Effect of dietary calcium concentration and exogenous phytase on inositol phosphate degradation, mineral digestibility, and gut microbiota in growing pigs.

Variations in the dietary Ca concentration may affect inositol phosphate (InsP) degradation, and thereby, P digestibility in pigs. This study assessed the effects of dietary Ca concentration and exogenous phytase on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs with ileal cannulation. In a completely randomized row-column design with four periods, eight ileal-cannulated barrows (initial body weight 27 kg) were fed four corn-soybean- and rapeseed meal-based diets containing 5.5 or 8.5 g Ca/kg dry matter (DM), with or without 1,500 FTU of an exogenous hybrid-6-phytase/kg diet. No mineral P was added and the P concentration in the feed was 4.8 g P/kg DM. Prececal InsP6 disappearance in pigs fed diets containing exogenous phytase was lower (P = 0.022) with additional Ca than without. Concentrations of InsP2-4 isomers and myo-inositol in the distal ileal digesta and prececal P digestibility were greater (P < 0.001) with exogenous phytase than without exogenous phytase. In feces, InsP6 disappearance was lower (P < 0.002) and concentration of InsP5 and InsP4 isomers was higher (P ≤ 0.031) with additional Ca compared to without additional Ca. The prececal amino acid digestibility, energy digestibility, and hindgut disappearance of energy did not differ. The Shannon diversity index of the microbiota in the distal ileal digesta and feces was similar among the diets but was lower in the distal ileal digesta than in the feces (P < 0.001). Permutation analysis of variance revealed no dietary differences between the bacterial groups within the ileal digesta and fecal samples (P > 0.05). In conclusion, additional Ca reduced the effect of exogenous phytase on prececal InsP6 degradation. Endogenous InsP degradation was impaired by additional Ca only in the hindgut but the abundance of bacterial genera in feces was not affected.

The dietary calcium concentration can influence the release of phosphorus from phytate in growing pigs. This study assessed the effects of dietary calcium and exogenous phytase on inositol phosphate (InsP) degradation and nutrient digestibility in ileal-cannulated, growing pigs. The phosphorus, calcium, and myo-inositol concentrations in the blood, microbiota composition in the ileal digesta and feces, and volatile fatty acid concentrations in the feces were also evaluated. Additional dietary calcium decreased prececal inositol hexakisphosphate (InsP6) disappearance, but only with exogenous phytase. Concentrations of InsP2-4 isomers and myo-inositol in the ileal digesta and prececal phosphorus digestibility were greater with exogenous phytase, but not affected by dietary calcium concentration. In contrast, fecal InsP6 disappearance was lower and the concentration of InsP4-5 isomers in feces was greater with additional dietary calcium. Regarding microbiota, the Shannon diversity index was lower in ileal digesta than in feces but was unaffected by dietary calcium concentration or exogenous phytase. In conclusion, dietary calcium concentration is relevant for phytate disappearance in feces, but not in the ileal digesta. However, when exogenous phytase is used, the dietary calcium concentration is important because prececal phytate degradation is changed.

The active core microbiota of two high-yielding laying hen breeds fed with different levels of calcium and phosphorus.

The nutrient availability and supplementation of dietary phosphorus (P) and calcium (Ca) in avian feed, especially in laying hens, plays a vital role in phytase degradation and mineral utilization during the laying phase. The required concentration of P and Ca peaks during the laying phase, and the direct interaction between Ca and P concentration shrinks the availability of both supplements in the feed. Our goal was to characterize the active microbiota of the entire gastrointestinal tract (GIT) (crop, gizzard, duodenum, ileum, caeca), including digesta- and mucosa-associated communities of two contrasting high-yielding breeds of laying hens (Lohmann Brown Classic, LB; Lohmann LSL-Classic, LSL) under different P and Ca supplementation levels. Statistical significances were observed for breed, GIT section, Ca, and the interaction of GIT section x breed, P x Ca, Ca x breed and P x Ca x breed (p < 0.05). A core microbiota of five species was detected in more than 97% of all samples. They were represented by an uncl. Lactobacillus (average relative abundance (av. abu.) 12.1%), Lactobacillus helveticus (av. abu. 10.8%), Megamonas funiformis (av. abu. 6.8%), Ligilactobacillus salivarius (av. abu. 4.5%), and an uncl. Fusicatenibacter (av. abu. 1.1%). Our findings indicated that Ca and P supplementation levels 20% below the recommendation have a minor effect on the microbiota compared to the strong impact of the bird's genetic background. Moreover, a core active microbiota across the GIT of two high-yielding laying hen breeds was revealed for the first time.

Methane Reduction Potential of Brown Seaweeds and Their Influence on Nutrient Degradation and Microbiota Composition in a Rumen Simulation Technique.

This study aimed to investigate the effects of two brown Icelandic seaweed samples (Ascophyllum nodosum and Fucus vesiculosus) on in vitro methane production, nutrient degradation, and microbiota composition. A total mixed ration (TMR) was incubated alone as control or together with each seaweed at two inclusion levels (2.5 and 5.0% on a dry matter basis) in a long-term rumen simulation technique (Rusitec) experiment. The incubation period lasted 14 days, with 7 days of adaptation and sampling. The methane concentration of total gas produced was decreased at the 5% inclusion level of A. nodosum and F. vesiculosus by 8.9 and 3.6%, respectively (P < 0.001). The total gas production was reduced by all seaweeds, with a greater reduction for the 5% seaweed inclusion level (P < 0.001). Feed nutrient degradation and the production of volatile fatty acids and ammonia in the effluent were also reduced, mostly with a bigger effect for the 5% inclusion level of both seaweeds, indicating a reduced overall fermentation (all P ≤ 0.001). Microbiota composition was analyzed by sequencing 16S rRNA amplicons from the rumen content of the donor cows, fermenter liquid and effluent at days 7 and 13, and feed residues at day 13. Relative abundances of the most abundant methanogens varied between the rumen fluid used for the start of incubation and the samples taken at day 7, as well as between days 7 and 13 in both fermenter liquid and effluent (P < 0.05). According to the differential abundance analysis with q2-ALDEx2, in effluent and fermenter liquid samples, archaeal and bacterial amplicon sequence variants were separated into two groups (P < 0.05). One was more abundant in samples taken from the treatment without seaweed supplementation, while the other one prevailed in seaweed supplemented treatments. This group also showed a dose-dependent response to seaweed inclusion, with a greater number of differentially abundant members between a 5% inclusion level and unsupplemented samples than between a 2.5% inclusion level and TMR. Although supplementation of both seaweeds at a 5% inclusion level decreased methane concentration in the total gas due to the high iodine content in the seaweeds tested, the application of practical feeding should be done with caution.

Composition of the ileum microbiota is a mediator between the host genome and phosphorus utilization and other efficiency traits in Japanese quail (Coturnix japonica).

BACKGROUND: Phosphorus is an essential nutrient in all living organisms and, currently, it is the focus of much attention due to its global scarcity, the environmental impact of phosphorus from excreta, and its low digestibility due to its storage in the form of phytates in plants. In poultry, phosphorus utilization is influenced by composition of the ileum microbiota and host genetics. In our study, we analyzed the impact of host genetics on composition of the ileum microbiota and the relationship of the relative abundance of ileal bacterial genera with phosphorus utilization and related quantitative traits in Japanese quail. An F2 cross of 758 quails was genotyped with 4k genome-wide single nucleotide polymorphisms (SNPs) and composition of the ileum microbiota was characterized using target amplicon sequencing. Heritabilities of the relative abundance of bacterial genera were estimated and quantitative trait locus (QTL) linkage mapping for the host was conducted for the heritable genera. Phenotypic and genetic correlations and recursive relationships between bacterial genera and quantitative traits were estimated using structural equation models. A genomic best linear unbiased prediction (GBLUP) and microbial (M)BLUP hologenomic selection approach was applied to assess the feasibility of breeding for improved phosphorus utilization based on the host genome and the heritable part of composition of the ileum microbiota. RESULTS: Among the 59 bacterial genera examined, 24 showed a significant heritability (nominal p ≤ 0.05), ranging from 0.04 to 0.17. For these genera, six genome-wide significant QTL were mapped. Significant recursive effects were found, which support the indirect host genetic effects on the host's quantitative traits via microbiota composition in the ileum of quail. Cross-validated microbial and genomic prediction accuracies confirmed the strong impact of microbial composition and host genetics on the host's quantitative traits, as the GBLUP accuracies based on the heritable microbiota-mediated components of the traits were similar to the accuracies of conventional GBLUP based on genome-wide SNPs. CONCLUSIONS: Our results revealed a significant effect of host genetics on composition of the ileal microbiota and confirmed that host genetics and composition of the ileum microbiota have an impact on the host's quantitative traits. This offers the possibility to breed for improved phosphorus utilization based on the host genome and the heritable part of composition of the ileum microbiota.

Microbiome Clusters Disclose Physiologic Variances in Dairy Cows Challenged by Calving and Lipopolysaccharides.

Dairy cows respond individually to stressful situations, even under similar feeding and housing conditions. The phenotypic responsiveness might trace back to their microbiome and its interactions with the host. This long-term study investigated the effects of calving, lipopolysaccharide (LPS)-induced inflammation, and l-carnitine supplementation on fecal bacteria and metabolites, dairy cow milk production, health, energy metabolism, and blood metabolites. Fifty-four multiparous Holstein dairy cows were examined over a defined period of life (168 days). The obtained data allowed a holistic analysis combining microbiome data such as 16S rRNA amplicon sequencing and fecal targeted metabolome (188 metabolites) with host parameters. The conducted analyses allowed the definition of three enterotype-like microbiome clusters in dairy cows which could be linked to the community diversity and dynamics over time. The microbiome clusters were discovered to be treatment independent, governed by Bifidobacterium (C-Bifi), unclassified (uncl.) Clostridiales (C-Clos), and unclassified Spirochaetaceae (C-Spiro). Animals between the clusters varied significantly in terms of illnesses, body weight, microbiome composition, and milk and blood parameters. C-Bifi animals were healthier and leaner with a less diverse but dynamic microbiome. C-Spiro animals were heavier, but the diversity of the static microbiome was higher. This pioneering study uncovered microbiome clusters in dairy cows, each contributing differently to animal health and productive performance and with a crucial role of Bifidobacterium. IMPORTANCE The health of dairy cows has to be carefully considered for sustainable and efficient animal production. The microbiome of animals plays an important role in the host's nutrient supply and regulation of immune functions. We show that a certain composition of the fecal microbiome, called microbiome clusters, can be linked to an animal's health at challenging life events such as calving and inflammation. Cows with a specific set of bacteria have coped better under these stressors than have others. This novel information has great potential for implementing microbiome clusters as a trait for sustainable breeding strategies.

Phytase Supplementation Effects on Amino Acid Digestibility in Broiler Chickens are Influenced by Dietary Calcium Concentrations but not by Acid-Binding Capacity.

BACKGROUND: Responses to dietary calcium (Ca) and supplemented phytase on prececal amino acid digestibility (pcAAD) in broiler chickens vary among studies. The variation may arise from the dietary acid-binding capacity (ABC) that influences the activity of enzymes in the digestive tract and from microbial activity. OBJECTIVE: This study aimed to investigate whether the ABC influences phytase effects on pcAAD and whether microbial activity contributes to this. METHODS: Male Ross 308 broiler chickens were provided 1 of 12 diets in 72 pens (15/pen) from day 16 of age until the end of the experiment on days 21 or 22. In a 3 × 2 × 2-factorial arrangement, the ABC was varied by replacing calcium carbonate (CaCO3) with Ca-formate or by adding formic acid to CaCO3-containing diets, and contained 5.6 or 8.2 g Ca/kg and 0 or 1500 phytase units/kg. The ileum content was collected for pcAAD measurement and microbial community composition was used to investigate whether changes in pcAAD are related to the microbiota. RESULTS: Three-factor ANOVA showed that reducing the ABC increased pcAAD (average 1.1 percentage points) and no significant interaction of the ABC with Ca concentration and phytase supplementation including 3-way interactions. Without phytase, increasing dietary Ca concentration decreased pcAAD (average 3.1 percentage points). Phytase supplementation increased pcAAD (average 2.1 and 5.0 percentage points at low and high Ca concentrations, respectively), to reach the same level for both Ca concentrations. Microbial functional predictions pointed towards an influence of the microbiota in the crop and ileum content on amino acid concentrations, as indicated by different relative abundances of predicted genes related to amino acid biosynthesis, degradation, and metabolism. CONCLUSIONS: Dietary Ca concentrations but not the ABC modulates the effect of supplemented phytase on pcAAD in broiler chickens. The microbiota might contribute to differences in pcAAD by changing the amino acid composition of the digesta. The extent of this effect is still unknown.

Relative phytase efficacy values as affected by response traits, including ileal microbiota composition.

The objective of this study was to compare the effects of graded inclusions of 2 phytase products and a mineral P source in broiler chickens using different response traits, including ileum microbiota composition. Eleven experimental diets were used. These were a low-P basal diet and diets supplemented with increasing levels of dicalcium phosphate (DCP), Natuphos E 5000 G (NE), or Natuphos 5000 G (N). The performance traits, prececal P digestibility, and tibia and foot ash results were subjected to regression analysis and slope ratios were used to compare the supplements based on the measured evaluation traits. In the microbiota analysis, total nucleic acids were extracted and the 16S rRNA gene was targeted for use in the amplicon sequencing process. Phylogenetic analysis was performed using Mothur, followed by a multivariate statistical analysis. The various response traits caused different estimates of relative efficacy. The mean results of all the response traits showed that a 1.75-fold increase in the activity of N was needed to achieve the same response as NE and the variability among the detected traits ranged from 1.59 (prececally digestible P intake) to 1.91 (amount of tibia ash). The mean slope ratio between DCP and NE was 311 and varied between 208 (ADG) and 349 (foot ash concentration). The mean slope ratio for phytase N with DCP was 552 and varied from 357 (ADG) to 640 (tibia ash concentration). The ileum microbiota composition was not different among the diets. A similar composition was driven in the abundance of Lactobacillus crispatus, Lactobacillus salivarius, and Lactobacillus gallinarum. The results suggest that different response traits cause markedly different estimates of relative phytase efficacy.

Effects of calcium level and source, formic acid, and phytase on phytate degradation and the microbiota in the digestive tract of broiler chickens.

BACKGROUND: Diet acidification, dietary calcium (Ca) level, and phytase supplementation are known influences on the microbial community in the digestive tract and on phosphorus (P) utilization of broiler chickens. Effects of dietary factors and microbiota on P utilization may be linked because microorganisms produce enzymes that release P from phytate (InsP6), the main source of P in plant feedstuffs. This study aimed to detect linkages between microbiota and InsP6 degradation by acidifying diets (i.e., replacing Ca carbonate (CaCO3) by Ca formate or adding formic acid to CaCO3-containing diets), varying Ca levels, and supplementing phytase in a three-factorial design. We investigated i) the microbial community and pH in the digestive tract, ii) prececal (pc) P and Ca digestibility, and iii) InsP6 degradation. RESULTS: All factors under investigation influenced digesta pH and the microbiota composition. Predicted functionality and relative abundance of microorganisms indicated that diets influenced the potential contribution of the microbiota on InsP degradation. Values of InsP6 degradation and relative abundance of the strains Lactobacillus johnsonii and Lactobacillus reuteri were correlated. Phytase supplementation increased pc InsP6 disappearance, with differences between Ca levels, and influenced concentrations of lower inositol phosphate isomers in the digestive tract. Formic acid supplementation increased pc InsP6 degradation to myo-inositol. Replacing CaCO3 by Ca-formate and the high level of these Ca sources reduced pc InsP6 disappearance, except when the combination of CaCO3 + formic acid was used. Supplementing phytase to CaCO3 + formic acid led to the highest InsP6 disappearance (52%) in the crop and increased myo-inositol concentration in the ileum digesta. Supplementing phytase leveled the effect of high Ca content on pc InsP6 disappearance. CONCLUSIONS: The results point towards a contribution of changing microbial community on InsP6 degradation in the crop and up to the terminal ileum. This is indicated by relationships between InsP6 degradation and relative abundance of phosphatase-producing strains. Functional predictions supported influences of microbiota on InsP6 degradation. The extent of such effects remains to be clarified. InsP6 degradation may also be influenced by variation of pH caused by dietary concentration and solubility of the Ca in the feed.

Genetic regulation and heritability of miRNA and mRNA expression link to phosphorus utilization and gut microbiome.

Improved utilization of phytates and mineral phosphorus (P) in monogastric animals contributes significantly to preserving the finite resource of mineral P and mitigating environmental pollution. In order to identify pathways and to prioritize candidate genes related to P utilization (PU), the genomic heritability of 77 and 80 trait-dependent expressed miRNAs and mRNAs in 482 Japanese quail were estimated and eQTL (expression quantitative trait loci) were detected. In total, 104 miR-eQTL (microRNA expression quantitative traits loci) were associated with SNP markers (false discovery rate less than 10%) including 41 eQTL of eight miRNAs. Similarly, 944 mRNA-eQTL were identified at the 5% False discovery rate threshold, with 573 being cis-eQTL of 36 mRNAs. High heritabilities of miRNA and mRNA expression coincide with highly significant eQTL. Integration of phenotypic data with transcriptome and microbiome data of the same animals revealed genetic regulated mRNA and miRNA transcripts (SMAD3, CAV1, ENNPP6, ATP2B4, miR-148a-3p, miR-146b-5p, miR-16-5p, miR-194, miR-215-5p, miR-199-3p, miR-1388a-3p) and microbes (Candidatus Arthromitus, Enterococcus) that are associated with PU. The results reveal novel insights into the role of mRNAs and miRNAs in host gut tissue functions, which are involved in PU and other related traits, in terms of the genetic regulation and inheritance of their expression and in association with microbiota components.

Citrulline supplementation attenuates the development of non-alcoholic steatohepatitis in female mice through mechanisms involving intestinal arginase.

Non-alcoholic fatty liver disease (NAFLD) is by now the most prevalent liver disease worldwide. The non-proteogenic amino acid l-citrulline (L-Cit) has been shown to protect mice from the development of NAFLD. Here, we aimed to further assess if L-Cit also attenuates the progression of a pre-existing diet-induced NAFLD and to determine molecular mechanisms involved. Female C57BL/6J mice were either fed a liquid fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C) for 8 weeks to induce early stages of NASH followed by 5 more weeks with either FFC-feeding +/- 2.5 g L-Cit/kg bw or C-feeding. In addition, female C57BL/6J mice were either pair-fed a FFC +/- 2.5 g L-Cit/kg bw +/- 0.01 g/kg bw i.p. N(ω)-hydroxy-nor-l-arginine (NOHA) or C diet for 8 weeks. The protective effects of supplementing L-Cit on the progression of a pre-existing NAFLD were associated with an attenuation of 1) the increased translocation of bacterial endotoxin and 2) the loss of tight junction proteins as well as 3) arginase activity in small intestinal tissue, while no marked changes in intestinal microbiota composition were prevalent in small intestine. Treatment of mice with the arginase inhibitor NOHA abolished the protective effects of L-Cit on diet-induced NAFLD. Our results suggest that the protective effects of L-Cit on the development and progression of NAFLD are related to alterations of intestinal arginase activity and intestinal permeability.

Phytate degradation, myo-inositol release, and utilization of phosphorus and calcium by two strains of laying hens in five production periods.

The objective of this study was to compare 2 laying hen strains in 5 production periods regarding phytase activity, phytate (InsP6) degradation, and myo-inositol (MI) release in the digestive tract and phosphorus (P) and calcium (Ca) utilization. One offspring of 10 nonrelated roosters per strain (Lohmann Brown-classic (LB) or Lohmann LSL-classic (LSL)) was placed in one of 20 metabolic units in a completely randomized block design in week 8, 14, 22, 28, and 58 of life. All hens were fed the same corn and soybean meal-based diet at one time, but the diet composition was adjusted to the requirements in the respective period. For 4 consecutive days, excreta were collected quantitatively at 24-hour intervals. In week 10, 16, 24, 30, and 60, the blood plasma, digesta of crop, gizzard, jejunum, ileum, and ceca, and mucosa of the jejunum was collected. The concentration of inorganic P in the blood plasma was higher in LB than in LSL hens (P = 0.026). Plasma Ca concentrations increased with each period (P < 0.001) in both strains. In jejunum digesta, the MI concentration did not differ between strains, but InsP6 concentration was higher in LB than in LSL hens (P = 0.002) and the highest in week 30 and 60. Total phosphatase and phytase activities were higher in LB than in LSL hens (P ≤ 0.009). Period effects were also significant for these enzymes. Concentrations of some constituents of the cecal content were different between the strains. The MI concentration in the egg albumen and yolk was higher in LB than in LSL hens. Differences in InsP6- and MI-related metabolism of the 2 hen strains existed. These differences were partly dependent of the period. Especially, week 24 was a period of remarkable change of metabolism. Great differences also existed among individuals, making it worth to have a closer look at the metabolism of individuals in addition to evaluating treatment means. Further studies on metabolic, genetic, and microbiome level may help explain these differences.

Identification of the Key Molecular Drivers of Phosphorus Utilization Based on Host miRNA-mRNA and Gut Microbiome Interactions.

Phosphorus is an essential mineral for all living organisms and a limited resource worldwide. Variation and heritability of phosphorus utilization (PU) traits were observed, indicating the general possibility of improvement. Molecular mechanisms of PU, including host and microbial effects, are still poorly understood. The most promising molecules that interact between the microbiome and host are microRNAs. Japanese quail representing extremes for PU were selected from an F2 population for miRNA profiling of the ileal tissue and subsequent association with mRNA and microbial data of the same animals. Sixty-nine differentially expressed miRNAs were found, including 21 novel and 48 known miRNAs. Combining miRNAs and mRNAs based on correlated expression and target prediction revealed enrichment of transcripts in functional pathways involved in phosphate or bone metabolism such as RAN, estrogen receptor and Wnt signaling, and immune pathways. Out of 55 genera of microbiota, seven were found to be differentially abundant between PU groups. The study reveals molecular interactions occurring in the gut of quail which represent extremes for PU including miRNA-16-5p, miR-142b-5p, miR-148a-3p, CTDSP1, SMAD3, IGSF10, Bacteroides, and Alistipes as key indicators due to their trait-dependent differential expression and occurrence as hub-members of the network of molecular drivers of PU.

Microbial and Functional Profile of the Ceca from Laying Hens Affected by Feeding Prebiotics, Probiotics, and Synbiotics.

Diet has an essential influence in the establishment of the cecum microbial communities in poultry, so its supplementation with safe additives, such as probiotics, prebiotics, and synbiotics might improve animal health and performance. This study showed the ceca microbiome modulations of laying hens, after feeding with dry whey powder as prebiotics, Pediococcus acidilactici as probiotics, and the combination of both as synbiotics. A clear grouping of the samples induced per diet was observed (p < 0.05). Operational taxonomic units (OTUs) identified as Olsenella spp., and Lactobacillus crispatus increased their abundance in prebiotic and synbiotic treatments. A core of the main functions was shared between all metagenomes (45.5%), although the genes encoding for the metabolism of butanoate, propanoate, inositol phosphate, and galactose were more abundant in the prebiotic diet. The results indicated that dietary induced-changes in microbial composition did not imply a disturbance in the principal biological roles, while the specific functions were affected.

Impact of coccidiostat and phytase supplementation on gut microbiota composition and phytate degradation in broiler chickens.

BACKGROUND: There is good evidence for a substantial endogenous phytase activity originating from the epithelial tissue or the microbiota resident in the digestive tract of broiler chickens. However, ionophore coccidiostats, which are frequently used as feed additives in broiler diets to prevent coccidiosis, might affect the bacterial composition and the abundance of phytase producers in the gastrointestinal tract. The aim of the present study was to investigate whether supplementation of a frequently used mixture of the coccidiostats Narasin and Nicarbazin alone or together with a phytase affects microbiota composition of the digestive tract of broiler chickens, characteristics of phytate breakdown in crop and terminal ileum, and precaecal phosphorus and crude protein digestibility. RESULTS: Large differences in the microbial composition and diversity were detected between the treatments with and without coccidiostat supplementation. Disappearance of myo-inositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate) (InsP6) in the digestive tract, precaecal P digestibility, inorganic P in blood serum, and the concentration of inositol phosphate isomers in the crop and ileum digesta were significantly affected by phytase supplementation, but not by coccidiostat supplementation. Crude protein digestibility was increased by coccidiostat supplementation when more phosphate was available. Neither microbial abundance and diversity nor any other trait measured at the end of the experiment was affected by coccidiostat when it was only supplemented from day 1 to 10 of age. CONCLUSIONS: The coccidiostats used herein had large effects on overall microbiota composition of the digestive tract. The coccidiostats did not seem to affect endogenous or exogenous phytase activity up to the terminal ileum of broiler chickens. The effects of phytase on growth, phosphorus digestibility, and myo-inositol release were not altered by the presence of the coccidiostats. The effects of phytase and coccidiostats on nutrient digestibility can be of significant relevance for phosphorus and protein-reduced feeding concepts if confirmed in further experiments.

High phosphorus intake and gut-related parameters - results of a randomized placebo-controlled human intervention study.

BACKGROUND: In recent years, high phosphate intakes were discussed critically. In the small intestine, a part of the ingested phosphate and calcium precipitates to amorphous calcium phosphate (ACP), which in turn can precipitate other intestinal substances, thus leading to a beneficial modulation of the intestinal environment. Therefore, we analysed faecal samples obtained from a human intervention study regarding gut-related parameters. METHODS: Sixty-two healthy subjects (men, n = 30; women, n = 32) completed the double-blind, placebo-controlled and parallel designed study (mean age: 29 ± 7 years; mean BMI: 24 ± 3 kg/m2). Supplements were monosodium phosphate and calcium carbonate. During the first 2 weeks, all groups consumed a placebo sherbet powder, and afterwards a sherbet powder for 8 weeks according to the intervention group: P1000/Ca0 (1000 mg/d phosphorus), P1000/Ca500 (1000 mg/d phosphorus and 500 mg/d calcium) and P1000/Ca1000 (1000 mg/d phosphorus and 1000 mg/d calcium). After the placebo period and after 8 weeks of intervention faecal collections took place. We determined in faeces: short-chain fatty acids (SCFA) and fat as well as the composition of the microbiome (subgroup) and cyto- and genotoxicity of faecal water (FW). By questionnaire evaluation we examined tolerability of the used phosphorus supplement. RESULTS: Faecal fat concentrations did not change significantly due to the interventions. Concentrations of faecal total SCFA and acetate were significantly higher after 8 weeks of P1000/Ca500 supplementation compared to the P1000/Ca0 supplementation. In men, faecal total SCFA and acetate concentrations were significantly higher after 8 weeks in the P1000/Ca1000 group compared to the P1000/Ca0 one. None of the interventions markedly affected cyto- and genotoxic activity of FW. Men of the P1000/Ca1000 intervention had a significantly different gut microbial community compared to the men of the P1000/Ca0 and P1000/Ca500 ones. The genus Clostridium XVIII was significantly more abundant in men of the P1000/Ca1000 intervention group compared to the other groups. Supplementations did not cause increased intestinal distress. CONCLUSIONS: The used high phosphorus diet did not influence cyto- and genotoxicity of FW and the concentrations of faecal fat independent of calcium intake. Our study provides first hints for a potential phosphorus-induced modulation of the gut community and the faecal total SCFA content. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov as NCT02095392 .

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.

Insights into Broilers' Gut Microbiota Fed with Phosphorus, Calcium, and Phytase Supplemented Diets.

Phytase supplementation in broiler diets is a common practice to improve phosphorus (P) availability and to reduce P loss by excretion. An enhanced P availability, and its concomitant supplementation with calcium (Ca), can affect the structure of the microbial community in the digestive tract of broiler chickens. Here, we aim to distinguish the effects of mineral P, Ca, and phytase on the composition of microbial communities present in the content and the mucosa layer of the gastrointestinal tract (GIT) of broiler chickens. Significant differences were observed between digesta and mucosa samples for the GIT sections studied (p = 0.001). The analyses of 56 individual birds showed a high microbial composition variability within the replicates of the same diet. The average similarity within replicates of digesta and mucosa samples across all diets ranged from 29 to 82% in crop, 19-49% in ileum, and 17-39% in caeca. Broilers fed with a diet only supplemented with Ca had the lowest body weight gain and feed conversion values while diets supplemented with P showed the best performance results. An effect of each diet on crop mucosa samples was observed, however, similar results were not obtained from digesta samples. Microbial communities colonizing the ileum mucosa samples were affected by P supplementation. Caeca-derived samples showed the highest microbial diversity when compared to the other GIT sections and the most prominent phylotypes were related to genus Faecalibacterium and Pseudoflavonifractor, known for their influence on gut health and as butyrate producers. Lower microbial diversity in crop digesta was linked to lower growth performance of birds fed with a diet only supplemented with Ca. Each diet affected microbial communities within individual sections, however, no diet showed a comprehensive effect across all GIT sections, which can primarily be attributed to the great variability among replicates. The substantial community differences between digesta and mucosa derived samples indicate that both habitats have to be considered when the influence of diet on the gut microbiota, broiler growth performance, and animal health is investigated.