Preparation of diets
Mechanically air-dried, whole plants of a proprietary strain of Chondrus crispus, cultivated intensively on-land, were obtained from Acadian Seaplants Limited (Dartmouth, Nova Scotia, Canada). The seaweed samples were finely ground and passed through a 60-mesh sieve (0.25 mm in diameter). The fructo-oligo-saccharide (FOS) powder, extracted from chicory roots, was provided by Cargill (Wayzata, MN), as Oliggo – Fiber™ DS2 inulin, with an average degree of polymerization less than 10. C. crispus and FOS were mixed with a standard basal feed (RMH 3000, LabDiet, St. Louis, MO, USA), respectively, at the ratio of 0.0 (plus 2.5 % corn starch), 0.5 (plus 2.0 % corn starch) or 2.5 % (dry w/w). The mixed feed was then pelleted (4.7 mm in diameter, 1.0-1.5 cm in length) using a feed mill facility located at the Faculty of Agriculture, Dalhousie University, Truro, Nova Scotia. Diets were prepared just before the trial and stored under dry and cool conditions.
Animals and sampling procedures
All animal protocols were approved by the University Committee on Laboratory Animals at Dalhousie University, Canada. Male Sprague–Dawley rats (21 days old; Charles River Laboratories Inc., Montreal, Canada) were individually housed in standard plastic cages with a 12-hour light–dark cycle, at 22 +/− 2 °C, with free access to food and water. Rats were randomly assigned to each of the five feeding groups (n = 6/group). Feed groups consisted ofthe basal diet control group (BF), the basal diet supplemented with 2.5 or 0.5 % (dry w/w) of cultivated Chondrus (C2.5 and C0.5, respectively), and the basal diet supplemented with 2.5 or 0.5 % (dry w/w) of FOS (F2.5 and F0.5, respectively). Environmental enrichment was provided by 50 mm diameter wooden pieces and 100 mm diameter plastic tubes. Feed intake and body weight for each animal were monitored weekly. After 21 days of feeding, fresh faeces were collected. One fecal portion was weighed immediately, dried thoroughly at 70 °C (for 24 h) and analyzed for moisture content. Another fecal portion was immediately frozen in liquid nitrogen and stored at −80 °C until further analysis. Rats were anesthetized with 3 % isoflurane, and euthanized by blood draining through cardiac puncture. Blood samples were collected into anticoagulant (K2-EDTA)-coated 5-ml tubes (BD, USA), and immediately centrifuged at 5000 × g for 15 min. The resulting plasma was stored at −20 °C until analysis. Liver, kidney, spleen and heart were collected, blotted on filter paper and weighed. Colon contents from each animal were squeezed into sterile micro-centrifuge tubes, snap-frozen and stored at −80 °C. The colon tissue was flushed with 0.9 % NaCl, and a 0.5-cm segment, excised 1-cm from the end of the proximal colon, was soaked in 10 % neutral, buffered formalin solution (Sigma).
Immunoglobulin enzyme-linked immunosorbent assay (ELISA)
The frozen plasma samples were thawed on ice and subjected to IgA and IgG assays as previously described [23], using the rat IgA and IgG ELISA kits, respectively (Genway, San Diego, USA), following the manufacturer’s instructions. Three technical replicates were performed for each plasma sample collected. Optical density was read on a microplate reader (BioTek) at 450 nm. A standard curve generated from serial dilution of the rat IgA or IgG, of a known concentration, as provided in the kit, was used to determine the sample concentration of IgA and IgG, respectively.
Colonic Histomorphology
After being fixed for 3 days in 10 % neutral formalin, the proximal colon samples were paraffin embedded, sectioned transversely and subjected to hematoxylin and eosin (H&E) staining, following standard procedures [24]. For each H&E section, at least 30 bright-field images were captured by a Motic 2500 digital camera (Motic, China), under 40–100 × magnification, using an Olympus BHS microscope (Olympus, Japan). The measurement function of the Motic Images Plus 2.0 ML software (Motic, China) was used to determine colonic crypt depth, and the thickness of the colonic mucosa, externa muscularis and total wall.
Bacterial DNA isolation from colon content
Bacterial DNA was extracted from 200 mg of colon digesta using the QIAamp DNA Stool Mini Kit (Cat # 51504, Qiagen, USA) following the supplier’s instructions. The DNA was quantified with a Nanodrop ND-2000 spectrophotometer (NanoDrop Technologies Wilmington, DE), and the integrity of DNA was determined by agarose gel electrophoresis. The DNA was stored at −20 °C until further analysis.
Colonic microbiota profiling and analyses
Colonic microbiota analysis was carried out using PhyloChip Arrays (Second Genome Inc., CA, USA). No less than 200 ng of colonic bacterial DNA (n = 4/group) was used as the template for bacterial 16S rRNA gene amplification. The PCR was run for 35 cycles at 95 °C for 30 sec for denaturing, 50 °C for 30 sec for annealing, and 72 °C for 2 min for extension, using the Ex Taq system (Takara Bio Inc., Japan). Primer sequences are as follows: forward primer, 5´-AGRG TTTG ATCM TGGC TCAG-3´; reverse primer, 5´-GGTT ACCT TGTT ACGA CTT-3´. The resulting PCR product from each sample was concentrated and quantified by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). PhyloChip Control Mix (Second Genome Inc.) was then added to label the PCR products, which were then fragmented, biotin labeled and hybridized to the G3 PhyloChip Array. PhyloChip arrays were washed, stained and scanned using a GeneArray scanner (Affymetrix, OH, USA) and each scan was captured using the GeneChip Microarray Analysis Suite (Affymetrix). The hybridization score derived from the fluorescence intensity (FI) (the mean log2 FI × 1,000) for each operational taxonomic unit (OTU) was used to denote abundance. OTUs were defined by >99 % similarity of the 16S rRNA sequence. A threshold based on perfect match and mismatch intensities of multiple probes per probe set [25] was used to determine the presence/absence of an OTU. Taxa-sample intersections were analyzed based on the abundance (AT) and binary matrices (BT). The Unifrac distance metric [26] and weighted Unifrac distance metric were used to compute the pairwise BT and AT dissimilarity scores; the weighted Unifrac metric reflects the abundance of and the phylogenetic distance between OTUs. Hierarchical clustering via average-neighbor (HC-AN) and principal coordinate analysis (PCA) were used to graphically summarize inter-sample relationships on the basis of AT and BT dissimilarity scores. Thereafter, we analyzed the abundance data and identified the taxa which showed significant/greatest changes between the control and the treated groups.
Gas chromatography analysis of short chain fatty acids (SCFAs)
The gut microbial metabolites, SCFAs, in rat faecal samples were quantified by gas chromatography (GC) following a previously described protocol [27], with modifications. A Bruker 430-GC system (Billerica, MA, USA), equipped with a flame ionization detector (FID) and an automatic liquid sampler, was used. A J&W DB-FFAP capillary column (Agilent Technologies Inc., USA; Part # J125-3232, 30 m × 0.53 mm × 1-μm film thickness) was used. An aliquot of 0.2 g of faeces, which was previously frozen and thawed on ice, was homogenized in 2 ml of extraction buffer (0.1 % (w/v) HgCl2 and 1 % (v/v) H3PO4) containing 0.045 g/l of 2-ethylbutyric acid (Sigma) as an internal standard. The resulting slurry was centrifuged, prior to the supernatant being passed through a 0.2 μm filter. The injection volume was 0.5 μl. Each sample run was preceded with a wash run of 1 % formic acid. The oven temperature was held at 80 °C for 1.2 min, then increased to 200 °C at 10 °C /min and held for 5 min. The temperature for the FID and the injection port was 220 °C and 180 °C, respectively. The flow rate of helium, hydrogen, and air was 25, 30, and 300 ml/min, respectively. Specific SCFAs were identified by running an external volatile acid standard mix (Supelco, USA). The concentration of SCFAs was quantified by running the internal standard and the external standard mix, as previously described [28]. All reagents were of GC grade and solutions were prepared with deionized water.
Statistics
The statistical analyses were performed using SPSS 15.0 (SPSS, USA). Data were presented as the mean ± SD or SE. Differences between the control group and the dietary supplemented groups were assessed using one-way ANOVA followed by the independent two-tailed t test or the Mann–Whitney test. Differences were considered as significant when p < 0.05.