The project underwent proper ethical standards and the experiments (KW-170519-1) were approved by the Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon, Republic of Korea.
Animals and Experimental Design. A total of 180 weaned pigs (28 day-old; Landrace × Yorkshire × Duroc; initial BW: 10.45 ± 0.03 kg) of mixed sex were randomly allotted to six treatments with 6 replicate pens in each treatment and 5 pigs per pen. The dietary treatments were: control diet (Con; with 1.1% Arg and without ZnO supplementation); Con + 2500 ppm Zn as ZnO (P-Zn); CON + 1.6% Arg (ARG); Con + 500 ppm of Zn as ZnO + 1.6% Arg (ZnArg1); Con + 1000 ppm of Zn as ZnO + 1.6% Arg (ZnArg2); P-Zn + 1.6% Arg (ZnArg3). All diets (Table 1) met or exceeded the nutrient requirements according to the NRC (2010). The crude protein, ether extract, lysine, methionine, arginine, calcium, and phosphorus content of the diets were analyzed by methods of AOAC . The treatment diets were fed in a meal form in 2 phases (d 0 to 7, phase Ⅰ; and d 8 to 14, phase Ⅱ). This experiment was conducted at the facility of Kangwon National University farm and the piglets were housed in slotted and concrete floor pens with a pen size of 1.90 m × 3.0 m. All pens were equipped with a self-feeder and nipple drinker to allow ad libitum access to feed and water. Individual weanling piglet weight and feed intake from each pen were recorded at the beginning of the experiment and at the end of every phase to calculate average daily gain (ADG), average daily feed intake (ADFI) and gain to feed ratio (G:F). All the weaned pigs were subjected to a mild heat stress condition at 35° C.
Microbial analyses. To study the effects of dietary treatments on small intestinal microbiota, representative piglets from each group (2 piglets per replicate; one male and one female) reflecting the average BW of the pen were selected and sacrificed by electrocution at d 14 and 28 of each phase. The digesta from the ileum and was collected in sterile plastic bottles for microbial analysis. The samples collected for microbial analysis were immediately placed on ice until analyses were conducted. The ileal digesta sample (one gram) was mixed with 9 ml peptone broth (1%) and the homogenized (Becton, Dickinson and, Franklin Lakes, NJ, USA). In the next step, serially 10‐fold dilutions in the pellets were used for Viable counts of bacteria. To determine the total anaerobic bacteria (Tryptic soy agar), Lactobacillus spp. (using MRS agar + 0.200 g/l NaN3 + 0.500 g/l L‐cystine hydrochloride monohydrate), Bifidobacterium spp. (MRS‐NPNL: MRS agar + nalidixic acid, paromomycin + neomycin sulphate + lithium chloride), Clostridium spp. (TSC agar) and coliforms (violet red bile agar) were used. The gas pack anaerobic system (BBL, No. 260678, Difco, Detroit, MI, USA) was used for preparing anaerobic conditions. The tryptic soy agar, MRS agar, and violet red bile agar were purchased from Difco Laboratories (Detroit), and TSC agar (CM0589) was purchased from Oxoid (Hampshire, UK). The bacterial concentrations were transformed (log) before statistical analysis.
Small intestinal morphology. The sacrificed pigs (two pigs per pen) were subjected to use for the morphological test. The intestinal morphology test was performed according to the procedure described by Hosseindoust et al., . In short, for each intestinal sample, three cross-sections were prepared after staining with azure A and eosin using standard paraffin‐embedding procedures. A total of 10 intact, well‐oriented crypt-villus units were selected in triplicate for each intestinal cross-section. The measurement of villus height was measured from the tip of the villi to the villus–crypt junction, while the crypt depth was defined as the depth of the invagination between adjacent villi and villus width was measured till the mid of the villus. All morphological measurements (villus height and crypt depth) were made in 10‐μm increments using an image processing and analysis system (Optimus software version 6.5, Media Cybergenetics, North Reading, MA, USA).
RNA Extraction and Real-time PCR of organ samples. Total RNA was isolated from the Jejunum (50 mg), livers (50 mg) and spleens (100 mg) samples using Trizol reagent (Invitrogen, Carlsbad, USA) according to manufacturer’s instruction. Extracted RNA was quantified to 1 μg/μl and cDNA synthesis was conducted using the Improm-II Reverse transcription system (Promega, Fitchburg, USA) and PCR was performed using Mx3000P real-time PCR (Stratagen, USA). The results were expressed as a relative expression by using the delta-delta method. The primers of interleukin-4 (IL-4), interleukin-6 (IL-6), interferon-γ (IFNγ), heat shock protein-27 (HSP27), toll-like receptor-4 (TLR4), and tumor necrosis factor-α (TNF-α) were presented in Table 2. In this process, the house-keeping gene, β-actin was introduced to adjust the quantity of input cDNA to maintain the role in internal control . A total of 20 μL reaction system included 10 μL SYBR Premix Ex Taq, 0.8 μL of forward and reverse primer (10 μM), 0.4 μL ROX Reference Dye II (50×), 2.0 μL cDNA template, and 6 μL dd H2O. Cycling conditions were as followed: 30 s at 95 °C, 40 cycles of denaturation step at 95 °C for 3 sec, 60 °C annealing step for 34 s and a 72 °C extension step for 15 s.
Blood parameters. The blood samples were collected from 2 piglets per pen on the last day of each phase. Five mL of EDTA-treated (Becton Dickinson, Franklin Lakes, NJ) and not-treated blood samples were collected from the jugular vein and stored on ice for immediate hematological analysis. The EDTA-treated blood was diluted by Natt-Herrick solution and mixed for 15 mins for white blood cells (WBC), red blood cells (RBC), lymphocytes, and monocytes were analyzed using Hemavet Multispecies Hematology Systems (Scientific Inc., Oxford, CT). The rest of the blood samples were centrifuged at 1,500 rpm for 20 minutes in a centrifuge, and plasma was separated and used for cortisol analysis. Cortisol was analyzed using an ELISA kit (ADI-900-70; Enzo Life Sciences, Farmingdale, NY).
Statistical Analysis. The data were analyzed as a completely randomized design using the GLM procedure of SAS (SAS Inst. Inc., Cary NC). The pen was the experimental unit for growth performance and feed intake, whereas individual piglet was an experimental unit for the microbial test, intestinal morphology, blood parameters, and gene expression analyses. The Turkey multiple range tests were applied for Treatment means separation by at P < 0.05 statistical level.