Twenty-four weaned pigs from the same littermates (Large White × Landrace × Duroc) with an average body weight (9.77 ± 0.11 kg) were randomly assigned into five groups, each treatment with six pigs, and fed with basal diet containing different levels of SPCI (0, 50, 75, 100, and 125 mg/kg). The basal diet ( table S1) with a low iron content (25.8 mg/kg) was formulated to meet the nutrient requirements recommended by the National Research Council 2012 [19]. The 0.7 m × 1.5 m metabolic cages placed in the heated room (25–28°C) were used to house pigs individually and each pig was given ad libitum. The room relatively humidity controlled at 55–65%. The SPCI (Fe ≥ 15.0%; chelate ratio ≥ 90%; MW of the small peptide: 180–500 Da) was kindly provided by Shuxing Biotechnology Co., Ltd (Jiangsu, china).
Growth performance
The average daily gain (ADG) was calculated according to the BW of each pig measured On day 1 and 22 after 12 h fasting. The feed intake of each pig was recorded daily to determine the average daily feed intake (ADFI), and the G:F was calculated according to ADG and ADFI (F/G equals to ADFI dividing ADG).
Sample Collection and Treatment
On d 18, we conducted four-day fresh fecal samples collection. On d 22, the bloods were obtained after 12 h fasting to obtain serum samples. The whole blood samples were used for the hematological analysis. Then the tissue samples (liver, gallbladder, kidney, tibia) were obtained following and stored at − 80°C immediately. Take approximately 4 cm of each duodenum, jejunum, and ileum tissues to fix in 4% paraformaldehyde solution for morphological analyses and immunofluorescence. Then Rinse central portion of duodenum, jejunum, and ileum tissues with 0.9% cold physiological saline and then the mucosa samples were scraped from duodenum, jejunum, and ileum segments.
Apparent total tract nutrient digestibility analysis
The processed feed and fecal samples were used to measure the digestibility of nutrients. uses acid insoluble ash (AIA) as endogenous indicators, a method described by the Chinese National Standard (GB/T23742-2009). The content of dry matter (DM), crude protein (CP), ether extract (EE) and Ash were measured according to AOAC, whereas the the gross energy (GE) content was determined by an adiabatic bomb calorimeter (LECO, St. Joseph, Michigan, USA). All contents were calculated by following formula: (100-A1F2/A2F1×100) [20]. A1: digesta nutrient; A2: digesta AIA; F1: diet AIA; F2: digesta AIA.
Iron content in fecal and tissue samples
The samples including fecal and tissues (liver, gallbladder, and kidney) were dried at 100°C for 24 h and ashed at 550°C for 10 h. Meanwhile the tibia samples were dried at same temperature and time but ashed at 550°C for 36 h. The pre-treated samples were finally used to determine iron content according to the method of Shelton and Southern [21]. The nitric acid-perchloric acid mixture (1:1) were used to dissolve the ashed samples and then the distilled deionized water were used for dilution and the flame atomic absorption spectrophotometry (AA-6300, Shimadzu Corp., Tokyo, Japan) were used for analysis of iron [22].
Serum parameter analysis
The Enzyme-linked kits (Jiangsu, China) can provide reliable indexes for the content determination of serum ferritin, transferrin, insulin-like growth factor-1, immunoglobulin subsets (IgG, IgM, IgA), D-lactate and diamine oxidase. All procedures correspond to the manual of the kits, respectively.
Catalase (CAT), malondialdehyde (MDA), glitathione peroxidase (GSH-PX), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) and urea nitrogen (BUN) in serum were determined using the commercial kits (Nanjing Jiancheng Biotechnology Co., Ltd, Nanjing, China).
Intestinal morphology analysis
The paraffin-embedded duodenum, jejunum, and ileum samples were cut and then stained with hematoxylin and eosin (H&E) for the intestinal morphology examination under the Eclipse CI-L photo microscope (Nikon, Japan). The height of 5 intact villi and crypt depths were recorded by image-Pro Plus 6.0 analysis software.
Immunofluorescence analysis
The immunofluorescence can provide reliable indexes for the detection of the ZO-1 protein distribution in jejunal tissues. The flushed samples were incubated with 1 mol/L ethylene diamine tetraacetic acid (EDTA, pH 9.0, Gooddbio Technology Co., Ltd., Wuhan, China) for antigen retrieval. Following this, the tissue sections were block with 3% bovine serum albumin and then incubate with rabbit anti-ZO-1 polyclonal antibody all night at 4°C. All procedures correspond to our previous research [23].
Flow cytometry assays
The proportion of jejunal apoptotic cells were determined by Flow cytometry and all procedures were conducted according to our previous report [23]. Briefly, isolate jejunal mucosal layer, followed by grinding and filtering to form a cell suspension. After that, take 100 µL of the cell suspension and put it into 5 mL tubes, and then add 5 µL PE Annexin V and 7-AAD in the tubes for incubation in the dark. Lastly, take Annexin V binding buffer (400 µL) to the reaction tubes then mixed it by a vortex. The jejunal apoptotic cells were detected by CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA) within 1 h.
Enzyme activity
The commercial kits (Nanjing Jiancheng Biotechnology Co., Ltd. Nanjing, China) were used to detect the digestive enzymes in duodenal, jejunal, and ileal mucosa including lactase, sucrase, and maltase.
RNA isolation, reverse transcription, and real-time quantitative PCR
Divalent metal transporter-1 (DMT1), peptide transporter-1 (PePT1), cationic amino acid transporter-1 (CAT1), zinc transporter-1 (ZnT1), Na+-dependent glucose transporter-1 (SGLT1), and glucose transporter-2 (GLUT2) mRNA levels of intestinal mucosa were analyze by quantitative real-time PCR and the experimental procedure refer to the method described by Wan et al [24]. The primer sequences synthesized commercially by Shenggong Bioengineering (Shanghai, China) were presented in Table S2.
Statistical analysis
The data were analysed by single factor variance of SPSS 24.0 (SPSS, Inc). Linear and quadratic orthogonal contrasts were used to determine the effects of inclusion of SPCI in the diets. P < 0.05 and 0.05 < P < 0.1 was considered significant and as trend respectively when compare the differences between the CON group and the SPCI groups.