Animal maintenance

This study was performed according to the guidelines provided by Canadian Council on Animal Care using protocols approved by the University of Alberta Animal Care and Use Committee. Pigs were managed according to approved protocols at the Swine Research and Technology Centre (SRTC), University of Alberta.

Four litters of crossbred piglets (Duroc × Large White/Landrace) born from 2nd or 3rd parity sows, were randomized to antibiotic (ANTI) and control groups (CON). Amoxicillin (30 mg/kg/day) or placebo (antibiotic flavoring) were administered by oral feeding twice daily (8 am and 4 pm) at birth through PND 14. Body weights were recorded weekly.

At PND 21, 8 pigs from each treatment were terminated for sample collection. The remaining pigs were weaned, penned by litter in a nursery room, and fed with a standard phase-feeding program. At PND 49, another 8 pigs from each treatment were terminated. All animals were maintained on a 12-hour light-dark cycle with room temperature of 22–25 °C and allowed free access to food and water. Ingredients of the piglet diets are shown in Supplemental Table S1.

Oral glucose tolerance test

Two weeks prior to OGTT, pigs were introduced to a standard meal consisting of 50 g ground pregrower fodder, and acclimated to handling and standing in a sling. OGTT was conducted after overnight fasting. An hour before OGTT, ears were cleaned and Lidocaine/prilocaine cream applied (EMLA Cream; AstraZeneca, Mississauga, Ontario) to reduce pain. After measuring fasting blood glucose (FBG) animals were given the standard meal (50 g) mixed with 2 g/kg glucose solution. Upon finishing the meal (time 0), glucose was measured with a glucometer (Accu-Check Compact Plus; Roche Diagnostics) in whole blood from ear vein at 15, 30, 45, 60, 90, 120, and 180 min. Additional blood samples were collected - during the first 45 min by 70 μl microhematocrit capillary tubes coated with ammonium heparin (Fisher Scientific). Blood samples were centrifuged at 1500 rpm for 10 min at 4 °C and plasma collected and stored at −80 °C until assayed for insulin by ELISA, according to the manufacturer’s instructions (Alpco Diagnostics, Salem, N.H., USA). Blood glucose and plasma insulin concentrations were plotted as a function of time, and area under the curve was calculated using established methods72.

Animal euthanasia and sampling

At PND 21 and 49, two pigs per treatment per litter were euthanized in the morning (between 0700–1100) for sample collection. Blood samples were collected from jugular vein, and serum samples were aseptically collected, snap frozen in liquid nitrogen, and stored at −80 °C for LPS assay. Pancreas was collected for pancreatic islets isolation [stored in ice-cold Hanks’ Balanced Salt Solution (HBSS; Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) supplemented with 0.2% bovine serum albumin (BSA), 25 mM HEPES, 100 mg/L L-glutamine and 0.35 g/L NaHCO 3 ], immunohistochemistry (10% formalin, Fisher Scientific) and RNA extraction (snap frozen in liquid nitrogen). The entire intestine was subsequently extracted. The ileum (20 cm proximal to the ileo-cecal junction), proximal colon and distal colon were carefully dissected; and mucosal samples were collected, immediately snap-frozen in liquid nitrogen and stored at −80 °C. Ileal, cecal and distal colon content were aseptically collected and snap frozen in liquid nitrogen until storage at −80 °C. In addition, at PND 7, fecal samples were collected before tissue collection using sterile dry swabs (BD Falcon™ SWUBE™, BD Canada, Mississauga, ON) and stored in −80 °C.

Pancreatic islet isolation and culture

Islets of Langerhans were freed from pancreas tissues after a two-time digestion (35 and 20 mins, respectively) in collagenase solution (1 mg/mL in HBSS + 0.2% BSA) at 37 °C, 130 rpm. Samples were then washed with HBSS + 0.2% BSA and filtered through 160 μm nylon filter. Each pellet sample was enriched for islets by means of a 27%, 23%, 14% dextran density gradient centrifuged at 1,500 rpm for 15 min at room temperature. Islets were harvested from the 23% gradient layer, washed and resuspended in HBSS + 0.2% BSA, and separated from exocrine tissue by hand picking under a dissection microscope. Islets were cultured overnight in CMRL1066 medium (Sigma) supplemented with 0.5% BSA at 37 °C in humidified air containing 5% CO 2 .

Glucose-stimulated insulin secretion

Cultured islets were washed with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Burlington, ON, Canada) with 0.1% BSA. To measure insulin release, quadruplicate samples of 3 islets/vial were incubated in DMEM with 2.8 and 16.5 mM glucose for 90 min at 37 °C gassed with 95% O 2 and 5% CO 2 . Supernatants were retained and insulin remaining in the islets was extracted with 3% acetic acid, and then stored at −20 °C for insulin radioimmunoassay (RIA)73. Total islet insulin content was calculated by adding insulin secreted into supernatant plus the remaining in the islet pellet, as determined by RIA. From this, the percentage of total insulin secreted was calculated for each data point to eliminate variance caused by islet size. Insulin release index was calculated as the ratio of insulin percentage release in response to 16.5 mM glucose versus 2.8 mM glucose.

Immunohistochemistry

Pancreas tissues were fixed in 10% phosphate buffered formalin for 24 h. The tissues were dehydrated and embedded in paraffin using standard procedures, and 5 μm sections cut and affixed to glass slides. After dewaxing and rehydration, pancreas sections were treated overnight at 4 °C with primary antibodies: guinea pig anti-insulin (Dako, Burlington, ON, CA), rabbit anti-glucagon (Millipore, Billerica, MA, USA) and rabbit anti-Ki67 (Abcam, Toronto, ON, CA), followed by a 2-hour incubation at room temperature with secondary antibodies: goat anti-guinea pig (IgG H&L, Alexa Fluor® 488; Invitrogen, Burlington, ON, CA) and goat anti-rabbit (IgG H&L, Alexa Fluor® 594; Invitrogen), respectively. All antibodies were diluted to 1:200 in PBS. Slides were then mounted with ProLong® Gold Antifade Mountant with DAPI (Invitrogen). TUNEL staining was performed using In Situ Cell Death Detection Kit following manufacturer instructions (Sigma-Aldrich Canada Co., Oakville, ON, CA), and double stained with insulin as described above.

Micrographs of pancreas sections were captured using an Axiovert microscope equipped with Axiovision 4.7 software (Carl Zeiss Microscopy GmbH, Jena, Germany). Pancreatic β-cell area was expressed as the percentage of insulin-positive area vs the entire pancreas area (excluding large ducts and veins) mounted on the slide, which was quantified using ImageJ.

For Ki67 and TUNEL staining, an EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific) was used to capture 30–40 randomly selected sections within the pancreas area. The number of nuclei of the insulin-positive areas and Ki67 and TUNEL positive cells within each of the insulin-positive areas was counted using Image J. The proportions of Ki67 and TUNEL positive β-cells relative to total β-cells were used to estimate the rates of proliferation and apoptosis.

Microbial composition analysis

Total DNA from fecal samples and ileal and distal colon contents was extracted with the QIAamp DNA Mini Stool Kit (Qiagen, Inc. Mississauga, ON, Canada) following the manufacturer’s instructions, with the addition of a bead beating step (FastPrep instrument, MP Biomedicals, Solon, OH). DNA concentrations and quality were determined by a NanoDrop 2000c. Extracted DNA was diluted to 20 ng/μl for PCR amplification.

The hypervariable regions (V1 to V3) of the bacterial 16 S rRNA gene were amplified with nucleotide barcoded primer pairs 27 F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and 519 R: 5′-GWATTACCGCGGCKGCTG-3′. The forward primer contained Roche/454 Titanium adaptor A (CCATCTCATCCCTGCGTGTCTCCGACTCAG) and unique 10-bp barcodes, and the reverse primer contained adaptor B (CCTATCCCCTGTGTGCCTTGGCAGTCTCAG). The PCR was performed on an S1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) using the following parameters: initial denaturation at 98 °C for 1 min, followed by 35 cycles of 98 °C for 10 s, 59 °C for 30 s and 72 °C for 30 s, with a final extension at 72 °C for 7 min. Then triplicate DNA amplification products were mixed and gel-purified (QIAquick gel extraction kit, Qiagen, Valencia, CA). Each amplicon (100 ng) was pooled and pyrosequenced using a 454 Titanium platform (Roche, Branford, CT).

Sequence data that passed Roche’s quality thresholds were processed according to the mothur 454 SOP74,75 accessed on June 16, 2015. Barcodes were trimmed and quality sequences were obtained by removing sequences containing ambiguous bases and quality read length < 200 bases. Sequences passing quality filter were aligned to the silva bacterial reference alignment. Sequences were clustered based upon 0.97 similarity using UClust into operational taxonomic units (OTUs) and hypothesis testing were performed with normalized data in mothur. Differences in overall community were tested by multivariate analysis of variance (MANOVA) with Bonferroni correction for multiple comparisons. Abundance of bacterial OTUs to phyla was compared using the Mann-Whitney U-test or student’s T-test. Inverse Simpson diversity index was used to ascertain differences in alpha diversity based on antibiotic exposure status. Weighted UniFrac distance matrices were calculated for beta diversity analyses.

Serum LPS assay

Serum LPS concentrations were measured by PYROGENT™–5000 Kinetic Turbidimetric LAL Assay (Lonza, Burlington, ON, CA), according to the manufacturer’s instructions.

SCFA measurement

SCFA concentrations in cecal content were analyzed by gas chromatography. One gram of distal colonic content was mixed with 4 ml of 25% phosphoric acid, vortexed thoroughly and centrifuged at 3,500 rpm for 10 min at 4 °C. Supernatant was transferred to 1.5 ml tubes and centrifuged at 15,000 rpm for 10 min at 4 °C. The supernatant was then filtered through 0.45 μm filters (Fisher), and mixed with internal standard solution (24.5 mmol/L isocaproic acid) at a ratio of 4:1. Samples were injected into a Stabilwax-DA column (30 m × 0.53 mm i.d. × 0.5 μm film thickness; Restek Corporation, Bellefonte, PA) on a Varian gas chromatograph (Model 3800; Varian Analytical Instruments, Palo Alto, CA) using an autosampler (Model 8400; Varian Inc., Walnut Creek, CA). Samples were run at a split vent flow of 20 mL/min with a column temperature gradient as follows: 80 °C held for 0.1 min, increased to 210 °C at 45 °C/min and held for 5 min at 210 °C. The temperature of the injector and the detector was 250 °C. Peaks were analyzed using Galaxie Software (Varian Inc., Palo Alto, CA) and concentrations of each SCFA were calculated. Total SCFA concentration was the sum of concentrations of the detected SCFA. Final results were normalized by the weight of each sample used.

RNA isolation and cDNA synthesis

Total RNA was extracted from the pancreas, ileum and proximal colon using the GeneJET RNA Purification Kit (Thermo Scientific). On column DNase (RNase-free DNase set; QIAGEN) was applied to eliminate genomic DNA contamination in the RNA samples. RNA concentrations and purity were determined by a NanoDrop 2000c (Thermo Scientific) RNA quality was further verified with gel electrophoresis (RNA loading dye, Thermo Scientific)76. Extracted RNA (1 μg) was then reverse transcribed using Maxima First Strand cDNA Synthesis Kit (Thermo Scientific).

Reverse transcription quantitative PCR

Gene expression was measured by RT-qPCR using PerfeCTa SYBR® Green SuperMix Rox (Quanta Biosciences Inc., Gaithersburg, MD) on a StepOnePlus real-time PCR System equipped with StepOne software v2.3 (Applied Biosystems, ON, Canada). A two-step thermal cycling protocol was performed as follows: initial denaturation for one cycle at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, and annealing or extension temperature for 30 s. Samples were analyzed in duplicates and glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the internal control. Relative gene expression was calculated using the comparative CT (2−ΔΔCT) method. Primers were designed in Beacon Designer 7.9 using sequences obtained from the ENSEMBL pig database (Supplemental Table S2), and the amplification products and annealing temperature were tested. Specificity of the amplification was verified by melt curve analysis and evaluation of efficiency (90–110%) of qPCR amplification and no reverse transcriptase control (NRT) as negative control for DNA contamination.

Intestinal alkaline phosphatase (IAP) activity

Please see the supplemental methods for full details.

Statistical analysis

Statistical analysis for the randomized block design with two treatments was carried out using SAS (version 9.3; SAS Institute Inc. Cary, NC) and GraphPad Prism v6.02 (La Jolla, CA). Comparisons between two treatment groups were analyzed using a proc-mixed model after normality tests unless otherwise stated. Classification variables included animal, treatment and litter. Treatment was included in the model as a fixed class variable, with litter as a random class variable. Differences between treatments means were determined by utilizing lsmeans treat litter/pdiff stderr statement. For OGTT and body weight data, classification variables included animal, treatment, litter and time. Treatment was included in the model as a fixed class variable, litter was included in the model as a random class variable and time was considered additionally as the experimental unit, and differences between treatments were determined by utilizing lsmeans treat time treat*time/pdiff statement. Data are expressed as mean ± SEM. Statistical significance was expressed as ***P < 0.001, **P < 0.01, and *P < 0.05. A P value between 0.1 and 0.05 was considered as a trend.