Changing Dietary Calcium-Phosphorus Level and Cereal Source Selectively Alters Abundance of Bacteria and Metabolites in the Upper Gastrointestinal Tracts of Weaned Pigs

Abstract

Several dietary ingredients may affect the bacterial community structure and metabolism in the porcine gut and may therefore influence animals' health and performance. This study investigated the effects of cereal source and calcium-phosphorus (CaP) level in the diet on bacterial microbiota and metabolites, nutrient intake, and gut environment in weaned pigs. Pigs (n ‫؍‬ 8/treatment) were fed wheat-barley-or corn-based diets with an adequate or high CaP level for 14 days. Effects on microbiota in the stomach, ileum, and midcolon were assessed using quantitative PCR. Data showed that Enterobacteriaceae, Campylobacter spp., and Helicobacter spp., which all contain highly immune reactive lipopolysaccharide (LPS), were abundant at all gut sites. Diet effects on bacteria and metabolites were moderate and occurred mainly in the upper gut, whereas no effects on bacteria, fermentation products, and LPS could be observed in the colon. Differences in carbohydrate intake with corn versus wheat-barley diets selectively stimulated Bifidobacterium in the stomach and ileum. There was a growth advantage for a few bacterial groups in the stomach and ileum of pigs fed the high versus adequate CaP level (i.e., gastric Enterobacteriaceae and ileal Enterococcus, Bacteroides-Prevotella-Porphyromonas, and Campylobacter). Interestingly, gastrointestinal pH was not affected by dietary CaP level. The present findings demonstrate the stability of the bacterial community and gut environment toward dietary changes even in young pigs. The results on stimulation of gastric and ileal Bifidobacterium by corn diets may be employed in nutritional strategies to support gut health after weaning.

Adaptation of the Cecal Bacterial Microbiome of Growing Pigs in Response to Resistant Starch Type 4

“…Based on the sequencing data, qPCR assays were designed and performed with the Stratagene Mx3000P qPCR system (Agilent Technologies, Santa Clara, CA) to quantify the abundance of 16S rRNA genes of total bacteria and five target bacterial groups (i.e., Lactobacillus group, Enterobacteriaceae, Clostridium cluster IV, Clostridium cluster I, and Campylobacter spp.) using previously reported primer sets and amplification conditions (20). Quantification of DNA in digesta samples was performed using Brilliant II SYBR green qPCR low ROX master mix (Agilent Technologies), forward and reverse primers (62.5 pmol/l), and 1 l of genomic DNA in a final volume of 25 l. All qPCR amplifications consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, annealing for 30 s, and elongation at 72°C for 30 s (20).…”

“…using previously reported primer sets and amplification conditions (20). Quantification of DNA in digesta samples was performed using Brilliant II SYBR green qPCR low ROX master mix (Agilent Technologies), forward and reverse primers (62.5 pmol/l), and 1 l of genomic DNA in a final volume of 25 l. All qPCR amplifications consisted of initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, annealing for 30 s, and elongation at 72°C for 30 s (20). Fluorescence was measured at the last step of each cycle.…”

“…Total DNAs were extracted from 250-mg digesta samples from cecum using a PowerSoil DNA extraction kit (MoBio Laboratories, Inc., Carlsbad, CA) as described by Metzler-Zebeli et al (20). To ensure proper lysis of bacteria, a heating step at 70°C for 10 min was introduced between mixing of the digesta sample with buffer C1 and the bead-beating step.…”

“…For quantification of bacterial 16S rRNA gene copies, standards were prepared by making serial dilutions (10 7 to 10 3 molecules/l) of the purified and quantified PCR products generated by standard PCR and genomic DNA from pig intestinal digesta using the universal primer set 27F-1492R and group-specific primers (20). Amplification efficiencies (E) were calculated according to the following equation: E ϭ 10 Ϫ1/slope (93 to 97%).…”

“…Linear relationships between quantification cycle (C q ) and log of DNA concentration were observed for each primer pair (R 2 ϭ 0.996 to 0.999). Gene copy numbers of total bacteria and target bacterial groups were determined by relating the C q values to standard curves (20). The final copy numbers of total bacteria and target bacterial groups per gram of digesta were calculated using the following equation: (QM ϫ C ϫ DV)/(S ϫ V), where QM is the quantitative mean of the copy number, C was the DNA concentration of each sample, DV is the dilution volume of extracted DNA, S is the DNA amount (nanograms) subjected to analysis, and V is the weight of the sample (grams) subjected to DNA extraction (20).…”