Feedstuffs

Three starchy feedstuffs (OSP, yam, and taro) and three fibrous feedstuffs (WMR, BBG, and MNC) were used in this study. These feedstuffs were selected based on their nutrient profile and potential to be used in the swine diets [4, 9].

In vitro enzymatic digestion

The 2-step in vitro digestion technique [10] simulates the digestion activities occurring in the upper gastrointestinal tract of the pig and provides information on the apparent ileal digestibility of dry matter (DM), gross energy (GE), and other nutrients.

All the six feed samples were ground to pass through a 1.0-mm mesh screen and subjected to 2-step in vitro digestion as described by Boisen and Fernandez [10]. Briefly, 2 g sample was weighed in a conical flask. A phosphate buffer solution (100 mL, 0.1 mol/L, pH 6.0) and an HCl solution (40 mL, 0.2 mol/L) was poured into the flasks. Two mL of chloramphenicol (Sigma C-0378, Sigma-Aldrich Corp., St. Louis, MO, USA) solution (0.5 g/100 mL ethanol) was added to prevent bacterial growth during hydrolysis. Fresh pepsin solution (4 mL, 20 g/L porcine pepsin, Sigma P-0609) was added, and the flasks were placed in a water bath at 39 °C for 2 h under gentle agitation (50 r/min). Afterward, 40 mL phosphate buffer (0.2 mol/L, pH 6.8) and 20 mL of 0.6 mol/L NaOH were added. Fresh pancreatin solution (12 mL, 100 g/L pancreatin; Sigma P-1750) was added, and hydrolysis was continued for 4 h under the same conditions. After hydrolysis, the residues were collected by filtration on a nylon cloth (42 μm), washed with ethanol (2 × 25 mL 95% ethanol) and acetone (2 × 25 mL 99.5% acetone), dried for 12 h at 60 °C and weighed. The enzymatic hydrolysis was repeated 3 times to obtain enough samples for the in vitro fermentation and their analysis. Hydrolyzed residues from the different replicates and batches of the same ingredients were pooled for subsequent analyses (DM and GE) and in vitro fermentation.

In vitro microbial fermentation

The in vitro fermentation technique simulates the microbial fermentation occurring in the large intestine of the swine [11]. It provides information on the total gas and fermentation metabolites produced by microbial fermentation, which are directly proportional to the amount of substrate fermented.

The fermentation rate of the hydrolyzed substrates was assessed in vitro, using a cumulative gas-production technique adapted to the pig as previously described [11]. Briefly, 200 mg samples were incubated at 39 °C (in a shaking water bath with 50 r/min) in a 125-mL glass bottle with 30 mL buffer solution containing macro- and micro-minerals and a fecal inoculum. Three growing swine from a local commercial farm herd fed a standard commercial diet devoid of antibiotics were used as donors for the fecal inoculums. The inoculum prepared from feces was diluted 20 times in the buffer solution, filtered through a 250-μm screen, and transferred into the bottle with fermentation substrates. Bottles were sealed with a rubber stopper and placed for incubation. An anaerobic environment was maintained throughout the experiment, from inoculum preparation until incubation, by flushing with CO2 gas. The gas generated by fermentation and CO2 released by buffering of SCFA produced during the fermentation was measured at 0, 2, 5, 8, 12, 18, 24, 36, 48, and 72 h using a pressure transducer (GP:50 SIN-54978, Grand Island, NY, USA), fitted with digital data tracker (Tracker 211, Intertechnology Inc., Don Mills, ON, Canada). The bottles were vented after every measurement. Fermentation was stopped at 72 h of incubation by quenching the bottles in ice water, and samples were collected from the bottles and stored frozen for SCFA analysis. Also, 5 mL of homogenous mix solution liquid after fermentation was collected for microbial analysis and processed as described below.

The experimental scheme for in vitro fermentation study was as follows: 6 samples × 6 replicates + 6 blanks (containing the only inoculum) repeated over 3 runs (batches).

Nutrient analysis

The feedstuffs were ground to pass through a 1.0-mm mesh screen using a laboratory mill. Ground samples were subjected to proximate analysis according to the Association of Official Analytical Chemists standard procedures [12] with specific methods as follows: DM (135 °C for 2 h, AOAC 930.15), ash (AOAC 942.05), CP by determining N using Kjeldahl method (AOAC 976.05, CP = N × 6.25), ether extract (AOAC 920.39; using Soxhlet apparatus and petroleum ether), ADF (AOAC 973.18), and NDF (AOAC 2002.04). Total starch content was determined using a commercial test kit (Megazyme International, Wicklow, Ireland). The GE content was determined using an oxygen bomb calorimeter (Parr Bomb Calorimeter 6200, Parr Instrument Co., Moline, IL, USA). Total and soluble NSP of fibrous feedstuffs with their constituent sugars were quantified by gas chromatography (GC) following procedures and calculations as previously described [13]. Chromatographic analysis was done using a GC system (TRACE™ 1300 gas chromatograph; Thermo Scientific, Waltham, MA, USA) equipped with a flame ionization detector and a fused silica capillary column (DB-17HT; Agilent Technologies, Wilmington, DE, USA), using 2-deoxy-D-glucose as an internal standard.

The residue after in vitro digestion was analyzed for DM (method 930.15) and GE (method 984.13A-D) using the standard procedure [12] and used to calculate the apparent in vitro digestibility.

Short-chain fatty acid analysis

Samples collected from the bottles at the end of fermentation were centrifuged, and supernatant from each bottle was analyzed for SCFA. Concentrations of SCFA in post-fermentation solution were determined using a GC system. Briefly, 0.8 mL of the test sample (supernatant from centrifuged at 2500×for 10 min at 4 °C) were added in a tube with 0.2 mL of 25% phosphoric acid and 0.2 mL of internal standard solution (150 mg of 4-methyl-valeric acid, S381810, Sigma-Aldrich) and vortexed thoroughly. Samples were analyzed for SCFA (i.e., acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and caproate) using a GC system (TRACE™ 1300 gas chromatograph; Thermo Scientific, Waltham, MA, USA) with a Stabilwax-DA column (30-m × 0.25-mm internal diameter; Restek, Bellefonte, PA, USA). A flame-ionization detector was used with an injector temperature of 170 °C and a detector temperature of 190 °C. Branched-chain fatty acids (BCFA) content was calculated as the sum of iso-butyrate and iso-valerate.

DNA extraction and metagenomic analysis

After the centrifugation of the fermentation product, the pellet residue from 9 replicates of each treatment from 3 batches were pooledin two tubes to make two replicates. Those two replicates of each treatment were used to extract genomic DNA using a Repeated Bead Beating Plus Column Method (RBB + C) with the QIAamp DNA Stool Mini Kit. Purified genomic DNA was isolated by removing the RNA and proteins using QIAamp Mini spin columns. Extracted DNA was then quantified using a GE NanoVue spectrophotometer, followed by examining its quality in a 0.8% (w/v) agarose gel. Metagenomic analysis of the 16S rRNA V3 and V4 regions was conducted using the Illumina MiSeq system in the John A. Burns School of Medicine, University of Hawaii at Manoa. The following primers were used before sequencing (in standard IUPAC nucleotide nomenclature): 16S Amplicon PCR Forward Primer = 5´-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3´.

16S Amplicon PCR Reverse Primer = 5′-

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3´.

For further processing, Quantitative Insights Into Microbial Ecology (QIIME™ version 2.0 release 2019.4) was used to import demultiplexed paired-end reads of 300 bp in length for all samples. After importing into QIIME2, the DADA2 pipeline was used to denoise, trim, and filter these paired-end sequences. The filtered sequences were subjected to align-to-tree-mafft-fasttree pipeline from the QIIME phylogeny plugin to generate an unrooted and rooted tree for phylogeny. A Naïve Bayes classifier pre-trained on the Greengenes 13.99% OTU was used for taxonomy analysis. The diversity plugin method named core-metrics-phylogenetic was used to conduct alpha and beta diversity analysis on sampling depth of 10,000 frequency. Alpha diversity results were presented as Shannon Index and observed OTUs, while Bray Curtis metrics and unweighted UniFrac were applied for beta diversity.

Statistical analysis

The in vitro dry matter and gross energy digestibility, total gas production, and SCFA production were compared among treatments using the MIXED procedure of SAS 9.2 software (SAS Institute Inc., Cary, NC, USA). Feedstuffs were the fixed factor in the model, and subsample (during both in vitro digestion and fermentation) and batch (only during in vitro digestion) as random factors. Means were separated by the Tukey method using the “pdmix” macro of SAS, and differences among variables were declared significant at a probability level of 0.05.

Statistical analysis of differentially abundant bacteria between fiber and starch-based feeds at the genus level was performed using a linear discriminant analysis (LDA) effect size (LEfSe) method.

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