Phytate degradation, intestinal microbiota, microbial metabolites and immune values are changed in growing pigs fed diets with varying calcium-phosphorus concentration and fermentable substrates.

The present study assessed effects of diets containing varying calcium-phosphorus (CaP) concentration and fermentable substrates on digestibility of diets, intestinal microbiota and immune system using 32 crossbred pigs (initial BW 54.7 kg). In a 2 × 2 factorial arrangement, pigs were fed either a corn-soybean meal (CSB) or corn-field pea (CFP) diet with either low [-] (4.4 g Ca/kg; 4.2 g total P/kg) or high [+] (8.3 g Ca/kg; 7.5 g total P/kg; supplemented with monocalcium phosphate) CaP content for a period of 9 weeks. In week 8, blood samples were taken, and at the end of the trial, all pigs were euthanized to collect digesta and mesenteric lymphoid tissue. Apparent total tract digestibility (ATTD) of P was greater (p < 0.05) for pigs fed the CaP+ and CFP diets than CaP- and CSB diets. The myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6 ) concentration in jejunal digesta was higher (p < 0.05) for CaP+ than in CaP- fed pigs. In addition, caecal and faecal InsP5 isomer concentration were greater (p < 0.05) for CSB than CFP diets. In the caecum, gene copy numbers of saccharolytic bacteria, such as Eubacterium rectale and Roseburia spp., as well as SCFA concentration were higher (p < 0.05) for CaP+ than CaP- diets. In particular, innate immune cell numbers, such as natural killer cells, dendritic cells, monocytes and neutrophils, were greater (p < 0.05) for CaP+ than CaP- fed pigs. Diets high in CaP resulted in higher abundance of potential beneficial bacteria and might promote the first line of defence enhancing the activation of the cellular adaptive immune response, thereby possibly decreasing the risk for intestinal disturbances. These results strongly suggest that both, CaP supply and dietary ingredients differing in fermentability, may beneficially affect gut health through increase in SCFA-producing bacteria and/or bacteria with anti-inflammatory properties.

Dietary changes in nutritional studies shape the structural and functional composition of the pigs' fecal microbiome-from days to weeks.

BACKGROUND: The possible impact of changes in diet composition on the intestinal microbiome is mostly studied after some days of adaptation to the diet of interest. The question arises if a few days are enough to reflect the microbial response to the diet by changing the community composition and function. The present study investigated the fecal microbiome of pigs during a time span of 4 weeks after a dietary change to obtain insights regarding the time required for adaptation. Four different diets were used differing in either protein source (field peas meal vs. soybean meal) or the concentration of calcium and phosphorus (CaP). RESULTS: Twelve pigs were sampled at seven time points within 4 weeks after the dietary change. Fecal samples were used to sequence the 16S rRNA gene amplicons to analyse microbial proteins via LC-MS/MS and to determine the SCFA production. The analysis of OTU abundances and quantification values of proteins showed a significant separation of three periods of time (p = 0.001). Samples from the first day are used to define the 'zero period'; samples of weeks 1 and 2 are combined as 'metabolic period' and an 'equilibrium period was defined based on samples from weeks 3 and 4. Only in this last period, a separation according to the supplementation of CaP was significantly detectable (p = 0.001). No changes were found based on the corn-soybean meal or corn-field peas administration. The analysis of possible factors causing this significant separation showed only an overall change of bacterial members and functional properties. The metaproteomic approach yielded a total of about 9700 proteins, which were used to deduce possible metabolic functions of the bacterial community. CONCLUSIONS: A gradual taxonomic and functional rearrangement of the bacterial community has been depicted after a change of diet composition. The adaptation lasts several weeks despite the usually assumed time span of several days. The obtained knowledge is of a great importance for the design of future nutritional studies. Moreover, considering the high similarities between the porcine and human gastrointestinal tract anatomy and physiology, the findings of the current study might imply in the design of human-related nutritional studies.

Effect of Bacillus subtilis and Bacillus licheniformis supplementation in diets with low- and high-protein content on ileal crude protein and amino acid digestibility and intestinal microbiota composition of growing pigs.

BACKGROUND: Bacillus spp. seem to be an alternative to antimicrobial growth promoters for improving animals' health and performance. However, there is little information on the effect of Bacillus spp. in combination with different dietary crude protein (CP) levels on the ileal digestibility and microbiota composition. Therefore, the objective of this study was to determine the effect of Bacillus spp. supplementation to low- (LP) and high-protein diets (HP) on ileal CP and amino acid (AA) digestibility and intestinal microbiota composition. METHODS: Eight ileally cannulated pigs with an initial body weight of 28.5 kg were randomly allocated to a row-column design with 8 pigs and 3 periods of 16 d each. The assay diets were based on wheat-barley-soybean meal with two protein levels: LP (14% CP, as-fed) and HP diet (18% CP, as-fed). The LP and HP diets were supplemented with or without Bacillus spp. at a level of 0.04% (as-fed). The apparent ileal digestibility (AID) and standardized ileal digestibility (SID) of CP and AA was determined. Bacterial community composition from ileal digesta was analyzed by Illumina amplicon sequencing and quantitative real-time PCR. Data were analyzed as a 2 × 2 factorial design using the GLIMMIX procedures of SAS. RESULTS: The supplementation with Bacillus spp. did not affect both AID and SID of CP and AA in growing pigs. Moreover, there was no difference in AID of CP and AA between HP and LP diets, but SID of cystine, glutamic acid, glycine, and proline was lower (P < 0.05) in pigs fed the HP diets. The HP diets increased abundance of Bifidobacterium spp. and Lactobacillus spp., (P < 0.05) and by amplicon sequencing the latter was identified as predominant genus in microbiota from HP with Bacillus spp., whereas dietary supplementation of Bacillus spp. increased (P < 0.05) abundance of Roseburia spp.. CONCLUSIONS: The HP diet increased abundance of Lactobacillus spp. and Bifidobacterium spp.. The supplementation of Bacillus spp. resulted in a higher abundance of healthy gut associated bacteria without affecting ileal CP and AA digestibility, whereas LP diet may reduce the flow of undigested protein to the large intestine of pigs.

Evaluation of DNA extraction kits and phylogenetic diversity of the porcine gastrointestinal tract based on Illumina sequencing of two hypervariable regions.

A robust DNA extraction method is important to identify the majority of microorganisms present in environmental microbial communities and to enable a consistent comparison between different studies. Here, 15 manual and four automated commercial DNA extraction kits were evaluated for their efficiency to extract DNA from porcine feces and ileal digesta samples. DNA yield, integrity, and purity varied among the different methods. Terminal restriction fragment length polymorphism (T-RFLP) and Illumina amplicon sequencing were used to characterize the diversity and composition of the microbial communities. We also compared phylogenetic profiles of two regions of the 16S rRNA gene, one of the most used region (V1-2) and the V5-6 region. A high correlation between community structures obtained by analyzing both regions was observed at genus and family level for ileum digesta and feces. Based on our findings, we want to recommend the FastDNA(™) SPIN Kit for Soil (MP Biomedical) as a suitable kit for the analyses of porcine gastrointestinal tract samples.