A total of 450 healthy 1-day-old AA broilers with similar body weight (40.0 ± 5.0 g) were randomly divided into 5 groups, with 6 replicates per group and 15 broilers per replicate. The animal protocols used in this study have been evaluated and approved by the Institutional Animal Care and Use Committee of Hebei University of Engineering (Identification code: HBUE2022, Date of approval: 16 March 2022). The control group was provided a basic diet, while the experimental groups were fed the basic diet supplemented with 0.25, 0.5, 1 and 2% CHM-JM113 (a Chinese herbal medicine formulation combined with JM113), respectively. During the feeding phase, all experimental groups were supplemented with JM113 powder (1 g/kg in the meal). The experiment lasted for 42 days, which was divided into an early stage (1–21 days old) and a late stage (22–42 days old). The basic diet composition was developed and granulated according to the Chinese broiler nutrition standard (NY/T 33-2004). The food composition and nutritional composition are shown in Table 1. During a 42 day test period, animals were fed in Gaobai Family Farm in Linzhang County, Handan City. Chinese herbal medicine was composed of Astragalus, tangerine peel, dandelion and thyme (brought from Anhui Bozhou Wuhua Food Co., Ltd.) in a ratio of 1:1:1:1. The viable count of Lactobacillus plantarum JM113 was 4.4 × 10⁹ CFU/g.

During the experiment, free diet, drinking water and artificial light were supplemented. The feeding method was imprisoned in the house. The illumination time was 24 h per day from 1 to 7 days of age, and subsequently the illumination was reduced by 1 h per week. After 4 weeks of age, the illumination time was switched to a standard illumination time of 20 h. The room temperature was 33°C on the first day of the experiment, and then decreased by approximately 2.5°C every week until it reached 19°C, while maintaining a humidity level of 60–70%.

On the 21st and 42nd days of the experiment, one AA broiler was randomly selected from each replicate to collect wing vein serum, which was then stored at −20°C for further use. After bloodletting the broiler’s neck, the neck and abdominal cavity were quickly opened, and the cardiac, liver, spleen, bursa of Fabricius, and other organs were completely removed. The fascia and adipose tissue adhered to the surface of the organs were removed, and the blood on the surface was sucked with filter paper. Accurately weigh and calculate organ index.

The duodenum, jejunum and ileum were excised approximately 2 cm and preserved in 4% paraformaldehyde. The intestinal tissue was dehydrated with ethanol, washed with xylene, soaked in wax, embedded, sectioned (μm), and stained with hematoxylin-eosin (H&E) for morphological measurement. The villus height (VH; measured from the villus-crypt junction to the villus tip) and the crypt depth (CD; measured from the crypt base to the villus-crypt junction) were estimated by the Image-Pro Plus 6.0 software, and the ratio of villus height to crypt depth (VH/CD) was calculated.

According to the manufacturer’s plan (Suzhou Grace Biotechnology Co., Ltd.), superoxide dismutase (SOD) detection kit (G0101W), catalase (CAT) detection kit (G0105W), total antioxidant capacity (T-AOC) detection kit (G0142W) and malondialdehyde (MDA) detection kit (G0109W) were used to determine the levels of SOD (U/mL), CAT (U/mL), T-AOC (U/mL) and MDA (nmol/mL) in serum of AA broilers.

Primers were designed according to the gene sequences of GADPH, claudin-1, occludin and GATA-3 from the GenBank database. Primers for quantitative reverse transcription polymerase chain reaction (qRT-PCR) were designed using Primer 5.0 and the National Center for Biotechnology Information (NCBI). Primer-related information is shown in Table 2. Total RNA was extracted from each segment of the small intestine, and the integrity of the RNA was confirmed through agarose gel electrophoresis. The entire RNA was selected for reverse transcription, and the expression levels of claudin-1, occludin and GATA-3 genes in the intestinal segments of AA broilers were detected by RT-PCR. The expression of the target gene relative to the internal reference gene was calculated by 2^−ΔΔCt method.

Data were analyzed using SPSS 20.0 (SPSS, Inc., Chicago, Illinois, United States) and compared using one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons. The data is expressed as mean ± standard deviation (SD). p < 0.05 was considered significant.

As shown in Table 3, dietary CHM-JM113 supplementation had no statistically significant impact (p > 0.05) on the ADG, ADFI and F/G during days 1 to 21, days 22 to 42, and during the whole period (days 1 to 42). Dietary supplementation with 0.25, 0.5, 1 and 2% CHM-JM113 significantly reduced (p < 0.01) mortality from days 1 to 42, and linearly (p < 0.01) and quadratically (p < 0.01) decreased the mortality. Additionally, dietary supplementation with 0.5, 1 and 2% CHM-JM113 increased the EBI (p < 0.05) for AA broiler breeding from days 1 to 42.

As shown in Table 4, at 21 days of age, the cardiac index of broilers was significantly increased (p < 0.05) in groups supplemented with 0.5 and 1% CHM-JM113. The liver index of 0.25, 0.5 and 2% CHM-JM113 was significantly higher than that of control group (p < 0.01), and the addition of 0.5% CHM-JM113 was found to be the most effective. The spleen index of the 0.5 and 1% CHM-JM113 groups was significantly higher than that of the control group (p < 0.01). The broilers fed with diet containing 1 and 2% CHM-JM113 also had a higher bursa of Fabricius index than those fed with basal diet (p < 0.05). Dietary supplementation with CHM-JM113 linearly increased the bursa index (p < 0.05) and quadratically increased the cardiac index, liver index, and spleen index of broilers at 21 days of age (p < 0.05). At 42 days of age, the cardiac index of 1% CHM-JM113 was significantly higher than that of the 0.5% CHM-JM113 group (p < 0.05). The 0.5% supplementation level of CHM-JM113 could significantly (p < 0.01) decrease the liver index of broilers, while 0.25 and 2% CHM-JM113 could also significantly (p < 0.05) increase the spleen index of broilers. The dietary addition of CHM-JM113 linearly increased the spleen index and quadratically increased the liver index of 42-day-old broilers.

The light microscope images of the intestinal morphology are shown in Figures 1, 2. The effects of CHM-JM113 on the intestinal morphology of broilers are shown on Tables 5, 6.

Table 5 demonstrates that in 21-day-old broilers, 2% CHM-JM113 resulted in increased VH and VH/CD ratio in the duodenum, as well as an increased VH/CD ratio in the jejunum (p < 0.05). These indices were also linearly elevated in comparison to the control group (p < 0.01). VH and VH/CD ratio in the ileum were significantly, linearly and quadratically enhanced (p < 0.01) when 0.5, 1, and 2% CHM-JM113 were added to the basal diet.

As observed in Table 6, at 42 days of age, 1 and 2% CHM-JM113 significantly (p < 0.05) increased VH and VH/CD ratio in the duodenum of broilers, while showing a linear effect (p < 0.01). The VH and VH/CD ratio in the jejunum of the 0.5, 1, and 2% CHM-JM113 groups was significantly higher than that of the control group. Additionally, the jejunal VH and VH/CD ratio values showed a linear increase with the addition of CHM-JM113 (p < 0.01). Meanwhile, the VH and VH/CD ratio of the ileum in CHM-JM113 treated groups with different levels of supplementation were significantly greater than those of the control group, and the addition of CHM-JM113 enhanced these indices in both quadratically and linearly (p < 0.05). Notably, the addition of 2% CHM-JM113 treatment group significantly and linearly increased CD values in the ileum of broilers (p < 0.05).

The impact of CHM-JM113 on the antioxidant properties of broilers is presented in Table 7. Compared with the control group, at 21 days of age, 0.5 and 1% CHM-JM113 significantly increased the serum SOD concentration of broilers (p < 0.01). Conversely, the addition of different levels of CHM-JM113 highly significantly decreased the serum MDA concentration (p < 0.01), and this reduction effect was dose-dependent. At 42 days of age, the SOD concentrations of 0.5, 1 and 2% CHM-JM113 were significantly higher than those of the control group (p < 0.01). The highest SOD concentration was found in 1% CHM-JM113. Additionally, the MDA concentrations in broiler serum were significantly reduced by different levels of CHM-JM113 (p < 0.01), and the best reduction observed in 0.5% CHM-JM113. The inclusion of 1 and 2% levels of CHM-JM113 notably increased the T-AOC level of broilers (p < 0.05). Furthermore, 2% CHM-JM113 significantly reduced the CAT level of broilers (p < 0.05).

As shown in Figure 3, adding 1% of CHM-JM113 to the duodenum of broiler chickens was found to significantly increase the level of occludin (p < 0.05) and to very significantly increase the levels of claudin-1 and GATA-3 (p < 0.01). Adding varying amounts of CHM-JM113 was found to promote the expression of GATA-3 in the duodenum (p < 0.01), with the 0.5% addition having the greatest effect in the duodenum (p < 0.01).

As can be seen from Figure 4, the addition of 0.25, 0.5 and 1% CHM-JM113 all significantly increased the expression level of occludin in the duodenum of broiler chickens extremely significantly (p < 0.01). The promotion effect increased linearly, but the intestinal occludin expression level of 2% addition level of CHM-JM113 was significantly lower than that of the control group (p < 0.01). The claudin-1 expression level of CHM-JM113 added at 0.25 and 0.5% was significantly higher than that of the control group (p < 0.01), and there was no significant difference in the promotional effect of the two addition levels of CHM-JM113 on claudin-1 expression in the jejunum (p > 0.05). The addition of 0.5% CHM-JM13 significantly increased the 42-day-old GATA-3 levels in broilers (p < 0.01), but the addition of 1 and 2% CHM-JM113 instead decreased GATA-3 levels (p < 0.01).

As can be seen from Figure 5, the adding of different proportions of CHM-JM113 had a linear effect on the expression of occludin in the intestines (p < 0.01). However, only at the addition of 2% CHM-JM113, the expression level was significantly higher than that of the control group (p < 0.01). The addition of 0.25% CHM-JM113 extremely significantly decreased the expression level of claudin-1 in the jejunum of 21-day-old broilers (p < 0.01). On the other hand, 0.5% CHM-JM113 significantly increased the expression of claudin-1 (p < 0.01), and 1 and 2% CHM-JM113 significantly decreased the level of GATA-3 expression in the jejunum (p < 0.05).

As can be seen in Figure 6, 0.5% CHM-JM113 significantly increased the occludin expression level in 42-day-old broilers (p < 0.01), while 2% CHM-JM113 significantly decreased its expression level (p < 0.01). Additionally, 0.5% CHM-JM113 significantly increased the claudin-1 expression level (p < 0.01); however, other levels of CHM-JM113 significantly decreased the expression of claudin-1 (p < 0.01). Furthermore, 0.25% CHM-JM113 significantly decreased the expression of GATA-3 in the jejunum of broilers (p < 0.01), and 1% CHM-JM113 highly significantly promoted the expression of GATA-3 (p < 0.01).

Chinese herbal medicine is able to promote the proliferation of probiotics and has a turnover effect on intestinal flora imbalance. Probiotics also facilitate the dissolution of active ingredients of Chinese herbal medicine, reduce the toxic and side effects of Chinese herbal medicine (30), and improve its efficacy. Wang et al. (31) showed that Lactobacillus plantarum JM113 has the ability to inhibit the growth of Salmonella in broilers, reduce the incidence of broiler chickens, and reduce the mortality. Liang et al. (32) found that the combination of Chinese herbal medicine and lactic acid bacteria could enhance the intestinal sensitivity of broilers, inhibit the growth of Escherichia coli, and reduce the diarrhea rate and mortality of broilers. Consistent with the results of this experiment, the addition of CHM-JM113 in the diet significantly reduced the mortality of broilers. Both herbs and probiotics have been shown to promote growth in animals (33, 34). Theoretically, the growth-promoting effects of herbs and probiotics in broilers should have a cumulative effect, but in fact, in this study, the compound herbs and JM113 did not have a significant growth-promoting effect on broilers (p > 0.05), which was similar to the results of Majekodunmi et al. (35), who found that there was no significant difference in feed intake and weight gain of Ross broilers with sweet citrus peel. Sun et al. (36) found that dietary supplementation with Bacillus amyloliquefaciens CECT 5940 did not significantly promote the growth of broilers fed a corn-soybean meal diet. This may be due to the antibacterial effect of compound Chinese herbal medicine and probiotics (37), rather than their growth-promoting effects. Several studies have demonstrated that thyme, Astragalus, tangerine peel, and dandelion have antioxidant and immune-promoting effects (38–40) and partially growth-promoting effects (24). Furthermore, the intricate and variability of the synergistic potential of plant extract combinations (41) may be a contributing factor to the lack of improvement in broiler growth performance in this experiment. The strain specificity of the host’s immune and gut health response to probiotics (42) may also be a contributing factor to the non-significant differences in growth performance in this experiment.

To a certain extent, organ index could reflect the development of animal organs, body weight changes and indirectly reflect the ability of animals to cope with external stress. The higher the index, the stronger the body’s immunity (43). The bursa of Fabricius is the site where B lymphocytes mature, and the spleen regulates the cellular immunity and humoral immunity of poultry by activating T lymphocytes, B lymphocytes and macrophages (44). Liang et al. (32) found that the combination of Chinese herbal medicine and probiotics improved the spleen index, thymus index and bursa index of broilers. This combined treatment had a more significant effect compared to using Chinese herbal medicine or probiotics alone. Li et al. (45) found that Astragalus polysaccharides significantly improved the thymus index and spleen index in vivo experiments, and were useful in restoring the damaged thymus and spleen tissue structure. In this experiment, the enhancement of the immune organ index in broilers in different degrees verified the previous perspective. The liver is the largest solid gland and an important metabolic organ, plays a vital role in excretion, metabolism, detoxification and the production of various coagulation factors (46). The growth performance of broilers may also decrease to a certain extent after liver damage (47). This experiment found that adding 0.3 g/mL of a compound Chinese herbal medicine with JM113 improved the liver index of broilers, indicating that Chinese herbal medicine is beneficial to liver health, which is consistent with previous studies (48). It is known that the combined use of compound herbs and JM113 promotes the development of the intestinal tract, thereby boosting the absorption of nutrients by the animal organism, which in turn supports the improvement of the organ index of broilers. The addition of CHM-JM113 in this experiment boosted the development of broiler organ indices to varying degrees, but was significantly influenced by the dosage, suggesting that the use of CHM-JM113 had a positive impact on the immunological performance of broilers.

The integrity of intestinal structure is very important for animals to absorb nutrients. Intestinal villus height (VH), crypt depth (CD) and VH/CD ratio are important indicators of intestinal structural integrity (49). Longer intestinal villus height and shallower crypt depth indicate better healthy digestion and absorption of microorganisms (10), improved intestinal mucosal differentiation, and enhanced capacity for digestion and absorption (50). Previous studies have found that adding probiotics, forsythia extract, natural pepper extract, and ellagic acid is an alternative to antibiotics in increasing the intestinal villus height of broilers (51, 52). This experiment found that compared with the control group, the combination of compound Chinese herbal medicine and JM113 improved the villus height and villus height ratio of the duodenum, jejunum and ileum of broilers, which is consistent with previous studies. This may be due to the fact that lactic acid bacteria, acting as probiotics, convert dietary fiber into short-chain fatty acids. They also promote the development of intestinal villi, improve the integrity of the intestinal barrier, and consequently improve the absorption of nutrients (53). Chinese herbal medicine is abundant in active ingredients, including lipids, unsaturated fatty acids, sugars, trace elements and vitamins, which provide sufficient nutrition for intestinal villus epithelial cells and support intestinal development (26). Studies have shown that there is a two-way communication between the liver and the intestine. Bile acids produced in the liver regulate the microbial composition and intestinal barrier function, while intestinal products regulate bile acid synthesis, as well as glucose and lipid metabolism in the liver. The intestinal barrier serves to restrict the systemic transmission of microorganisms and toxins, while facilitating the passage of nutrients into the portal vein circulation for delivery to the liver (54). Based on the gut-liver relationship, the increase of liver index is also benefits the absorption of nutrients by the intestine. In this experiment, the addition of compound Chinese herbal medicine and JM113 not only increased the liver index but also increased the absorption of nutrients in the small intestine, possibly as a result of the bidirectional communication between the liver and the intestine. The combined use of compound Chinese herbal medicine and JM113 can be observed to promote the development of the intestine, thereby enhancing the absorption of nutrients by the animal body and consequently improving the organ index of broilers.

Oxidative stress refers to the imbalance between antioxidants and free radicals, which may lead to the production of various reactive oxygen species (ROS) such as hydroxyl radicals and superoxide anions in the body (55). Excess ROS can disrupt animal metabolism, damage cell structure, and accelerate oxidation, leading to tissue damage and various diseases (56). Antioxidant enzyme activity and oxidation product concentration are reliable biomarkers for assessing the antioxidant status of animals (50). SOD catalyzes the disproportionation of superoxide anions into oxygen and H₂O₂ (57). CAT is a common enzyme that catalyzes the decomposition of H₂O₂ into water and oxygen. In addition, previous studies have shown that the removal rate of H₂O₂ removal in cells is partially dependent on GSH levels (58). The reduction reaction of lipid peroxide is catalyzed by glutathione peroxidase (GSH-Px), and the overall antioxidant capacity is indicated by the level of T-AOC (59). MDA is one of the end products of peroxidation derived from membrane lipids, reflecting the extent of lipid peroxidation and damage to antioxidant capacity in animals (60). Yin et al. (59) found that adding LBPs to the diet could increase the serum total antioxidant capacity (T-AOC) and GSH-Px activity, and decrease the MDA level (p < 0.05). Abo-Samaha et al. (61) found that adding licorice (0.4 g/L and 0.8 g/L) to drinking water resulted in antioxidant activity, leading to a reduction in MDA levels and an increase in GSH levels and CAT activity. Various active ingredients in thyme, Astragalus, tangerine peel and dandelion have been shown to reduce the effects of peroxides on animal bodies and enhance the body’s antioxidant capacity (62–64). In this experiment, the concurrent administration of Chinese herbal medicine and JM113 resulted in increased levels of SOD, T-AOC and MDA in 21-day-old broilers. Additionally, the SOD level of 42-day-old broilers was also improved. The liver is the primary site of free radical formation. Shati et al. (65) found that aqueous extracts of thyme and ginger have detoxifying and antioxidant effects on the liver and brain of mice. Antioxidants like thyme and rosemary have been utilized as therapeutic agents to alleviate liver damage (66). Additionally, dandelion root is also believed to have a beneficial effect on the gastrointestinal system by stimulating digestion and liver function (67). The increase in the liver index in this experiment also supports the positive impact of CHM-JM113 on the antioxidant performance of broilers from another perspective.

Nutrients entering the animal intestine will be selectively digested and absorbed by the intestine. This is due to the physical barrier on the intestinal surface, which prevents harmful substances from entering the bloodstream through the intestinal mucosa and reduces the damage caused by these substances to the body (68). The intestinal physical barrier is composed of tight junction structures (TJ) and mucosal epithelial cells. TJ regulate the permeability of the intestinal barrier by sealing the paracellular space between adjacent epithelial cells, thereby preventing the spread of microorganisms and antigens (69). This is also a key indicator for assessing intestinal health. The regulation of intestinal tight junctions is mediated by related proteins, such as occludin, claundin-1 and MUC2. Occludin and claudin-1 play an important role in maintaining epithelial cell polarity and regulating intestinal barrier permeability (70). The up-regulation of occludin may indicate an increase in cell permeability to nutrients, which facilitates the digestion and absorption of nutrients (71). GATA-3 is a significant regulator of T-cell differentiation and a Th2 cell-specific transcription factor (72). Gene-targeting studies have demonstrated that GATA-3 plays a vital role in the development and function of T cells, B cells, CD1 natural killer cells (NKT cells), natural killer cells (NK), and innate lymphoid cells (ILCs) (73). This experiment revealed that the addition of CHM-JM113 significantly enhanced the expression of occludin and claudin-1 in the intestine and to varying extents, improved the integrity of the intestinal barrier and organ indices. This may be due to the fact that the intestinal mucosal immune system influences the development of immune organs, including the spleen, thymus, and secondary immune organs, as well as immune responses (74), which in turn promotes the expression of occludin, claudin-1, and GATA-3 in the intestine, consistent with previous research findings (75).

In summary, CHM-JM113 may be considered as an alternative to antibiotics for broiler production, but it does not improve the performance of the animal and economic considerations need to be taken into account.