All birds in this study feeding program were approved by the Animal Ethics Committee of Shangqiu Normal University (2023–1,109).

Chinese yam and Rehmannia glutinosa were obtained from Wuzhi county, Jiaozuo city, Henan Province, China in 2024. At the time, all samples were dried and crushed. Chinese yam passed through an 80-mesh sieve, and Rehmannia glutinosa with a particle size pass 40 mesh. Then prepared the medicine pair (CYRG) by mixing Chinese yam powder and Rehmannia glutinosa powder in a 1:1 (m/m) ratio. Then, CYRG was stored in a cool, dry place until use.

Three hundred healthy 23-day-old Arbor Acres female broilers with similar body weight were randomly placed into five groups with 6 replicates, with 10 broilers per replicate. In the control group (CON), broilers received the basal diet, and other treatment groups (0.5% CYRG, 1% CYRG, 1.5% CYRG, and 2% CYRG) received the basal diet supplemented with 0.5, 1, 1.5, and 2% CYRG, respectively. The feeding experiment last for 19 days, until the birds were 42 days old. The basic diet was formulated to reference to the National Research Council (1994) (15), and Table 1 shows the ingredients of the basal diet. Broilers were caged, and had free access to diet and water during the experiment. Birdhouse temperature was kept at 24 ± 2 °C and under constant light for 24 h until end of the trial.

At 23 and 42 days of age (day 1 and 19 of the experiment), live- weight and feed weight were measured per replicate to calculate average daily gain (ADG), average daily feed intake (ADFI), average daily feed intake/average daily gain (F/G). On day 42, after 12 h of fasting, one bird was randomly selected from each replicated to measure the body size trait. Body slanting length (BSL) was gauged from the shoulder to the ipsilateral ischial tuberosity. Keel length (KL) was the distance, between the front and rear ends of the keel. Shank girth (SG) was the circumference of the middle of the shin. Shank length (SL) was determined as the straight-line distance from the supra metatarsal joint to the middle of the third and fourth toes. Chest width (CW) was calculated as the distance between two shoulder joints. Chest depth (CD) was measured from the first thoracic vertebra to the front of the keel. Hip bone width (HBW) was the length between the two side waist corners. Waist edge width (WEW) was the maximum width of the two hip joints on the left and right sides. Here, caliper was used to measure shank length, chest width, chest depth, and hip bone width and a measuring tape was used to record body slanting length, keel length, shank girth and waist edge width. All operations were carried out following agricultural standard NY/T823-2020 (16).

Thirty broilers (one per replicate), the venous blood was drawn from the wing veins of birds, and placed in centrifuge tubes for overnight at 4 °C. Serum was collected after centrifugation (3,000 g, 10 min, at 4 °C), placed into sterile tubes, and kept at −80 °C until analysis. Then, birds were slaughtered. After bloodletting, feather extraction, beak shell and foot skin removed, the weight of carcass, evisceration, semi-evisceration, abdominal fat, breast muscle, leg muscle, wing (a pair), legs (two) were recorded. Next, the slaughter percentage, eviscerated weight percentage, semi-eviscerated weight percentage, abdominal fat percentage, breast muscle percentage, leg muscle percentage, wing weight percentage and leg weight percentage were calculated. The determination method is in accordance with the agricultural industry standard NY/T 823-2004 “Terms and Measurement and Statistical Methods for Poultry Production Performance”.

Thirty broilers (one per replicate), the heart, liver, spleen, lung, kidney, thymus, pancreas, intestine, glandular stomach, muscular stomach and bursa of Fabricius were immediately dissected, blotted and weighed. Relative weight of internal organs = weight of organ/live weight × 100%.

Interleukin 4 (IL-4), Interleukin 6 (IL-6), and Interleukin 1β (IL-1β) levels were measured in serum using the enzyme-linked immunosorbent assay (ELISA) kits specific for poultry according to the manufacturer’s instructions (Jiancheng Biotechnology Co. Ltd., Nanjing, China). At the same time, the immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) levels in serum were determined with ELISA kits specific for chicken according to the manufacturer’s instructions (Shanghai Mlbio Co., Ltd. Shanghai China).

The oxidation-stress related indicators were determined as described by Guo et al. (17). The level of total antioxidant capacity (T-AOC) in bird serum was detected with ELISA kits specific for poultry (Jiancheng Biotechnology Co. Ltd., Nanjing, China). The catalase (CAT) level was measured using the molybdate colorimetric method. The glutathione peroxidase (GSH-Px) level was detected using the 5,5′-dithio-nitrobenzoic acid (DTNB) chromogenic method. Superoxide dismutase (SOD) in serum was measured by the modified pyrogallol autoxidation method. Malondialdehyde (MDA) was measured by the modified thiobarbituric acid reactive substances assay.

The cecal contents were collected and sent to Beijing Biomarker Technologies Co. Ltd. (Beijing, China) for 16S rRNA gene sequencing. Three samples each for five groups.

Total bacterial DNA was extracted from ceca contents (100 mg) using the E.Z.N. ATM Mag-Bind Soil DNA Kit (Omega Bio-Tek, USA), and the extracted DNA was detected by 1% gel electrophoresis. Qubit R 4.0 (ThermoFisher Scientific, USA) was used to determine the DNA concentration. The synthesized primers (338F: ACTCCTACGGGAGGCAGCA; 806R: GGACTACHVGGGTWTCTAAT) were used for PCR amplification of the 16S r RNA gene V3/V4 regions. All of the PCR reactions were conducted in triplicates. The amplified products were quality evaluated by using 2% agarose gel electrophoresis and Qubit^® 4.0 fluorescence quantifier. Sequencing libraries were using the TruSeq^® Nano DNA Kit (Illumina, USA). The qualified libraries were used for paired-end sequenced (2 × 250 bp) on the Illumina NovaSeq 6,000 platform.

Trimmomatic v0.33 and cutadapt 1.9.1 software was used to remove the adapters, primers, and low-quality sequences from the raw reads, and denoise, double-ended sequence splicing, and removal of chimeric sequences was by the DADA2 plug-in. The processed reads were clustered into Operational Taxonomic Unit (OTUs) with 99% sequence identity. Then, phylogenetic analysis and multiple sequences were performed based on the obtained OTUs. Before conducting the diversity analysis, the rarefaction curves were generated to reflect sequencing depth before diversity analysis. q2-diversity plug-in in QIIME2 2020.6 software was used to analysis microbiota diversity and composition. The significance of differences between groups were analyzed using the Wilcoxon rank-sum test, and the p-values were corrected by FDR. The Linear discriminant analysis (LDA) effect size (LEfSe) was conducted by Python Lefse package to search for biomarkers with statistical differences between groups based on LDA > 4 and p < 0.05. Statistical analysis was performed using One-way ANOVA in SPSS26.0 (SPSS Inc., USA), and multiple comparisons was conducted by Duncan’s method. Using the ‘corrr’ and ‘ggplot2’ packages in R (R version 4.5.2), the ‘pearson’ method was employed to calculate the microbial correlation matrix and generate a correlation heatmap.

Table 2 shows the effects of dietary CYRG on broilers’ growth performance in this study. In the trail period, the highest body weight gain was determined in the 0.5% CYRG group (1669.20 g), while body weights between 1429.40 and 1598.60 g were obtained in the 2% CYRG, 1% CYRG, 1.5% CYRG and CON groups, respectively. No difference of ADG, ADFI and F/G between the treatment and the CON groups were observed during the trail periods (P > 0.05). However, the 0.5% CYRG and 1.5% CYRG treatment groups had higher ADG, and 0.5% CYRG group had the highest ADG in all trail periods.

The effects of dietary CYRG on body size measurements are presented in Table 3. No difference of the measured body size data was observed among the treatment groups in this study (P > 0.05). Several body size measurements, including BSL, KL, AND CD of broilers in the CON group were higher than those in the supplementation CYRG groups, while SG and SL in the dietary CYRG treatment groups were higher than those in the CON group.

Table 4 shows the effects of dietary CYRG on the slaughter performance in broilers. Slaughter percentage and semi-evisceration weight percentage of broilers in the dietary addition of CYRG groups were significantly higher than those in the CON group (P < 0.05). Meanwhile, the CYRG treatment groups had a higher evisceration weight percentage and leg weight percentage compared with the CON group. Furthermore, abdominal fat percentage, breast muscle percentage, leg muscle percentage, and wing weight percentage were not different among all groups (P > 0.05).

As shown in Table 5, no significant difference among the five groups was found for all of the detected of the relative internal organs weight in this study (P > 0.05). The relative weight of lung and kidney of the broilers in the CYRG treatment groups were higher than those in the CON group, while glandular stomach and muscular stomach in the in the CYRG treatment groups were lower compared to the CON group.

As shown in Table 6, the serum concentrations of IL-4 in the 0.5, 1, and 1.5% CYRG treatment groups were significantly higher than those in the CON, and 2% groups (p < 0.05). The serum concentration of IL-6 in the CON group were significantly higher than those in the 0.5, 1, and 2% CYRG groups (p < 0.05). In addition, the serum concentration of IL-1β in the 2% CYRG group were significantly higher than those in the other four groups (p < 0.05).

The effects of dietary CYRG on the concentrations of serum immunoglobulins in broilers are shown in Table 7. The serum concentration of IgG in the 1.5% CYRG group was significantly higher than that in the CON group (p < 0.05). In addition, the serum IgM concentrations in the 1.5 and 2% CYRG groups were higher than that in the CON group (p < 0.05). Meanwhile, no differences in serum IgA concentrations were found among all the groups (p > 0.05).

As shown in Table 8, the activities of CAT, GSH-Px, and T-AOC in the serum of all experimental groups were higher than those of the CON group, while the activity of MDA was on the contrary. Meanwhile, the activity of CAT in the 0.5% CYRG group was significantly higher than that in the CON group (p < 0.05). The activities of GSH-Px and T-AOC in the 0.5% CYRG and 2% CYRG groups were significantly higher than those in the CON group (p < 0.05). The level of MDA in the 0.5% CYRG group was significantly lower than that in the CON group (p < 0.05). Among all five groups, no differences in serum SOD were determined (P > 0.05).

The analysis revealed that IL-4 level in the serum of broiler was negatively associated with IL-1β and MDA levels (p < 0.05) (Figure 1). Moreover, serum IL-6 level was negatively correlated with antioxidant indexes, such as CAT, GSH-Px, and T-AOC levels (p < 0.05). And CAT level has been positively associated with SOD, GSH-Px, and T-AOC levels (p < 0.05). GSH-Px level was positively correlated with T-AOC level (p < 0.05). CYRG intervention significantly elevated the levels of CAT, GSH-Px, and T-AOC, combined with the MDA level declined. Meanwhile, CYRG intervention significantly elevated the level of anti-inflammatory factor IL-4 and immunoglobulin (IgA, IgG, and IgM), and declined the inflammatory factors IL-6, and IL-1β. Therefore, CYRG inhibit the release of inflammatory factors, and elevate the immune and antioxidant activities.

Clustering of microbes in the ceca microbiota was observed among the five groups (Figure 2). An average of 58,078 clean reads obtained from each ceca sample. Figure 2A showed that the sequence number was more than 50,000 reads, while the OTU number kept unchanged, indicated that the sequencing depth was sufficient to adequately reflect the microbial community composition of the ceca samples. Among the 6,773 clustered OTUs, 248 OTUs were shared by all the five treatment groups (Figure 2B), and the CON, 0.5% CYRG, 1% CYRG, 1.5% CYRG, and 2% CYRG groups were found to contain 1758, 1,297, 1817, 2016, and 2021 distinct OTUs, respectively. And as shown in Figure 2B, 1,165, 738, 1,163, 1,387, and 1,382 unique OTUs were identified in the CON, 0.5% CYRG, 1% CYRG, 1.5% CYRG, and 2% CYRG groups, respectively. The alpha diversity parameters, including Ace, Chao, Shannon, and Simpson index (Figure 2C and Table 9), were not different among the five treatment groups in this paper (p > 0.05). R value calculated by ANOSIM was above 0, indicating there were greater difference between groups. The principal components analysis (PCA) (Figure 2D) showed that there was greater difference between groups (p = 0.01), and CYRG supplementation had distinguishable clustering with the CON group while the principal component axes PC1, PC2, and PC3 explained 46.77, 13.50, and 9.18% of the total variation, respectively.

Taxonomic unit analysis revealed that the dominant phyla (Figure 3A and Table 10) of the five groups included Firmicutes (97.39, 98.33, 98.87, 99.14, 99.23%), which is in absolute dominance, followed by Actinobacteriota (1.34, 1.01, 0.67, 0.60, 0.53%), and Proteobacteria (0.93, 0.55, 0.37, 0.11, 0.12%). The relative abundance of Actinobacteriota in 1.5% CYRG and 2% CYRG groups were significantly declined compared with that in the CON, 0.5% CYRG, and 1% CYRG groups (P < 0.05) (Table 10). And CYRG supplementation significantly declined the relative abundance of Cyanobacteria compared to the CON group. The relative abundance of Firmicutes, Proteobacteria, and Bacteroidota were not affected by CYRG supplementation (p > 0.05) (Table 10). At the genus level (Figure 3B and Table 11), the dominant microorganisms in the five groups were Faecalibacterium (8.34, 12.16, 7.59, 9.20, 12.69%), Lachnoclostridium (12.16, 7.38, 6.87, 9.59, 8.35%), [Ruminococcus]_torques_group (6.36, 7.47, 8.10, 6.52, 5.72%), Christensenellaceae_R_7_group (7.70, 6.67, 6.91, 5.59, 5.04%), and Blautia (4.53, 7.90, 7.89, 4.94, 4.62%). Compared with the CON group, 0.5% CYRG and 1% CYRG supplementation significantly declined the relative abundance of Lachnoclostridium, and increased the abundance of unclassified_Clostridia_UCG_014 (p < 0.05) (Table 11). The birds of 1.5% CYRG group had more Ligilactobacillus compared to the CON and 1% groups (p < 0.05). Contrast to the CON, 1% CYRG and 1.5% CYRG birds had fewer Sellimonas, and 1.5% CYRG and 2% CYRG birds had fewer Romboutsia (p < 0.05). In addition to that, we can find that compared to the CON and 0.5% CYRG groups, 1% CYRG supplementation decreased the number of unclassified_Lachnospiraceae in the caecum of broilers, while increased significantly the number of Limosilactobacillus (p < 0.05) (Table 11).

The LEfSe results (Figure 3C) showed that Lachnoclostridium, Clostridium hylemonae, Peptostreptococcales Tissierellales, Ruminococcaceae bacterium AM2, unclassified Hungateiclostridiaceae, and Hungateiclostridiaceae were enriched in the CON group. The microbiota in the 0.5% CYRG group were more abundant in Peptostreptococcaceae, Romboutsia ilealis, Enterococcus cecorum, Enterococcaceae, and Enterococcus. The relative abundance of unclassified Christensenellaceae R7 group and uncultured_rumen_bacterium was increased in the 1% CYRG group. The 1.5% CYRG group was significantly populated with Ruminococcus sp. 16,442, unclassified Family XII UCG 001, and Family XII UCG 001. While Candidatus Dorea massiliensis AP6 and unclassified Incertae Sedis were highly represented in 2% CYRG group.

Figure 3D showed the variation in species composition and their species abundance distribution trends among groups. The CON group was significantly populated with Lachnoclostridium, Faecalibacterium, Christensenellaceae_R_7_group, [Ruminococcus]_torques_group, Erysipelatoclostridium, and Blautia. Lactobacillus, Faecalibacterium, Blautia, Lachnoclostridium, and [Ruminococcus]_torques_group were greatly enriched in the 0.5% CYRG group. While [Ruminococcus]_torques_group, Blautia, Faecalibacterium, Christensenellaceae_R_7_group, and Lachnoclostridium were highly represented in 1% CYRG group. The 1.5% CYRG group were populated with Ligilactobacillus, Lachnoclostridium, Faecalibacterium, Lactobacillus, and [Ruminococcus]_torques_group. The relative abundance of Faecalibacterium, Lachnoclostridium, Lactobacillus, and Ligilactobacillus was increased in 2% CYRG group.

The correlation analysis revealed that Clostridium hylemonae in the cecal of broilers was negatively associated with anti-inflammatory cytokines IL-4, and positively correlated with inflammatory factors IL-6 (P < 0.01) (Figure 4). Sellimonas intestinalis has been negatively associated with IL-4 (P < 0.05). IgA was positively associated with Lactobacillus salivarius and Lactobacillus_crispatus (P < 0.05), and negatively correlated with unclassified Ruminococcaceae and Faecalibacterium prausnitzii (P < 0.01). Meanwhile, Lactobacillus salivarius was positively associated with IgG (P < 0.05). Unclassified Clostridia ucG 014 and Ruminococcaceae bacterium were positively correlated with CAT and GSH-Px, while Unclassified Clostridia ucG 014 was negatively correlated with MDA. Moreover, Clostridium hylemonae was negatively correlated with antioxidant indexes, such as CAT, GSH-Px, and T-AOC. CYRG intervention significantly elevated the relative abundance of Unclassified Clostridia ucG 014 and Limosilactobacillus, combined with the structural changes of intestinal flora. Therefore, we speculate that CYRG may be by increasing the proportion of beneficial bacteria in the intestine to enhance the anti-inflammatory cytokines and immunoglobulin production, inhibit the release of Pro-inflammatory cytokines, and elevate antioxidant activity. The mechanism of CYRG relieving intestinal inflammation in broiler needs to be further analyzed.

Pan et al. (24) found that supplemental 2% Chinese yam significantly increased ADG and decreased F/G in weaned piglets. Basal diet supplemented with Radix rehmanniae preparate polysaccharide (RRPP) did not affect the broilers’ growth performance during 1–21 days, while during 22–35 days and 1–35 days periods, 600 mg/kg and 900 mg/kg RRPP linearly improved the body weight gain and feed conversion ratio. Rehmannia glutinosa oligosaccharides significantly increased the body weight, pancreatic index, thymus index, and colon length of mice by lipopolysaccharide (LPS)-induced (25). Radix rehmanniae preparate significantly decreased the liver, spleen, kidney, pancreas and fat indexes of diabetic mice, and there was no significant difference in heart and lung indexes (26). 0.5 g/kg Chinese yam polysaccharides (CYP) improved the thymus index and spleen index of broilers at 28 and 48 days, and improved the thymus index at 48 days (27). Yam treatment significantly decreased the liver index of carbon tetrachloride-induced hepatic fibrosis rats (28). Gavaged administration with 800 mg/kg/d polysaccharides fraction, DOP-2, prepared from Dioscorea opposita thunb significantly improved the thymus and spleen indexes of immunosuppressed mice (29). Zhang et al. (3) reported that 0.1% Chinese yam polysaccharide copper complex increased remarkedly ADG, slaughter percentage, semi-evisceration weight percentage, evisceration weight percentage, breast percentage, leg muscle percentage and decreased FCR in broilers. 500 mg/kg Chinese yam polysaccharide improved live weight, half-eviscerated carcass percentage, eviscerated carcass percentage, and thigh muscle percentage of broilers (7). 250 mg/kg and 500 mg/kg Chinese yam polysaccharide improved the thigh muscle percentage (9). Genistein extracted from soy plants increased the broilers’ body weight gain, tibial length, tibial width and slaughter performance and decreased the feed conversion ratio (FCR) (30). Supplementation betaine reduced the feed intake, and 2.25 g/kg betaine improved the weight gain, the carcass weight, breast yield, intestinal length and weight and reduced fat weight of Japanese quails. And they also found there were no difference among groups in body length, shank length, shank diameter, and keel bone length or breast width (31). We found dietary addition CYRG improved the broilers’ slaughter percentage and semi-evisceration weight percentage, while the growth performance, body size trait, and relative organ weight were not altered in this study. We speculated that the reasons for the differences in the above research results might be related to factors such as the species of the test subjects, the duration of the test, and the dose of additives used.

Studies indicate that, compared with single drug administration, the combined use of ‘medicine pair’ can promote the growth, immunity and antioxidant capacity of animals. This is because there is a synergistic effect between the medicine pair, and it can also enhance intestinal health and nutrient absorption by promoting the proliferation of beneficial bacteria, thereby inhibiting the growth of pathogenic bacteria.

In this study, the addition of CYRG in diet can enhance the immunity of broilers by improving the concentrations of IL-4, IgA, IgG, and IgM in serum and declining the levels of serum IL-6 and IL-1β, and we recommend the supplementation of 0.5% CYRG in broiler diets. Deng et al. (9) found that 0.50 g/kg Chinese yam polysaccharides supplementation in diets improved significantly the serum IL-4, IL-6, IgA, IgG, and IgM levels at 28 days. The levels of serum immunoglobulin IgG, IgM and Newcastle disease antibody were significantly reversed by Chinese yam peel supplementation with diets in intramuscular injection of cyclophosphamide (CTX) broilers (32). Zhang et al. (33) found that a non-starch polysaccharide (CYP-A) isolated from Chinese yam suppressed pro-inflammatory cytokine production in colitis symptoms mice induced by dextran sulfate sodium (DSS) and reduced oxidative stress. Rehmannia glutinosa polysaccharide (RGP) reduced the gene and protein expression levels of pro-inflammatory factors such as TNF-α, IL-1β, IL-6, and caspase-1, while upregulated the gene expression levels of the anti-inflammatory factor IL-10 in LPS-induced mice (34). Similar to our research findings. Chinese yam and Rehmannia glutinosa could increase the levels of serum cytokines and immunoglobulin, thereby enhancing the body’s disease resistance and immune function. Numerous studies indicate that natural botanical are potential antioxidants (35, 36) and antioxidants are the substances that protect organisms against damage caused by oxidation. In present study, an evaluation about the effects of the supplementation CYRG in broilers’ diets on serum antioxidative indicators demonstrated that dietary CYRG significantly increased the levels of CAT, GSH-Px, T-AOC, and decreased the levels of MDA of broilers (0.5% CYRG group), which revealed that CYRG had strong antioxidant activity. A previous study shown that dietary supplementation with 500 mg/kg Chinese yam polysaccharide can significantly improve the contents of serum T-AOC, T-SOD, glutathione peroxidase (GPX), and glutathione s-transferase (GST) in broilers (p < 0.05) (7). Chen et al. (37) found that supplementation with 1.6% Chinese yam by-product in juvenile fish diets could significantly improve the levels SOD and GSH (p < 0.05), meanwhile, 0.4 and 1.6% Chinese yam by-product significantly declined the levels of MDA, thereby, Chinese yam by-product could protect liver and intestine health. Rehmannia glutinosa oligosaccharides elevated significantly the activities of intestine SOD, GSH-Px, and CAT, and decreased the content of MDA in LPS-induced mice (25). Qiao et al. (34) found LPS challenge induced pronounced oxidative damage in mice, elevating MDA and CAT levels, and Rehmannia glutinosa polysaccharide treatment significantly attenuated these changes, reduced MDA accumulation and normalized CAT activity, improved the redox balance. These studies have demonstrated that both Chinese yam and Rehmannia glutinosa can enhance the antioxidant function of the animal body. In this study, it was found that the combination of Chinese yam and Rehmannia glutinosa also has the ability to enhance the animal’s antioxidant capacity, moreover, the ‘medicine pair’ can exert a synergistic effect. Our research found adding 0.5% CYRG could improve the antioxidant function of broilers, mainly manifested in that 0.5% CYRG significantly increased the total antioxidant capacity, glutathione peroxidase and hydrogen peroxide enzyme levels in the serum, and significantly reduced the malondialdehyde level.

Composition of intestinal microbiota is a vital determinant of intestinal health. The intestinal microbiota was responsible for converting food into nutrients and energy (38), and modulate the overall health and productiveness in poultry (21, 39). The chicken cecum is considered to be the most important part in the distal intestine and is the greatest concentration of intestinal microorganisms in mature chickens (40). Digestion in the cecum is associated with cecal microbes, therefore, the diversity and composition of the intestinal microbiota are key elements to maintain the intestine health (41–43). In this study, the alpha diversity was not affected by dietary CYRG, while the results of beta diversity analysis showed significant differences between groups. Similar to our research results, Chen et al. (44) found that the alpha diversity indexes of caca microbes were not affected by supplementation with 300 and 600 mg/kg Chlorogenic acid in a basal diet in Hy-line brown pullets. Our results of the PCA analysis indicted a distinction in the ceca microbial community composition among the five treatment groups. Firmicutes was the most abundant phylum with the largest proportion in this study, followed by Actinobacteriota and Proteobacteria, which consistent with the other report (38, 45). The result support other study (46) that Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Cyanobacteria were the major microbial groups. Microbiota analysis revealed that birds fed diets supplementation with 0.5, 1, 1.5 and 2% CYRG increased the abundance of cecal Firmicutes by 0.97, 1.52, 1.80, and 1.89% and decreased the proportion of Actinobacteriota, Proteobacteria, and Cyanobacteria by 24.63, 50, 55.22, and 60.45%, 40.86, 60.22%, 88.17, and 87.10, 66.67, 75.76, 57.58, and 66.67%, respectively, compared with the CON group. El Kaoutari et al. (47) reported that many members of Firmicutes could encoded carbohydrate-active enzymes to the hydrolysis and the utilization of carbohydrates. And, members of Firmicutes are involved in the degradation of insoluble fibers in food digestion (48). Meanwhile, Turnbaugh et al. (49) found Firmicutes can generate more harvestable energy. In this study, CYRG supplementation increased the abundance of Firmicutes, which indicate that CYRG may enhance the energy intake capacity of broiler chickens. Reid et al. (50) discovered that Proteobacteria members are associated with cellulose activity. Proteobacteria is the largest phylum of bacteria and members of Proteobacteria are all Gram-negative bacteria, including many opportunistic pathogens (51), such as Escherichia coli, Escherichia, Salmonella, Vibrio, Helicobacter, Shigella, Pseudomonas aeruginosa, Vibrio cholerae, Yersinia pestis, Neisseria meningitidis, Neisseria gonorrhoeae, Campylobacter jejuni, Helicobacter pylori, etc. and its abundance can significantly increase in the case of a disease. In the present study, the CYRG diet reduced the relative abundance of Proteobacteria, indicating that CYRG can improve intestinal health by reducing Proteobacteria in the cecum. Wu et al. (52) reported that certain bloom-forming cyanobacteria produced microcystins, which can induce toxicity in various organs, such as renal toxicity, reproductive toxicity, cardiotoxicity, and immunosuppressive effects. And they also found the toxicity of microcystins on the gastrointestinal tract is multidimensional, it not only can affect gastrointestinal barrier function but also shift the gut microbiota structure in different gut regions, and it can inhibit the release of inflammatory cytokines, then affects the expression of immune-related genes in the intestine (52). In this study, we found that adding CYRG to the diet reduced the relative abundance of cyanobacteria, indicating that CYRG can improve the intestinal microbial environment of broilers and protect intestinal health.

When intestinal diseases and inflammation occurs, they can cause an imbalance of intestinal flora. Most members of the Firmicutes are beneficial bacteria, such as Lactobacillus and Fecal bacilli. They produce acetic acid and butyric acid in the intestines, which helps promote the growth of intestinal epithelial cells and prevent pathogens from interfering with intestinal health (25). In this study, the results showed that CYRG could regulated the intestinal floral structure, which may be facilitated by increasing the relative abundance of intestinal probiotics (Firmicutes), and decreasing the numbers of harmful bacteria such as Proteobacteria and Cyanobacteria. Thus, CYRG affects the structure of intestinal microbiota, regulates the intestinal microecological balance, while promoting the health of broilers. Meanwhile, correlation analysis revealed that beneficial bacteria in the intestine (including Lactobacillus salivarius and Lactobacillus crispatus) were positively associated with intestinal IgA and IgG. Moreover, Ruminococcaceae bacterium were positively correlated with CAT and GSH-Px activities, and Unclassified Clostridia ucG 014 was negatively correlated with MDA. Thus, CYRG may improve intestinal function, enhance immunity and maintain intestinal health by adjusting the intestinal flora.

Therefore, CYRG supplementation was beneficial to growth performance, slaughter performance, immunity and antioxidant capacity of broilers. At the same time, CYRG can regulate the cecal floral structure in broilers, increase the abundance of beneficial bacteria and decrease the quantity of harmful bacteria, reduce the risk of intestinal inflammation and maintain the intestinal barrier. CYRG has potential application value in promoting the growth of broiler chickens and preventing intestinal inflammatory diseases.

It’s generally considered that traditional Chinese medicines are characterized by “multi-components” and “multi-targets.” Therefore, the growth performance, body size trait, relative organ weight, slaughter performance, cytokines and antioxidant indicators in serum were detected in this paper. However, traditional Chinese medicine contains numerous ineffective and unknown ingredients (53–55), which brings about great difficulty in clarifying its application effect and in accurately applying it in actual production. So, the results in this study indicated that CYRG is effective in improving slaughter performance of broilers, which provides an objective data for the application of CYRG in poultry breeding. Nevertheless, there are still some shortcomings in this research. For example, if the feeding experiment could be conducted for a longer period of time, would it be possible to have a positive impact on the body size and growth performance of the broilers? This will be explored in more depth in future research, to ensure the assessment of the application potential of Chinese yam–Rehmannia glutinosa ‘medicine pair’ in the livestock industry more objective and impartial.