRP), and 1 g/kg (High dose BRP, HBRP), respectively, while the chlortetracycline group (CTC) received 0.05 g/kg of chlortetracycline added to the basal feed. The experimental period lasted for 42 days. The yellow-feathered quails were housed in stainless steel, four-tiered battery cages situated within a ventilated, dust-free room previously sterilized with formaldehyde gas. Adequate provisions of water and feed were ensured to facilitate unrestricted weight gain. At the age of 7 days, the quails were administered water immunization against Newcastle disease and infectious bursal disease through vaccination. At 28 days of age, they received intramuscular immunization with an avian influenza vaccine. The room housing the yellow-feathered quails was illuminated for 17–20 h daily. For the initial three days, the indoor temperature and humidity were maintained at 38 °C and 60%, respectively. Subsequently, the temperature was reduced by 1 °C daily from days 4 to 10, followed by a reduction of 3 °C per day, eventually stabilizing at an optimal 22 °C until the conclusion of the experiment.

Growth performance

On days 21 and 42 of the experiment, the final body weights and feed intake per cage of the yellow-feathered quails were measured. The average daily gain (ADG), average daily feed intake (ADFI), and feed/gain ratio (F/G; grams of feed consumed per gram of weight gained) were calculated.

Collection of blood samples

At the ages of 21 and 42 days, after a fasting period of 12 h (with ad libitum access to water), a single yellow-feathered quail was randomly selected from each replicate within each group. Initial anesthesia was administered using pentobarbital, dosed at 50 mg/kg body weight via intramuscular injection, followed by the collection of blood samples. Blood was collected using single-use vacuum anticoagulant tubes, approximately 5 ml of blood was extracted from the heart of each quail, deposited into tubes, and allowed to coagulate at room temperature for 40 min before being centrifuged at 3000 g for 10 min at 4 °C. The serum supernatants were then stored at -20 °C for subsequent analysis of immunological and antioxidant parameters. The immunological markers included Immunoglobulin G (IgG), Immunoglobulin M (IgM), Immunoglobulin A (IgA), Interleukin-1β (IL-1β), Tumor Necrosis Factor (TNF), and Interleukin-10 (IL-10). The antioxidant indices measured were Total Superoxide Dismutase (T-SOD), Catalase (CAT), and Glutathione Peroxidase (GSH-Px). All assays were performed using reagent kits procured from Shanghai Enzyme-linked Biotechnology Co., Ltd., following the protocols specified by the manufacturer.

Measurement of relative immune organ weight

Following blood collection, subjects were weighed, euthanized via cervical dislocation for humane reasons, and then dissected to harvest the liver, pancreas, thymus, spleen, and bursa of Fabricius. The weights of these organs were recorded and expressed as a percentage of the total body weight (relative organ weight = (organ weight/body weight) * 100%).

16 S rRNA sequencing and analysis

Bacterial 16 S rRNA sequencing was performed as previously described11. Genomic DNA was extracted from the cecal contents using the Mag-Bind Soil DNA Kit (Omega Bio-tek, Norcross, GA). Amplification of the V3 to V4 hypervariable regions of the bacterial 16 S ribosomal RNA gene was carried out with primers 341 F (5’-CCTACGGGNGGCWGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) using an ABI GeneAmp 9700 PCR thermocycler (Thermo Fisher Scientific, Waltham, MA). Sequencing was conducted on the Illumina MiSeq platform at Majorbio Biopharm Technology Co., Ltd., Shanghai, China. Raw data were demultiplexed, quality filtered, and merged, and operational taxonomic units (OTUs) were clustered and annotated using UPARSE software (version 7.1; http://drive5.com/uparse/) with a 97% similarity cutoff. Representative sequences for each OTU were classified using the RDP Classifier (http://rdp.cme.msu.edu/). Alpha diversity metrics (ACE, Chao1, Shannon and Simpson) and beta diversity distances based on UniFrac metrics (inter-group) were calculated and utilized for Principal Coordinates Analysis (PCoA), with Similarity Analysis (ANOSIM) employing the unweighted UniFrac full tree method. Differential abundant taxa among treatments were identified through Linear Discriminant Analysis Effect Size (LefSe) analysis, with a significance threshold of α = 0.05 and an LDA score > 3.

Ethical statement

The study was approved by the Animal Experimentation Ethics Committee of the School of Animal Science and Technology, Shihezi University. The code of ethical inspection was A2021-14. All Quail were kept experimentally and euthanized strictly followed the ARRIVE guidelines. During the test, all efforts were made to minimize the suffering of the animals.

Statistical analysis

Statistical analyses were conducted using SPSS software version 20.0 (SPSS Inc., Chicago, IL, USA) was used for One-way ANOVA analysis, and Duncan’s method was used for multiple comparisons. P < 0.01 was considered difference or significant regression effect, and P < 0.05 was considered difference or significant regression effect. 0.05 < P < 0.1 means difference or regression effect tends to be significant, and P > 0.1 means difference or regression effect is not significant.

Result

Effects of Brassica rapa L. Polysaccharides on the growth performance of yellow-feathered quail

Table 2 delineates the effect of BRP supplementation in the diet on the growth metrics of yellow-feathered quails. At the outset, on day 1, body weight across all experimental groups did not exhibit significant disparities (P > 0.05). By the 42nd day, the MBRP group’s body weight significantly surpassed that of the CON group (P < 0.05), and a notable differentiation in body weight was observed between the LBRP group and the CTC group (P < 0.05). However, the body weight differences between the MBRP and HBRP groups in comparison to the CTC group were not statistically significant (P > 0.05). During the initial growth phase spanning days 1–21, the LBRP, MBRP, and HBRP groups did not show significant variations in average daily gain, average daily feed intake, and feed-to-gain ratio when compared to the CON and CTC groups (P > 0.05). Conversely, the CTC group demonstrated significant differences in average daily gain, average daily feed intake, and feed-to-gain ratio in contrast to the CON group (P < 0.05). In the subsequent growth period from days 22–42, no significant differences were identified in average daily gain, average daily feed intake, and feed-to-gain ratio among the LBRP, MBRP, HBRP, and CTC groups relative to the CON group (P > 0.05).

Table 2 The impact of Brassica rapa L. Polysaccharides on the production performance of yellow-feathered quails.
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Effects of Brassica rapa L. Polysaccharides on the Immune organs of yellow-feathered quail

Table 3 presents the impact of BRP supplementation on the relative weights of immune organs. At 21 days of age, the MBRP exhibited significantly higher average weights for the thymus and bursa of Fabricius compared to the CON (P < 0.05), while no significant differences were observed in spleen average weights across all groups (P > 0.05). By day 42, the average weight of the thymus in the MBRP group was significantly greater than that in the CON (P < 0.05); however, the relative weights of the spleen and bursa of Fabricius showed no significant differences among the groups (P > 0.05).

Table 3 Brassica rapa L. polysaccharides’ effect on the immune organs of yellow-feathered quails.
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The impact of Brassica rapa L. Polysaccharides on the serum immunological properties of yellow-feathered quail

The effects of BRP supplementation on the concentrations of immunoglobulins and cytokines in immune organs are detailed in Table 4. At 21 days of age, significant differences were observed in the concentrations of IgA, IgG, IL-1β, IL-10, and TNF between the MBRP group and the CON group (P < 0.05). However, when compared to the CTC group, the differences in concentrations of IgA, IgG, IL-1β, IL-10, and TNF in the MBRP group were not significant (P > 0.05). By day 42, no significant differences were found in the concentrations of IgA, IgG, IgM, and the cytokines IL-1β, IL-10, and TNF-α between the LBRP, MBRP, HBRP groups, and the CTC group when compared to the CON group (P > 0.05).

Table 4 Effect of Brassica rapa L. polysaccharides on the serum immune function of yellow-feathered quails.
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Effects of Brassica rapa L. Polysaccharides on the serum antioxidant capacity of yellow-feathered quail

The impact of BRP supplementation on the concentrations of serum antioxidants in yellow-feathered quails is presented in Table 5. At 21 days of age, levels of CAT, GSH-Px, and T-SOD were significantly higher in the LBRP, MBRP, and HBRP treatment groups compared to the CON group (P < 0.01). By day 42, no significant differences were observed in the levels of CAT, GSH-Px, and T-SOD between the LBRP, MBRP, HBRP, and CTC groups when compared to the CON group (P > 0.05).

Table 5 Effect of Brassica rapa L. polysaccharides on the antioxidant capacity of serum in yellow-feathered quails.
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The impact of Brassica rapa L. Polysaccharides on the cecal microbial community in yellow-feathered quails

Species venn diagram analysis

As depicted in Fig. 1A, the cecal microbiota of 21-day-old yellow-feathered quails shared a total of 322 operational taxonomic units (OTUs) across the LBRP, MBRP, HBRP, and CON groups. Specifically, unique OTUs numbered at 125 for the LBRP group, 105 for the MBRP group, 62 for the HBRP group, and 134 for the CON group. Figure 1B illustrates that at 42 days of age, the common OTUs across the LBRP, MBRP, HBRP, and CON groups increased to 383. Within this age bracket, unique OTUs were identified as 37 for LBRP, 106 for MBRP, 97 for HBRP, and significantly higher at 206 for the CON group.

Figure 1
figure 1

Venn diagrams 1(A) and (B) show the distribution of microbial OTUs in the ceca of yellow-feathered quails at ages 21 days and 42 days, respectively. Orange represents the LBRP group, light blue represents the MBRP group, green represents the HBRP group, and gray represents the CON group.

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Analysis of alpha diversity

At 21 days of age, compared to the CON group, there were no significant differences in the intestinal microbiota abundance (Ace index, Chao index and Shannon index) of the LBRP, MBRP, and HBRP groups as shown in Fig. 2(A-D) (P > 0.05). As depicted in Fig. 2(E-H) at 42 days of age, the intestinal microbiota abundance (Ace index, Chao index, and Shannon index) of the LBRP, MBRP, and HBRP groups were significantly lower than that of the CON group (P < 0.05), indicating that BRP altered the cecal microbiota abundance of yellow-feathered quails.

Figure 2
figure 2

Analysis of α-diversity of the microbial flora in the ceca of yellow-feathered quails at 21 and 42 days old, using ACE index (A and E), Chao index (B and F), Shannon index (C and G), and Simpson index (D and H). Red represents the CON group, light blue represents the LBRP group, green represents the MBRP group, and dark blue represents the HBRP group. a−bWithin a figure, different su-perscript letters indicate significant differences (p < 0.05).

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Analysis of beta diversity

Principal Coordinates Analysis (PCoA) of Cecal Microbiome β-Diversity at 21 Days of Age, as Illustrated in Fig. 3A: the distributions of LBRP, MBRP, and HBRP groups exhibit partial overlap with the CON group across the two major principal components, yet also display discernible separation. By day 42, Fig. 3B illustrates a pronounced segregation in microbial community compositions between the CON group and the LBRP, MBRP, and HBRP groups. These findings indicate that the alterations in microbial community structures attributable to the LBRP, MBRP, and HBRP treatments are measurable and significant when compared to the control.

Figure 3
figure 3

The β-diversity of the microbial flora in the ceca of yellow-feathered quails at 21 and 42 days was measured using PCoA (A and B). Red dots represent the LBRP group, light blue represents the MBRP group, green represents the HBRP group, and dark blue represents the CON group.

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The top 5 microorganisms in relative abundance at the phylum level

As illustrated in Fig. 4A, at 21 days of age, the top five phyla in terms of relative abundance within the cecal microbiota of yellow-feathered quails were Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and an unidentified phylum of bacteria. Figure 4B indicates that at 42 days of age, the leading phyla based on relative abundance in the ceca of yellow-feathered quails were Firmicutes, Bacteroidetes, Actinobacteria, Campilobacterota, and Desulfobacterota.

Figure 4
figure 4

The effect of adding BRP to the diet on the types of microbial communities in the ceca of yellow-feathered quails. Note: The top 5 microbial groups at the phylum level in the ceca of yellow-feathered quails at 21 and 42 days of age were measured, respectively (A and B).

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The LEfSe analysis of the cecal microbiota (LDA score > 3)

LEfSe analysis, known as LDA Effect Size analysis, allows for comparisons between multiple groups to identify species with significant differences in abundance between groups. Figure 5 displays species with LDA Score > 3, representing microbiota species with significant differential abundance. The length of the bar chart indicates the importance of the impact of the different species. As depicted in Fig. 5A, at 21 days of age in the ceca of yellow-feathered quails, the LBRP group harbored two differentially abundant microbial species at the genus level, specifically Lactococcus and Weissella. The MBRP group exhibited one differentially abundant microbial species at the genus level, identified as Parabacteroides. The HBRP group contained only one species with higher relative abundance at the family level, Tannerellaceae. In the CON group, three differentially abundant microbial species were observed, including two at the genus level, Faecalicoccus and Enoma, and one at the family level, Erysipelotrichaceae. At 42 days of age in the ceca of yellow-feathered quails, differential microbial species are illustrated in Fig. 5B. In the LBRP group, five differentially abundant microbial species were identified, encompassing Streptococcaceae at the family level, Streptococcus at the genus level, Lactobacillales at the order level, Bacilli at the class level, and Leuconostocaceae at the family level. The MBRP group presented only one differentially abundant microbial species at the genus level, Oscillibacter. The HBRP group was characterized by four differentially abundant microbial species, including Clostridia_UCG-014 at the order level, norank_o_Clostridia_UCG-014 at the family level, norank_f_norank_o_Clostridia_UCG-014 at the genus level, and norank_f_Ruminococcaceae. The CON group exhibited eleven differentially abundant microbial species, namely norank_f_Lachnospiraceae, Eubacterium_nodatum_group, unclassified_c_Clostridia, Mucispirillum, and Colidextribacter at the genus level; Deferribacteres at the class level; Deferribacteraceae and unclassified_c_Clostridia at the family level; Deferribacterales and unclassified_c_Clostridia at the order level; and Deferribacterota at the phylum level.

Figure 5
figure 5

LEfSe analysis of the differences in the cecal microbial communities of yellow-feathered quails at 21 days (A) and 42 days (B) of age. Note: (LDA Score > 3). Red represents the LBRP group, blue represents the MBRP group, green represents the HBRP group, and pink represents the CON group.

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Analysis of the correlation between microbial species diversity and measurement parameters

The Spearman correlation analysis between the relative abundance of dominant bacterial taxa and various production performance metrics, immune organ indices, and serum immune and antioxidant parameters is illustrated in Fig. 6. The results indicate that, with respect to immune parameters, the bursa of Fabricius index, and the immunoglobulins IgA, IgM, and IgG are significantly positively correlated with the abundance of genus Lactobacillus (R = 0.72, P < 0.01; R = 0.86, P < 0.001; R = 0.91, P < 0.001; R = 0.90, P < 0.001). IgA, IgM, and IgG are also significantly positively correlated with the abundance of genus Lactococcus (R = 0.76, P < 0.05; R = 0.84, P < 0.001; R = 0.84, P < 0.001). TNF is significantly negatively correlated with the abundance of genus Bifidobacterium (R = 0.72, P < 0.01). At the same time, IgA, IgM, and IgG exhibit significant negative correlations with the abundance of genus Enterococcus (R = 0.79, P < 0.01; R = 0.68, P < 0.05; R = 0.83, P < 0.001), and with the genus Faecalibacterium (R = 0.68, P < 0.05; R = 0.70, P < 0.05; R = 0.73, P < 0.01).In terms of antioxidant parameters, the enzyme catalase (CAT) shows significant positive correlations with the abundance of genera Lactococcus, Lactobacillus, and Bifidobacterium (R = 0.59; R = 0.62; R = 0.62, all at P < 0.05). T-SOD is significantly positively correlated with the abundance of genus Lactobacillus (R = 0.59, P < 0.05).

Figure 6
figure 6

Spearman correlation heatmap of cecal microbiota and the measured parameters. Figure 6 presents a heatmap of Spearman’s correlation analysis between dominant bacterial taxa and various production, serum immunological, and antioxidative parameters. Positive correlations are indicated in red, and negative correlations are denoted in blue, with significance levels marked as (*P < 0.05), (**P < 0.01), and (***P < 0.001). Abbreviations include: ADG, Average Daily Gain; ADFI, Average Daily Feed Intake; Thymus (%), relative thymus organ index; Bursa of Fabricius (%), relative bursa of Fabricius organ index; IgA, Immunoglobulin A; IgG, Immunoglobulin G; IgM, Immunoglobulin M; IL-1β, Interleukin-1 beta; IL-10, Interleukin 10; TNF, Tumor Necrosis Factor; CAT, Catalase; GSH-Px, Glutathione Peroxidase; T-SOD, Total Superoxide Dismutase.

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Discussion

Herbal polysaccharides exhibit significant biological activity and are currently a major focus of research. They hold potential as alternatives to antibiotic growth promoters in animal husbandry12. Limited studies have explored the use of BRP as a feed additive. This study investigates the effects of BRP supplementation in feed on growth performance, immune response, antioxidant capacity, and intestinal microbiota in quails. The objective of this study was to assess the potential improvements in growth performance, immunity, and antioxidant status in yellow-feathered quails following the dietary inclusion of BRP, and to establish the optimal addition level.

Extensive controlled studies have elucidated the impact of traditional Chinese herbal polysaccharides on animal growth. Guo et al.13 reported that a diet supplemented with Artemisia annua L polysaccharides significantly improved the average daily gain and apparent crude protein digestibility from days 36 to 42. Mahmoud et al.14 found that adding Astragalus in the feed could improve the feed conversion rate of quail. In our study, we observed that BRP significantly increased the body weight of yellow-feathered quails. Meanwhile, the differences in ADG and ADFI compared to the antibiotic group were not statistically significant and exhibited a dose-dependent relationship, indicating that BRP enhances the production performance of yellow-feathered quails. This finding aligns with the results reported by Zhang et al.15, who noted that Glycyrrhiza polysaccharides significantly improved average daily weight gain and reduced the feed-to-weight ratio in quails. However, Al-Sagan et al.16 reported that the main non-starch polysaccharide (NSP) in lupin seeds is galactan, which is made up of different proportions of arabinose and galactose monosaccharides. on-starch polysaccharides can negatively impact gut ecology and reduce the digestibility of poultry diets. Diets based on lupin have been observed to lower feed intake and growth rates in birds, which contrasts with our experimental findings. This difference might be due to the variations in the monosaccharide composition of Brassica rapa L. Polysaccharides. Therefore, BRP may promote growth by enhancing the synthesis and secretion of growth-related hormones, facilitating lipid and carbohydrate metabolism, and increasing the synthesis of amino acids and proteins.

The bursa of Fabricius, thymus, and spleen serve as principal immune organs in the development, differentiation, and antibody production of immune cells. Their relative weights often act as a metric for evaluating avian immune functionality, where higher indices of immune organ suggest robust organ development17. In our experiment, BRP significantly enhanced the immune organ index from days 1 to 21. Similar findings have been documented in previous investigations on traditional Chinese herbal polysaccharides. Liu et al.18 demonstrated that Lycium barbarum polysaccharides increased the weights of the spleen, thymus, and bursa of Fabricius in broilers. Nonetheless, some studies have reported no impact of Chinese herbal polysaccharides on the weight of immune organs19. The discrepancies with prior findings may be attributed to the source of polysaccharides, the health status of the animals, and their rearing conditions. The outcomes of this experiment indicate that BRP can stimulate the growth and development of immune organs, enhancing the resistance of quails to diseases, thereby improving their immunity.

Serum immunoglobulin assays represent the most commonly employed method for evaluating humoral immune function. Optimal immune functionality is crucial for the defense against and neutralization of pathogens20. IgA, IgG, and IgM constitute the primary antibodies in animal humoral immunity21, with serum levels of IgA, IgM, and IgG serving as valuable indicators of immune system capacity. Numerous studies have reported that Chinese herbal polysaccharides can enhance serum immune markers. Qiao et al.22 found that dandelion leaf polysaccharides provided immunoprotective effects in black bone silk chickens treated with cyclophosphamide (Cy). These effects included enhanced growth performance, increased spleen, thymus, and bursa indices, as well as promoted proliferation of blood lymphocytes. Additionally, it stimulated the secretion of cytokines (IL-2, IL-6, and INF-γ) and serum immunoglobulins (IgA, IgG), ultimately improving immune protection. Luo et al.23 discovered that Astragalus polysaccharides enhanced the immune capability of goslings with intestinal inflammation by increasing serum levels of IgM, IgG, and IgM. In our experiment, BRP enhanced the immunoglobulin levels in the serum of yellow-feathered quails between days 1 and 21. This intervention significantly reduced pro-inflammatory factors while increasing anti-inflammatory factors. However, by 42 days of age, changes in serum immune factor levels were minimal, and the levels of immunoglobulins and immune factors appeared to stabilize. This stabilization may be linked to the maturation and development of the immune system. In a report on a vegetable related to Nozawana in the Brassicaceae family, pectin extracted from the stems of Brassica oleracea var. italica induced the production of the anti-inflammatory substance IL-10 but did not induce macrophages to produce NO, IL-1β, and IL-1224. This may be attributed to BRP may enhance the activity and function of immune cells and promote their secretion of cytokines by binding to receptors on the surface of immune cells, thereby activating signaling pathways. These cytokines play an important role in regulating immune responses and promoting the development of immune organs.

The antioxidative enzyme system, serving as the first barrier in animal antioxidant defenses, reflects the metabolic level of reactive oxygen species and the extent of tissue damage. This enzymatic system encompasses SOD, catalase, and GSH-Px, among others25. The health of an organism and its defense system’s antioxidative capacity are intimately linked. A robust antioxidant system, by scavenging free radicals and thus preventing the formation of lipid peroxides in biological membranes, plays a pivotal role in delaying aging and preventing chronic diseases26. An increase in the levels of SOD, GSH-Px, and CAT signifies an improvement in the antioxidative state27. This study demonstrates that adding BRP to the feed significantly increases the levels of antioxidant enzymes in the serum of yellow-feathered quails. Our findings closely align with those of Yu et al.28, who demonstrated that Artemisia argyi leaf polysaccharides significantly elevated SOD and GSH-Px levels in the serum, liver, and muscle of mice, exhibiting a clear dose-dependent effect. This result indicates that plant polysaccharides promote healthy growth by scavenging free radicals, protecting immune cells from oxidative damage, and maintaining their normal function.

The gut microbiota establishes a multilevel intestinal microbial barrier, participating in the host’s nutrition and intestinal defense, and modulating the immune function of the host29. Under normal circumstances, the distribution of intestinal microbiota is relatively stable, maintaining a balance within the microbial community30. The diversity of the gut microbiota is closely linked to the health of the host, with a reduction in microbial diversity being a significant indicator of dysbiosis31. Our study found that BRP significantly increased the diversity and variability of cecal microorganisms in yellow-feathered quails. Notably, BRP exhibited a certain inhibitory effect on Campylobacter, a major cause of acute gastroenteritis globally since its identification as a human pathogen in the 1970s32. These findings suggest that BRP may have potential for the prevention and treatment of human acute gastroenteritis.

Additionally, as quails age, BRP promotes the growth of the microbial population on the posterior wall of the cecum in yellow-feathered quails. Guan et al.33 demonstrated that supplementing the diet with 8 g/kg Yupingupinus polysaccharide significantly enhanced the abundance of Firmicutes and increased the proportion of beneficial bacteria, aligning with our experimental findings. Previous research has demonstrated that Firmicutes are associated with the capability to harvest energy34, and the ratio of Firmicutes to Bacteroidetes is of significant importance for maintaining homeostasis in the internal environment and the utilization of nutrients. Increasing the abundance of Firmicutes while reducing Bacteroidetes in the cecal contents can enhance the efficiency of nutrient absorption. It has been reported that herbal polysaccharides can increase the secretion of gastrointestinal digestive enzymes, and alter the quantity of intestinal microbiota to promote health and growth35.

To explore the impact of dominant intestinal microbiota on phenotypic characteristics, we employed Spearman’s rank correlation coefficient to assess the associations between the relative abundance of dominant bacterial taxa and various measurement parameters. Our research shows that the addition of BRP to the feed significantly positively correlates with the immune organ index and immunoglobulin in yellow-feathered quail, while it significantly negatively correlates with harmful bacteria such as Enterococci and fecal bacilli. Furthermore, the abundance of Lactic acid bacteria and Bifidobacteria shows a significant positive correlation with antioxidant enzymes and a significant negative correlation with pro-inflammatory factors (such as TNF). Lactic acid bacteria and Bifidobacteria have multiple health benefits, such as preventing pathogenic microbial infections, reducing the risks of hypertension, inflammation, diabetes, and oxidative stress. They also play a role in regulating gut microbiota, immune modulation, and lowering cholesterol36. Additionally, bifidobacteria and lactic acid bacteria can metabolize to produce organic acids such as lactic acid, acetic acid, propionic acid, and butyric acid, thereby lowering intestinal pH and inhibiting the growth of acid-sensitive pathogens like Escherichia coli and Salmonella37,38.

It is evident that the appropriate supplementation of BRP in the diet can enhance the growth performance, immune capability, antioxidant function, and improve the gut microbiota of yellow-feathered quails. Taking cost into consideration, it is recommended to add 0.5 g of BRP per kilogram of feed. This suggests that BRP has the potential to serve as a feed additive and could act as an alternative to antibiotics.