Twelve neonatal GF pigs were delivered by hysterectomy from two multiparous Bama sows (a native breed of China). At 112 days of gestation (full-term, 114 days), pregnant Bama sows were anesthetized with 4% isoflurane, the uterus was excised from the anesthetized sow and was transferred into a sterile isolator (DOSSY Experimental Animals Co., Ltd, Chengdu, China) through a tank including 120 L of 0.1% peracetic acid for decontamination of the uterus. Then the pigs were taken from the uterus in the isolator and the twelve neonatal pigs were transferred to six rearing isolators (Class Biologically Clean Ltd., Madison, Wisconsin, USA) depending on the litter of origin and sex. The isolator has a checkboard and pigs were fed separately. The rearing isolators had been sterilized by spraying with 1% peracetic acid in advance and maintained in sterile environments as described previously . The sterile environments, pig’s skin, oral mucosa, and rectal swabs were checked by anaerobic (thioglycollate medium) and aerobic (brain-heart infusion broth) culture of samples at least every week as described by Chinese National Standard (GB/T 14926-41-2001). After 14 days of culture, microbial growth was assessed by gram dyeing microscopy and together with the culture in blood agar base for 48 h at 37°C of colonic digesta collected at the end of the experiment, further confirmed the sterile status.
Experimental treatments and diets
For the six isolators, three of them were treated as the GF group (n = 6), and the other three isolators were designated as the FA group (n = 6). These pigs in GF and FA group were hand-fed Co60-γ-irradiated sterile milk powder prepared by our laboratory (Table S1) and diluted with sterile water (1:4) for 21 days. A corn-soybean feed formulated according to NRC (2012) requirements and Chinese feeding standards for local pigs (2004) (Table S2), and sterilized by Co60-γ-radiation, then introduced to the GF and FA pigs for another 21 days. In the second 21-days period, the GF group orally infused with 25 mL/kg sterile saline per day, FA group orally infused with 25 mL/kg SCFAs mixture (acetic, propionic, and butyric acids, 45, 15, and 11 mM, respectively) per day. The concentrations and dose of acetic, propionic, and butyric acids conducted in present experiments were according to the preliminary test on conventional Bama pigs. In the preliminary test, we had observed that orally infused with excessive concentration (acetic, propionic, and butyric acids, 60, 20, and 15 mM, respectively) and dose (35 mL/kg) of SCFAs led to the death of pigs. In addition, the SCFAs mixture was prepared in the laminar airflow clean benches, the acetic, propionic, and butyric acid (analytically pure) were filtered by a 0.22 um membrane and mixed into sterile water. In the two 21-days periods, all pigs were allowed ad libitum access to sterile water. When the sterile milk, feed, and bottled water in the isolators were consumed, a replacement container was provided via the transfer port, in which containers for sterile feed, milk, or water, and the container were sterilized by spraying with 1% peracetic acid.
Before the pigs were euthanized, blood samples were obtained from anterior vena cava before euthanized via isoflurane anesthesia on the 42th day of the experiment, centrifuged at 3,000 g for 15 min, and stored at -80 °C for further analysis. The abdomen was opened in the laminar airflow clean benches, and the tissues of the colon, liver, and longissimus dorsi were immediately collected in liquid nitrogen and stored at -80 °C for further measurements.
Serum biochemical analyses
The concentrations of adiponectin, insulin, glucagon, glucagon-like peptide 1, and leptin in serum were detected by commercial enzyme-linked immunosorbent assay (ELISA) kits from Chenglin Co. Ltd. (Beijing, China) according to the manufacturer's instructions. The concentrations of total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-c), low density lipoprotein-cholesterol (LDL-c), and glucose in serum were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following with the manufacturer’s instructions. Each parameter was measured in triplicate simultaneously on the same plate. And the differences among parallels must be small (coefficient of variation was less than 10%) to guarantee the reproducibility of repeated measurements.
Determination of enzyme activity
For the enzyme activity assessment, about 1 g frozen sample of liver and longissimus dorsi were homogenized in ice-cold saline solution (1:9, wt/vol) and then centrifuged at 3,000 g for 15 min at 4 ℃. The supernatant was collected for further analysis. The activities of carnitine palmitoyltransferase 1 (CPT-1), lipoprotein lipase (LPL), hepatic lipase (HL), and malate dehydrogenase (MDH) in liver and longissimus dorsi were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The total protein content of liver and longissimus dorsi homogenates was detected by Bradford brilliant blue method . Each parameter was determined in triplicate simultaneously on the same plate.
Real-time quantitative PCR
Total RNA was isolated from frozen colon, liver, and longissimus dorsi, using Trizol reagent (TaKaRa) according to the manufacturer’s instructions. The concentration and purity of the RNA were determined using a NanoDrop ND-2000 Spectrophotometer (NanoDrop, Germany). The ratio of OD260:OD280 ranging from 1.8 to 2.0 in all samples was regarded as suitable for further analysis. The integrity of RNA was detected by agarose gel electrophoresis and the 28S:18S ribosomal RNA band ratio was determined as ≥1.8. RNA was reverse transcribed into cDNA using the PrimeScriptTM RT reagent kit (TaKaRa) according to the manufacturer’s guidelines. Primers for the selected genes (Table S3) were designed by Primer 6 Software (PREMIER Biosoft International, Palo Alto, CA, USA) and synthesized commercially by Sangon Biotech Limited (Shanghai, China). The Quantitative real-time PCR was performed on an ABI Prism 7000 detection system in a two-step protocol with SYBR Green (Applied Biosystems, Foster City, CA, USA). Each reaction was performed in a volume of with 1 μL of cDNA, 5 μL of SYBR Premix Ex Taq TM (2×), 0.2 μL of ROX reference dye (50×), 0.4 μL of each forward and reverse primer, and 3 μL of PCR-grade water. The PCR conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 25 s, and a 72 °C extension step for 5 min. A melting curve analysis was generated following each Quantitative real-time PCR assay to verify the specificity of the reactions. The housekeeping gene β-actin was chosen as the reference gene to normalize mRNA expression of target genes. Gene expression data of replicate samples was calculated using the 2–ΔΔCT method . The relative expression of the target genes in the GF group was set to be 1.0. Each sample was measured in triplicate.
Analysis of adenosine
The sample of liver or longissimus dorsi (100 mg) was homogenized with 5 mL of 0.4 M perchloric acid at 0 ℃ for 1 min, and with ultrasonic treatment for 30 min. Then, the mixture was centrifuged at 3,000 g for 10 min. and the supernatant immediately neutralized to pH 6.5 with 50 mM monopotassium phosphate. After that, monopotassium phosphate was removed by filtration through the sintered glass and stored at -80 ℃ for subsequent analysis. The high-performance liquid chromatography (HPLC, U3000, Thermo Fisher Scientific, USA) was used to determine the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in liver and longissimus dorsi .
Determination of protein levels by western blot
The antibodies against β-actin, GPR43, p-AMPK, AMPK, CPT-1B, and ACC were obtained from Cell Signaling Technology (Davers, MA), Abcam (Cambridge, MA, USA), and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), respectively. Protein levels for the β-actin, GPR43, p-AMPK, AMPK, CPT-1B, and ACC in liver were measured by western blot analysis according to the instructions described by Suryawan et al. (2001) .
Ultrahigh-performance liquid chromatography equipped with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF/MS) analysis
The UHPLC-Q-TOF/MS analysis was performed with an Agilent 1290 UHPLC system (Agilent, Palo Alto, USA) and combined with a Q-TOF mass spectrometer (ESI/Triple TOF 5600; AB Sciex, Concord, Canada) were used to measure the serum metabolites. For the serum sample, the pretreatment, extraction, and identification were according to the procedure described by Hu et al. (2019) . The raw data (whiff scan files) were converted into mzXML format using ProteoWizard , and were imported to the XCMS software for peak matching, retention time alignment, and peak area extraction . Metabolite structure identification was performed by comparing the accuracy of m/z values (< 25 ppm), and MS/MS spectra were interpreted with an in-house database (Shanghai Applied Protein Technology Co. Ltd, China) established with authentic standards. For the XCM data, the ion peaks that were missing values greater than 50% in the group were filtered and excluded and data were normalized to total peak intensity. Then, statistical analyses were performed using SIMCA-P software (version 14.1, Umetrics, Umea, Sweden), where could subjected to multivariate data analysis, including partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), which were carried out to uncover and extract the statistically significant metabolite variations. The PLS-DA and OPLS-DA models were validated based on multiple correlation coefficient (R2) and cross-validated (Q2) in cross-validation and permutation test by applying 2000 iterations . The R2 value in the permutated plot described how well the data fit the derived model, whereas the Q2 value described the predictive ability of the constructed model and was a measure of model quality . The significance of the biomarkers was ranked using the variable importance in the projection (VIP) score (>1) from the OPLS-DA model. Metabolites with the highest VIP score are the most powerful group discriminators, VIP score > 1 are significant . The procedure of metabolites identification and pathway analysis was according to Wang et al. (2017) .
Metabolites with a VIP score >1 was further analyzed by the Student’s t-test at the univariate level to measure the significance of each metabolite. The univariate data analysis also included a fold-change analysis. Other data were analyzed in SAS 9.2 (SAS Institute, Inc., Cary, NC, USA) and analyzed using Student’s t-test, and were presented as means ± SEMs. The individual pig as the statistical unit. P < 0.05 was considered to be statistically significant, and a tendency was declared with 0.05 < P < 0.10.