Fresh plants were harvested from the mountainous area of ShaoYang, Hunan, China, based on advice from local older individuals. The plants, including the root, stem, and leaves, were sun-dried for five to seven days under strong sunlight on the ground. The plants were then cut into 5–10 cm-long segments using a chainsaw. The plant segments were sun-dried again, crushed using a traditional Chinese medicine grinder, and sieved through a 60-mesh (ø250 μm) screen. The powders of Sarcococca ruscifolia Stapf, Hedera nepalensis var. sinensis (Tobl.) Rehd, and Clematis chinensis Osbeck were mixed in a ratio of 1:1:1 to form the TCM mixture. The nutrient contents of the TCM plant powder, based on air-dried matter, were as follows: dry matter 90.98 ± 0.60%, total protein 13.81 ± 2.41%, crude fat 0.29 ± 0.05%, crude fiber 32.85 ± 0.50%, Ca 0.24 ± 0.04%, total phosphorus 0.14 ± 0.01%, and crude ash 3.95 ± 0.99%. Active ingredients, including polysaccharides (concentration 1.26 mg/mL, extraction rate 0.99%), total flavonoids (concentration 0.07 mg/mL extraction rate 0.57%), polyphenols (concentration 0.09 mg/mL, extraction rate 0.35%), and total protein (concentration 0.09 mg/mL, extraction rate 0.07%), were identified in the plant water extracts.

The research was approved by the Animal Care and Use Committee at Hengyang Normal University under project number HNUACUC-B202111001.

A total of 100 10-day-old male native Xianghuang broilers, with an average initial body weight of 99.33 ± 4.41 g, were randomly assigned to two replicated experimental groups: control (C) and experimental (E), with five replicates per group and 10 birds per replicate. A corn-soybean-based diet was formulated to exceed the nutritional requirements of the yellow-feathered broilers (14) (Table 1) and was fed to the birds in the control group. The birds in the experimental group were supplemented with 2% of the TCM plant powder in the control diet. All birds were housed in pens (0.10 m²/bird) with padded rice hulls located at Yimin Farm and had ad libitum access to water and feed. The temperature of the room was maintained at around 29.0°C for the first week (when the broilers were 11 days old) and then gradually reduced by 3°C every week to 24°C until the end of the study using an air conditioner. The light was provided for 12 h at 20 lux using LED lights throughout the experimental period.

All broilers were individually weighed after 12 h of feed deprivation. The body weight (BW) and feed leftovers were recorded per replicate weekly through the experiment. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain (F:G) ratio (the ratio of ADFI to ADG) were calculated.

On day 28, 10 broilers were randomly selected from each of the control and experimental groups for sample collection. Blood was individually collected via the wing vein using a 22 g needle, a venous blood collection needle, and a red vacuum blood collection tube. After standing at room temperature for 3 h, 5 mL of blood was centrifuged at 3,000 rpm at 4°C for 15 min. The supernatants (serum) were collected and stored at −80°C until further analysis. After that, the broilers were euthanized by carbon dioxide followed by cervical dislocation. Organ samples, including the liver, spleen, intestine, kidney, bursa, and heart, were weighed, and the organ index was calculated as follows, Organ index = Organ weight*100%/live body weight. Approximately 5 cm segments of the proximal duodenum, mid jejunum, and distal ileum were washed with 0.9% saline to remove the intestinal contents and then carefully fixed in 4% paraformaldehyde for 48 h for histological examination. The remaining segments of the small intestine were washed with 0.9% sterile saline and scraped with a glass slide to collect the mucosa. The mucosa and spleen samples were frozen in liquid nitrogen and then stored at −80°C for further analysis.

The intestinal segments fixed in 4% paraformaldehyde were dehydrated with increasing concentrations of ethanol, vitrified with dimethylbenzene, embedded in paraffin, and then cut into 5-μm-thick slides. These were stained with hematoxylin and eosin according to the method mentioned in the report (15). An image of the villus was captured using a microscope (Leica RM2135, Wetzlar, Germany). The width of the baseline, villus height, villus width, and crypt depth of the duodenum, jejunum, and ileum were measured from 50 villi per broiler using Image-Pro Plus (Media Cybernetics Inc., Silver Spring, MD, USA).

Antioxidant parameters, including the activities of T-SOD, CAT, total antioxidant activity (T-AOC), and malonaldehyde (MDA) concentration in the serum and the supernatants of the mucosal samples, were determined according to the method mentioned in the report (15, 16). Approximately 0.1 g of the mucosa was homogenized with 0.9 mL of 0.9% sterile saline using a tissue grinder (SCIENTZ-12, Xinzhi Biotech logy, Ningbo, China) and then centrifuged at 2,500 r/min for 15 min to collect the supernatant. The activity of T-AOC (mmol/L) was analyzed using the OD 593 nm value of the supernatant compared with a standard curve of FeSO₄.7H₂O. The unit of CAT was defined as the amount of hydrolyzed H₂O₂ in 1 min per mg of protein in the sample. One unit of T-SOD was defined as the amount that caused 50% inhibition of the nitroblue tetrazolium light reaction rate. The supernatant was extracted with 10% trichloroacetic acid and then used to test MDA concentration.

The mRNA expression levels of inflammation, intestinal barrier function, and apoptosis-related genes were examined using quantitative real-time PCR. Total RNA from the mucosa and spleen samples was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The integrity of the RNA was determined by 2% agarose gel electrophoresis. The concentration and purity of the RNA were measured using the NanoDrop ND-2000 spectrophotometer system (Thermo Fisher Scientific). One μg of the total RNA was reverse transcribed into DNA using an RT reagent kit (Aikerui, Changsha, China) according to the manufacturer’s instructions. The RT-PCR was performed on a QuantStudio3 device (Thermo Fisher Scientific, Waltham, MA, USA) using a SYBR Green quantitative PCR mix (Aikerui, Changsha, China) following the manufacturer’s protocols. The primer sequences for the target and reference (β-actin) genes are listed in Table 2. The mRNA expression levels of the target genes were calculated using the 2^−ΔΔCT method (17).

The data were analyzed using the one-way ANOVA procedure of SAS 8.0. The individual broiler was the experimental unit for all data, except for growth performance, where the replicate was the experimental unit. Differences between the treatments were evaluated using a t-test. The data were presented as means ± standard deviation. In this study, a p-value <0.05 was considered significant, and 0.05 ≤ p ≤ 0.10 was considered a tendency.

The effect of the TCM supplementation on the growth performance of the Xianghuang broilers is shown in Table 3. No significant differences in the ADFI, ADG, and F:G ratio between the control and experimental groups were observed (p > 0.05).

The TCM plant powder supplementation had no significant effect on the weight and indices of the liver, intestine, kidney, bursa, spleen, and heart (p > 0.05) (Table 4).

The effects of the TCM supplementation on the small intestinal morphology of the broilers are presented in Table 5 and Figure 1. The duodenal villus height exhibited an increasing tendency in the TCM group compared to the control group (p = 0.092). However, the baseline width, villus width, and crypt depth of the duodenum remained unaffected by the treatment (p > 0.05) (Figures 1DA,DB). The supplementation with TCM notably augmented the baseline and villus widths in the jejunal segment compared to the control group (p < 0.05). Within the jejunal segment, there was an increasing trend toward the villus height (p = 0.068) and the ratio of the villus height to the crypt depth (p = 0.061) in the TCM group compared to the control group (Figures 1JA,JB). The birds in the TCM group had higher villus height and a higher ratio of the villus height to the crypt depth in the ileum compared to their control counterparts (p < 0.05). Nonetheless, the crypt depth in both the jejunum and ileum, along with the baseline and villus widths in the ileum, did not exhibit significant alterations due to the TCM treatment (p > 0.05) (Figures 1IA,IB).

As shown in Table 6, the activities of T-AOC, SOD, and CAT and the concentration of MDA in the ileal mucosa and serum were not affected by the treatment (p > 0.05). T-AOC activity and MDA concentration were not affected by the treatment in the jejunal mucosa (p > 0.05); however, the jejunal mucosa from the TCM group had higher activities of SOD and CAT compared to the control group (p < 0.05).

The mRNA expression levels of the genes associated with apoptosis, inflammation, and tight junctions in the jejunal mucosa, ileal mucosa, and spleen are shown in Figure 2. In the jejunal mucosa, the expression of the genes tumor necrosis factor alpha (TNF-α) and toll-like receptor 4 (TLR4) was significantly downregulated in the birds supplemented with the TCM plant powder (p < 0.05). In addition, there was a tendency for lower expression of the claudin-1 (CLDN1) and B-cell lymphoma-2 (BCL2) genes (p = 0.081 and 0.091, respectively) in the TCM group compared to the control group. The expression of the IL-1β gene was not affected by the treatment (p > 0.05) (Figure 2A).

In the ileal mucosa, the expression of the BCL2 and IL-8 L2 genes was significantly upregulated (p < 0.05) in the TCM group compared to the control group. The Ileal TLR4 gene tended to be higher (p = 0.079) in the TCM group compared to the control group (p < 0.05). Interestingly, the mRNA expression of the IL-1β gene was lower in the TCM group compared to the control group (p < 0.05). No significant differences were observed in the expression of the CLDN1 and TNF-α genes in the ileal mucosa between the two groups (p > 0.05) (Figure 2B).

In the spleen, upregulation of BCL2 (p < 0.01), IL8L2 (p < 0.05), and TLR4 (p < 0.01) was observed in the TCM group compared to the control group. The mRNA expression of TNF-α (p < 0.05) was downregulated, and the expression of IL-1β (p = 0.091) tended to be lower in the TCM group compared to the control group. No effect was observed for the CLDN1 expression in the spleen (p > 0.05) (Figure 2C).