The LCE product was provided by Centre Technology Co., Ltd. (Beijing, China). The LCE consisted of extracts from Lonicera flos and Curcuma longa L. in certain proportion. The final concentrations of chlorogenic acid and curcumin were 100 mg/kg and 20 g/kg, respectively.

A total of 480 one-day-old healthy female broilers (WOD168) were obtained from Beijing Huadu Yukou Poultry Industry Co., Ltd. (Beijing, China). The birds were weighed on arrival and randomly assigned to 3 treatments with 5 replicates of 32 birds. The control group (CON) was fed an antibiotic-free basal diet. The antibiotic group (ANT) was fed the basal diet supplemented with 50 mg/kg spectinomycin hydrochloride and 25 mg/kg lincomycin hydrochloride (Sichuan Hengtong Animal Pharmacy Co., Ltd., Neijiang, China). The LCE group was fed the basal diet supplemented with 500 mg/kg LCE. The concentrations of chlorogenic acid and curcumin in the LCE diet were 50 μg/kg and 10 mg/kg, respectively. The feeding program was divided into 2 phases: starter (days 1–21) and finisher (days 22–35). The basal diets for each phase were formulated to meet the nutrient requirements recommended by the National Research Council (NRC, 1994) and manufactured in mash form. The composition and nutrient levels of basal diets were shown in Table 1.

All birds were housed in two-tier cages (1.00 m × 1.25 m × 0.45 m) in an environmentally controlled room. A light schedule of 23 h light and 1 h dark was used throughout the experimental period. Room temperature were maintained at 33°C for the initial 3 days and then gradually decreased by 3°C every week to reach a final temperature of 24°C. The relative humidity was maintained at 70% within the first 3 days and at 60% afterward. Ad libitum water and feed were provided throughout the experimental period. Birds were vaccinated against Newcastle disease (ND), Infectious bronchitis, Avian influenza (H9 subtype; AI) and Infectious bursal disease on day 1.

At 1, 21 and 35 days of age, body weight (BW) and feed intake were recorded on a cage basis to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR). Mortality was recorded daily to modify the performance parameters.

At 21 and 35 days of age, 5 birds (1 birds per replicate cage) from each treatment were randomly selected after 12 h fasting. Blood samples were collected from the wing vein into tubes with or without EDTA to yield whole blood and serum, respectively. Whole blood was immediately transported to the lab for peripheral blood lymphocyte proliferation analysis. Serum was obtained by centrifugation at 3,000 × g for 15 min at 4°C and stored at −80°C until analysis. Subsequently, birds were sacrificed by cervical dislocation. The liver was collected, snap frozen in liquid nitrogen, and stored at −80°C until analysis.

The liver tissue (1 g) was homogenized in 9 mL of ice-cold phosphate buffer saline to prepare the liver tissue homogenate and then centrifuged at 3,000 × g for 15 min at 4°C. The supernatant was collected for further analysis. Total protein concentration in liver tissues was measured by the bicinchoninic acid (BCA) method as described previously (26).

Total-antioxidant capacity (T-AOC, No. A015-2-1), malondialdehyde (MDA, No. A003-1-2) concentration, activities of glutathione peroxidase (GSH-Px, No. A005-1-2), superoxide dismutase (SOD, No. A001-1-2) and catalase (CAT, No. A007-1-1) in serum and liver tissues were determined with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Results in liver tissues were normalized against the corresponding total protein concentrations and expressed as units/mg protein.

Interleukin-6 (IL-6, No. ml059839), interleukin-10 (IL-10, No. ml059830), tumor necrosis factor-α (TNF-α, No. ml002790), interferon-γ (IFN-γ, No. ml042758) and prostaglandin E2 (PGE-2, No. ml058373) concentrations in serum and liver tissues were analyzed by commercial ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., China) following the manufacturer’s instructions. Values were normalized to total protein concentrations in liver tissues, and given as units/g protein.

Peripheral blood lymphocytes were isolated from whole blood using density gradient centrifugation (Solarbio, Beijing, China). Then collected lymphocytes were washed three times with RPMI 1640 medium and suspended in RPMI 1640 medium (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Cell number and viability were determined using trypan blue staining (Sigma, Germany), and the final concentration of lymphocytes was adjusted to 1 × 10⁷ viable cells/mL.

Lymphocyte proliferation was measured by 3-(4,5-dimethylthiazol-2 yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Solarbio, Beijing, China). Briefly, 100 μL of lymphocyte suspension and 100 μL of RPMI 1640 medium with or without 90 μg/mL concanavalin A (Con A; Sigma Chemical Co., St. Louis, MO) was added to each microwell in a 96-well plate in triplicate. After incubation for 72 h at 39°C in a 5% CO₂ incubator, 10 μL MTT was added into each well, incubated for 4 h, and then 100 μL of dimethyl sulfoxide was incorporated into each well and shaken until complete dissolution. Light absorbance serving as an index of lymphocyte proliferation was determined at 570 nm using a microplate reader (Bio-Tek, United States) and expressed as a mean stimulation index.

The phagocytic activity of reticuloendothelial system was evaluated by carbon clearance assay as described previously (27). Briefly, the black ink (Pelikan, Hanover, Germany) was centrifuged at 3000 × g for 30 min to collect colloidal carbon. Then 10 birds from each treatment were weighed and injected (0.1 mL/300 g of BW) with colloidal carbon via wing vein at 21 and 35 days of age. Blood samples were collected at the time intervals of 3 and 15 min post injection, and then were immediately suspended into 4 mL solution of 0.1% sodium carbonate solution. The absorbance was measured at 640 nm in a NanoDrop ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE, United States). After then, birds were sacrificed, and spleen and liver were weighed. Phagocytic index was calculated using the following equations (27):

where OD3 is the absorbance at 3 min, OD15 is the absorbance at 15 min, T1 is the first time point of blood collection, and T2 is the last time point of blood collection.

At 21 and 35 days of age, serum antibodies against NDV were determined by hemagglutination inhibition (HI) assays. HI tests were carried out by using serial 2-fold dilutions of serum and 4 hemagglutination units of the NDV antigen (Harbin Weike Biotechnology Co. Ltd., China). The geometric mean titer was expressed as reciprocal log2 values of the highest dilution that displayed HI.

Date were subjected to a one-way ANOVA using the GLM procedures of SAS (version 9.2; SAS Inst. Inc., Cary, NC, United States) followed by Tukey’s tests. Each cage was defined as an experimental unit. Treatment was considered a fixed effect and bird was as a random effect. Significant difference was declared at p < 0.05.

The results of growth performance in broiler chickens are presented in Table 2. There was no significant difference in initial BW among treatments. However, birds in ANT and LCE groups had similar BW, but greater (p < 0.01) than those in CON group at day 21 and 35. From day 1 to 21, dietary supplementation of LCE increased (p < 0.05) ADG and decreased (p < 0.01) FCR, while dietary supplementation of ANT only increased (p < 0.01) ADG when compared with CON. No difference was observed in ADFI among treatments. From day 22 to 35, these performance parameters did not differ significantly among treatments. During the entire experimental period, dietary ANT and LCE supplementation resulted in a significant increase (p < 0.01) in ADG and a significant decrease (p < 0.05) in FCR compared with CON.

The effects of LCE on serum antioxidant capacity in broiler chickens are shown in Table 3. At day 21, serum MDA concentration, CAT activity and T-AOC were not influenced by dietary treatments. However, birds in LCE group had higher (p < 0.05) serum activities of GSH-Px and SOD than those in CON group, but similar with those in ANT group. At day 35, dietary LCE supplementation decreased (p < 0.05) serum MDA concentration, and increased (p < 0.05) serum SOD activity and T-AOC compared to other groups. Besides, supplementation of ANT and LCE increased (p < 0.05) serum GSH-Px activity when compared with CON. Serum CAT activity was not influenced by dietary treatments.

Effects of dietary LCE supplementation on peripheral blood lymphocyte proliferation in broiler chickens at day 35 are presented in Figure 1. There was no significant difference in stimulation index of peripheral blood lymphocytes among treatments at day 35.

Effects of dietary LCE supplementation on phagocytic index in broiler chickens are presented in Figure 2. At day 21, dietary LCE supplementation significantly increased (p < 0.05) phagocytic index when compared with the other groups. At day 35, birds fed LCE diet had higher (p < 0.05) phagocytic index than those fed CON, but not different from those fed ANT diet.

Effects of dietary LCE supplementation on serum antibody titers to NDV in broiler chickens are shown in Figure 3. At day 21, dietary supplementation of ANT and LCE significantly increased serum antibody titers to NDV when compared with CON. At day 35, dietary treatments significantly (p < 0.05) affected serum antibody titers to NDV. The highest serum antibody titers to NDV were observed in birds fed LCE, followed by those fed ANT, and then those fed CON (p < 0.05).

Effects of dietary LCE supplementation on serum inflammatory cytokines in broiler chickens are presented in Table 4. At day 21, serum IL-6 concentration was reduced (p < 0.01) in birds fed LCE diet than those fed CON diet, but it was similar to those fed ANT diet. Both ANT and LCE supplementation decreased (p < 0.01) serum concentrations of TNF-α, IFN-γ and PGE-2. Supplementation of LCE increased (p < 0.01) serum IL-10 concentration compared with the other groups. At day 35, birds in ANT and LCE groups showed lower (p < 0.01) serum concentrations of IL-6 and IFN-γ than those in CON group. Supplementation of LCE increased (p < 0.01) IL-10 concentration and decreased (p < 0.01) PGE-2 concentration in serum compared with CON and ANT groups. Serum TNF-α concentration was lower (p < 0.01) in birds fed ANT diet than those fed CON and LCE diets.

The results of liver antioxidant capacity in broiler chickens are shown in Figure 4. Birds in LCE group had lower (p < 0.05) MDA concentration in liver than those in CON group at day 21, and than those in CON and ANT groups at day 35 (Figure 4A). Dietary LCE supplementation significantly increased (p < 0.05) liver SOD activity compared with CON and ANT groups at day 21 and 35 (Figure 4B).

The results of Inflammatory cytokine concentrations in liver are presented in Table 5. At day 21, liver IL-6 concentration was the lowest in birds fed LCE diet, followed by those fed ANT diet, and then those fed CON diet (p < 0.01). Liver IL-10 concentration was the highest for LCE group, but the least for CON group with intermediate values for ANT group (p < 0.01). Liver TNF-α concentration in birds fed LCE diet was lower (p < 0.01) than those fed either CON diet or ANT diet, which did not differ from each other. At day 35, supplementation of LCE and ANT reduced (p < 0.01) liver concentrations of IL-6 and TNF-α compared with CON group. On the other hand, birds in LCE group had higher (p < 0.01) liver concentration of IL-10 than those in CON and LCE groups.