The animal protocol for this experiment was approved by the Animal Care Committee of the Anhui Science and Technology University. Animals were maintained and processed in accordance with the Anhui Science and Technology University Guide for the Care and Use of Laboratory Animals.
Animals, diets, and management
Three hundred and sixty 1-day-old healthy broiler chicks (Ross 308) were allotted to 5 groups with 6 replicates in each group and 12 broilers in each replicate. Dietary treatments were as follows: (1) basal diet without supplemental Cu (control); (2)basal diet+50 mg Cu/kg dry matter (DM) as copper sulphate (CS-50); (3) basal diet+100 mg Cu/kg DM as copper sulphate (CS-100); (4) basal diet+50 mg Cu/kg DM as copper citrate (CC-50) and (5) basal diet+100 mg Cu/kg DM as copper citrate (CC-100). The experiment lasted for 42 days, and diets were formulated in two stages (1 ~ 21 days old and 22 ~ 42 days old). Ingredients and chemical composition of the basal diets are presented in Table 1. The final Cu content of the diets are presented in Table 2.
The single-layer cage (200 cm long × 100 cm wide × 40 cm high, with concrete floors of 0.167 m2/bird) was adopted in the experiment. The chicken house and related appliances were cleaned and disinfected before the experiment. The temperature of the chicken house was controlled by human industrial temperature control three days before entering the chicken house. Diets and water (less than 0.01 mg/L Cu by analysis) were available ad libitum.
Sample collections
On the d 1, d 21 and d 42, the weight was measured (stop feeding for 12 hours before weighing and drink freely), and the feed intake was recorded at the same time. Average daily gain (ADG), average daily feed intake (ADFI) and the feed/gain ratio (F/G) were calculated based on the recorded data.
On d 42, 60 birds (2 chicks per replicate) were randomly selected and weighed after feed deprivation for 12 h. Blood samples were collected from the wing vein and centrifuged at 3,500×g for 10 min. Serum was separated and stored at −80°C for further analyses.
After blood sampling, birds were killed by cervical dislocation immediately and the pectorals and liver were excised in situ. The pH of the digesta in the jejunum and ceca was subsequently measured with a calibrated digital pH meter with a glass-tipped probe (model IQ120, IQ Scientific Instruments Inc., Carlsbad, CA). Two independent pH readings were taken in situ in each location along the digestive tract. The cecal samples and the content samples of the ileal (Meckel’s diverticulum up to 40 mm above the ileo-cecal junc-tion) digesta were aseptically collected and placed on ice for transportation to the laboratory, where fresh cecal samples were diluted 10-fold by weight in buffered peptone wate and homogenized using a stomacher. Viable counts of bacteria in the samples were then determined by plating serial 10-fold dilutions (in 10 g/L peptone solution) onto MacConkey agar, Lactobacilli MRS agar, and Bifidobacterium agar plates (Beijing Luqiao technology Limited by Share Ltd) to verify the E. coli i, Lactobacillus, and Bifidobacterium, respectively. Agar plates were incubated at 37°C for 36 or 48 h, after which bacterial colonies were counted. Concentration of microflora was finally expressed as log10 colony-forming units per gram of intestinal content.
At the 39th to 42nd day of the experiment, the feed intake was recorded and all excreta was collected for 4 consecutive days. Excreta were collected from plastic trays placed under the cages 6 times per day (06:00, 9:00, 12:00, 15:00, 18:00 and 21:00 h) and stored at −20℃. Finally, feces from each cage were collected, mixed and weighed. Fecal samples were dried in an oven at 65°C for 48 h. Feed and excreta samples were ground through a 0.45 mm screen and stored for further analysis.
Chemical analysis
The nutrient contents of feed were analyzed by the methods of the Association of Official Analytical Chemists (AOAC)[11]. Copper concentrations of feed, tissue and plasma were analyzed by Flame Atomic Absorption Spectroscopy (Shimadzu Scientific Instruments, Kyoto, Japan). The activities of serum Cu-Zn superoxide dismutase (Cu/Zn SOD), and ceruloplasmin were determined by using respective assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, P. R. China).
Statistical analysis
All data were analyzed as a 2 × 2 + 1 factorial experiment based on a completely randomized design using the General Linear Model procedure of the SAS software (Statistical Analysis System, Version 9.13, 2002). Data were analyzed as repeated measures with a model containing source, level, and source × level. Means were compared using Duncan’s multiple range test and P < 0.05 was considered as the significant level, and P > 0.05 and < 0.10 was considered a trend.