M. Witzig

A note on sampling digesta from the ileum of broilers in phosphorus digestibility studies

The responses of broilers to increments of dietary P concentration in P retention and P prececal digestibility were studied in 2 experiments using Ross 308 broilers in their fourth week of age. The low-P basal diet was mainly based on corn, potato protein, and corn starch. Titanium dioxide was used as the indigestible marker. Monobasic calcium phosphate was added in 6 (experiment 1) or 5 (experiment 2) graded levels up to a maximum of 8.1 g of P/kg of the diet. Five (experiment 1) and 6 (experiment 2) replicated pens of 10 birds were used. Excreta were collected from trays underneath the pens. Birds were asphyxiated by carbon dioxide exposure and the ileum (from Meckels diverticulum to 2-cm anterior to the ileo-ceca-colonic junction) was dissected. Digesta was collected from the entire section (experiment 1) or from 3 subsections of equal lengths (experiment 2). Excretion of P increased linearly with increasing dietary P concentration up to a level of about 5.2 g of P/kg of the diet and increased nonlinearly with further increase in dietary P. In contrast, P flow in the ileum increased linearly over the entire range of P intake. Up to the level of 5.2 g of P/kg of the diet, P excretion was slightly lower than P flow in the ileum, but responses in P excretion and P flow in the ileum were similar, suggesting that P excretion with urine is very low and unaffected by P intake when the birds are supplied with P below their requirement. Between the 3 subsections of the ileum, calculated digestibility was significantly different for the basal diet and the diet with the 3 lowest levels of P supplementation. We concluded that the response in P prececal digestibility to increments in dietary P concentration is linear over a wider range of dietary P than the response in P retention. When digesta is collected from the ileum for determination of P prececal digestibility, the first third of the ileum should not be considered.

Effects of the composition of the basal diet on the evaluation of mineral phosphorus sources and interactions with phytate hydrolysis in broilers

The objectives of this study were to determine the availability of P from mineral phosphate sources by using different basal diets and measurement of P retention and prececal (pc) P digestibility as well as pc myo-inositol phosphate (InsP) degradation in broilers. Semi-synthetic and corn-soybean meal-based basal diets were used in experiment 1, and corn-based and wheat-based basal diets were used in experiment 2. Anhydrous monosodium phosphate (MSPa) or monocalcium phosphate monohydrate (MCPh) was supplemented to increment the P concentration by 0.05, 0.10, and 0.15% or by 0.075 and 0.150% in experiments 1 and 2, respectively. Titanium dioxide was used as an indigestible marker. Diets were pelleted through a 3-mm screen. In experiment 1, retention was measured based on total excreta collection from 20 to 24 d of age using 7 replicated birds per diet. In experiment 2, digesta from the terminal ileum was collected from 22-d-old broilers penned in groups of 19 with 5 replicated pens per diet. The P retention response to supplemented MSPa did not differ between the 2 basal diets in experiment 1. The response in pc P digestibility to MCPh supplements also did not differ between the 2 basal diets in experiment 2, as calculated by linear regression analysis. Hydrolysis of InsP6 measured on both the excreta and pc levels was high in the basal diets without a mineral P supplement. Mineral P supplementation significantly decreased (P < 0.05) InsP6 hydrolysis from the InsP-containing diets in both experiments. Thus, the choice of the basal diet did not affect the evaluation of the supplemented mineral P source. However, calculated values for mineral P sources need to be adjusted for the decline in hydrolysis of InsP contained in the basal diet that results from the P supplement.

Changes in Rumen Microbial Community Composition during Adaption to an In Vitro System and the Impact of Different Forages

This study examined ruminal microbial community composition alterations during initial adaption to and following incubation in a rumen simulation system (Rusitec) using grass or corn silage as substrates. Samples were collected from fermenter liquids at 0, 2, 4, 12, 24, and 48 h and from feed residues at 0, 24, and 48 h after initiation of incubation (period 1) and on day 13 (period 2). Microbial DNA was extracted and real-time qPCR was used to quantify differences in the abundance of protozoa, methanogens, total bacteria, Fibrobacter succinogenes, Ruminococcus albus, Ruminobacter amylophilus, Prevotella bryantii, Selenomonas ruminantium, and Clostridium aminophilum. We found that forage source and sampling time significantly influenced the ruminal microbial community. The gene copy numbers of most microbial species (except C. aminophilum) decreased in period 1; however, adaption continued through period 2 for several species. The addition of fresh substrate in period 2 led to increasing copy numbers of all microbial species during the first 2–4 h in the fermenter liquid except protozoa, which showed a postprandial decrease. Corn silage enhanced the growth of R. amylophilus and F. succinogenes, and grass silage enhanced R. albus, P. bryantii, and C. aminophilum. No effect of forage source was detected on total bacteria, protozoa, S. ruminantium, or methanogens or on total gas production, although grass silage enhanced methane production. This study showed that the Rusitec provides a stable system after an adaption phase that should last longer than 48 h, and that the forage source influenced several microbial species.

Comparison and evaluation of bone measurements for the assessment of mineral phosphorus sources in broilers

The main objective of this study was to compare different bone measurements in response to supplements of mineral P sources. Comparisons were also made with P retention and digestibility responses determined in a companion study and with blood inorganic phosphate (P(i)) responses. A corn-soybean meal-based basal diet was used (0.35% total P on DM basis). Anhydrous monosodium phosphate (MSP(a)) or anhydrous dibasic calcium phosphate (DCP(a)) was supplemented to increment the P concentration by 0.08%, 0.16%, or 0.24%. Each of the 7 diets was fed for 10 d starting 11 d (period 1) or 25 d posthatch (period 2). Bone ash and P were determined, and density criteria were measured using quantitative computed tomography. Responses were evaluated and compared based on linear regression analysis. In general, responses to MSP(a) had a greater slope than DCP(a) for all criteria studied. In period 1, differences between the slopes were significant (P < 0.05) for almost all bone criteria. In period 2, the slopes significantly differed for the amounts of ash and P of all bones studied, for tibia, tarsometatarus, and foot ash percentage, for total and cortical density of tibiae, but not for the other criteria. For the different bones, the ratio of slopes for MSP(a) and DCP(a) was very similar based on the amount of ash in both periods. Foot ash proved to be as sensitive as tibia ash for evaluation of mineral P sources in both periods. Ninety-four percent of the variance of the corticalis content based on quantitative computed tomography measurements could be explained by the amount of tibia ash in period 1. Blood serum P(i) and BW gain were not suitable for P evaluation. We concluded that the ranking of mineral P sources based on bone criteria differed from the ranking that was based on P retention or prececal digestibility. This underlines the need for developing a standard protocol of determination of available P in poultry.

Wheat and barley differently affect porcine intestinal microbiota

BACKGROUND Diet influences the porcine intestinal microbial ecosystem. Barrows were fitted with ileal T-cannulas to compare short-term effects of eight different wheat or barley genotypes and period-to-period effects on seven bacterial groups in ileal digesta and faeces by qPCR. RESULTS Within genotypes of wheat and barley, there was no difference (P > 0.05) in contents of analysed NSP, yet cereal types differed (P < 0.001) except for soluble arabinoxylans. Genotypes showed no effect on bacterial gene copy numbers. In ileal digesta of barley- compared to wheat-fed pigs, log10 copy numbers were lower (P < 0.05) for total eubacteria (9.6-9.8), Bacteroides-Prevotella-Porphyromonas (6.5-6.8), Clostridium cluster IV (6.7-6.9), and Roseburia spp. (6.6-7.2), while higher copy numbers were found for Lactobacillus spp. (9.4-8.8). Enterobacteriaceae (7.0-7.8) and Bifidobacterium spp. (7.0-7.7) were lower (P < 0.001) in faeces of barley compared to wheat-fed pigs. Ileal eubacteria, Clostridium cluster IV and Roseburia spp. linearly increased from period 1 to 8 for both cereals (P < 0.05). CONCLUSION Wheat and barley differently influence microbial composition particularly in the small intestine, with barley increasing the Lactobacillus spp.:Enterobacteriaceae ratio, underlining its potential to beneficially manipulate the intestinal microbial ecosystem.

Effect of the corn silage to grass silage ratio and feed particle size of diets for ruminants on the ruminal Bacteroides-Prevotella community in vitro

This study examined whether different corn silage to grass silage ratios in ruminant rations and different grinding levels of the feed affect the composition of the ruminal Bacteroides-Prevotella community in vitro. Three diets, composed of 10% soybean meal as well as of different corn silage and grass silage proportions, were ground through 1mm or 4mm screened sieves and incubated in a semi-continuous rumen simulation system. On day 14 of the incubation microbes were harvested by centrifugation from the liquid effluent of fermenter vessels. Microbial DNA was extracted for single strand conformation polymorphism (SSCP) analysis of 16S rRNA genes followed by sequencing of single SSCP bands. Fluorescence in situ hybridization (FISH) and real-time quantitative (q) PCR were used to quantify differences in the relative abundance of Bacteroides-Prevotella and Prevotella bryantii. SSCP profiles revealed a significant influence of the forage source as well as of the feed particle size on the community structure of the Bacteroides-Prevotella group. Different, phylogenetically distinct, so far uncultured Prevotella species were detected by sequence analysis of several treatment-dependent occurring SSCP bands indicating different nutritional requirements of these organisms for growth. No quantitative differences in the occurrence of Bacteroides-Prevotella-related species were detected between diets by FISH with probe BAC303. However, real-time qPCR data revealed a higher abundance of P. bryantii with increasing grass silage to corn silage ratio, thus again indicating changes within the community composition of the Bacteroides-Prevotella group. As P. bryantii possesses high proteolytic activity its higher abundance may have been caused by the higher contents of crude protein in the grass silage containing diets. To conclude, results of this study show an influence of the forage source on the ruminal community of Bacteroides-Prevotella. Furthermore, they suggest an effect of the feed particle size on this bacterial group.

Results of an international phosphorus digestibility ring test with broiler chickens

&NA; The objective of this ring test was to investigate the prececal phosphorus (P) digestibility of soybean meal (SBM) in broiler chickens using the trial protocol proposed by the Worlds Poultry Science Association. It was hypothesized that prececal P digestibility of SBM determined in the collaborating stations is similar. Three diets with different inclusion levels of SBM were mixed in a feed mill specialized in experimental diets and transported to 17 collaborating stations. Broiler chicks were raised on commercial starter diets according to station‐specific management routine. Then they were fed the experimental diets for a minimum of 5 d before content of the posterior half of the ileum was collected. A minimum of 6 experimental replicates per diet was used in each station. All diets and digesta samples were analyzed in the same laboratory. Diet, station, and their interaction significantly affected (P < 0.05) the prececal digestibility values of P and calcium of the diets. The prececal P digestibility of SBM was determined by linear regression and varied among stations from 19 to 51%, with significant differences among stations. In a subset of 4 stations, the prececal disappearance of myo‐inositol 1,2,3,4,5,6‐hexakis (dihydrogen phosphate)‐P; InsP6‐P) also was studied. The prececal InsP6‐P disappearance correlated well with the prececal P digestibility. We hypothesized that factors influencing InsP6 hydrolysis were main contributors to the variation in prececal P digestibility among stations. These factors were probably related to the feeding and housing conditions (floor pens or cages) of the birds in the pre‐experimental phase. Therefore, we suggest that the Worlds Poultry Science Association protocol for the determination of digestible P be should extended to the standardization of the pre‐experimental period. We also suggest that comparisons of P digestibility measurements among studies are made only with great caution until the protocol is more refined.

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

Influence of the maize silage to grass silage ratio and feed particle size of rations for ruminants on the community structure of ruminal Firmicutes in vitro

Aims:  To investigate the effect of the forage source and feed particle size (FPS) in ruminant rations on the composition of the ruminal Firmicutes community in vitro.

Variations of Phosphorous Accessibility Causing Changes in Microbiome Functions in the Gastrointestinal Tract of Chickens.

The chicken gastrointestinal tract (GIT) harbours a complex microbial community, involved in several physiological processes such as host immunomodulation and feed digestion. For the first time, the present study analysed dietary effects on the protein inventory of the microbiome in crop and ceca of broilers. We performed quantitative label-free metaproteomics by using 1-D-gel electrophoresis coupled with LC-MS/MS to identify the structural and functional changes triggered by diets supplied with varying amount of mineral phosphorous (P) and microbial phytase (MP). Phylogenetic assessment based on label-free quantification (LFQ) values of the proteins identified Lactobacillaceae as the major family in the crop section regardless of the diet, whereas proteins belonging to the family Veillonellaceae increased with the P supplementation. Within the ceca section, proteins of Bacteroidaceae were more abundant in the P-supplied diets, whereas proteins of Eubacteriaceae decreased with the P-addition. Proteins of the Ruminococcaceae increased with the amount of MP while proteins of Lactobacillaceae were more abundant in the MP-lacking diets. Classification of the identified proteins indicated a thriving microbial community in the case of P and MP supplementation, and stressed microbial community when no P and MP were supplied. Data are available via ProteomeXchange with identifier PXD003805.