The objective was to characterise degradation of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) and formation of inositol phosphate (InsP) isomers in different segments of the broiler digestive tract. Influence of an Aspergillus niger (PhyA) and two Escherichia coli-derived (PhyE1 and PhyE2) phytases was also investigated. A total of 600 16-d-old broilers were allocated to forty floor pens (ten pens per treatment). Low-P (5·2 g/kg DM) maize–soyabean meal-based diets were fed without (basal diet; BD) or with a phytase added. On day 25, digesta from different digestive tract segments were pooled per segment on a pen-basis, freeze-dried and analysed for P, InsP isomers and the marker TiO2. InsP6 degradation until the lower ileum (74 %) in BD-fed birds showed a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP patterns in different gut segments suggested the involvement of phosphatases of different origin. Supplemented phytases increased InsP6 hydrolysis in the crop (P < 0·01) but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time. In the intestinal segments, specifically formed InsP4–5 isomers of supplemented phytases were still present, indicating further activity of these enzymes. Myo-inositol tetrakisphosphate (InsP4) accumulation differed between PhyE1 and PhyE2 compared with PhyA in the anterior segments of the gut (P < 0·01). Thus, the hydrolytic cleavage of the first phosphate group is not the only limiting step in phytate degradation in broilers.

Phytate breakdown in the digestive tract of broilers is affected by supplements of mineral phosphorus (P) and phytase with unknown interactions between the 2 factors. It was the objective to study phytate hydrolysis and the presence of inositol phosphate isomers (InsPs) as affected by supplements of mineral P and phytase in the small intestine of broilers. Fifteen-day old broilers were assigned to 48 pens of 20 broilers each (n = 8 pens/treatment). Two low-P corn-soybean meal-based diets without (BD-; 4.4 g P/kg dry matter) or with monocalcium phosphate (MCP; BD+; 5.2 g P/kg dry matter) were supplied without or with added phytase at 500 or 12,500 FTU/kg. On d 24, digesta from the duodenum/jejunum and lower ileum was pooled per segment on a by-pen basis, freeze-dried, and analyzed for P, InsPs, and the marker TiO2. Another 180 broilers (n = 6 pens/treatment, 10 birds each) were fed the 3 BD+ diets from d 1 to 21 to assess the influence of supplemented phytase on tibia mineralization and strength. Significant interactions between MCP and phytase supplements on myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) hydrolysis (duodenum/jejunum: P ≤ 0.001; ileum: P = 0.004) and level of specific lower InsPs were detected. Supplementation with 12,500 FTU/kg phytase resulted in 92% InsP6 hydrolysis and strong degradation of InsP5. This treatment resulted in higher P net absorption, affirmed by higher BW gain, tibia strength, and mineralization compared to treatments without or with 500 FTU/kg phytase (P ≤ 0.05). MCP supplementation reduced the degradation of InsP6 and specific lower InsPs in birds fed diets without or with 500 FTU/kg of phytase (P ≤ 0.05), but did not reduce InsP6 hydrolysis or degradation of InsP5 at the high phytase dose. Effects of added MCP on phytase efficacy depend on the dose of supplemented phytase. Differences in the concentrations of lower InsPs indicated that the initial step of InsP6 hydrolysis is not the only catabolic step that is influenced by MCP or phytase levels.

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.

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.

Dietary phosphorus (P) is known as a main modulator of phosphate (Pi) transporter expression. The effect of supplemented mineral P with or without phytase on protein expression of two sodium-dependent Pi (NaPi) transporters and a calcium channel was studied in the small intestine of broilers. Thirty-six broilers were randomly assigned to six different diets at 15 days of age. Two levels of total P (tP, adjusted by monocalcium phosphate (MCP) supplementation), 0.39% (BD-) and 0.47% (BD+) were fed until day 25; and at each tP level, three levels of phytase were used with 0, 500, and 12,500 FTU/kg of an E. coli phytase. Mucosa samples from jejunum and ileum were taken and apical membranes were isolated by MgCl2 precipitation. Protein expression of NaPi IIb, NaPi type III (PiT1) and the calcium channel TRPV6 were semiquantitatively measured by Western blotting and jejunal mucosal phytase activity by measurement of Pi release. The jejunal NaPi IIb transporter was expressed with two distinct bands, which were modulated differently by diet. NaPi IIb Band1 increased (P < 0.05) and Band2 decreased (P < 0.05) with phytase supplementation but was not affected by MCP supplementation. This inverse modulation of Band1 and Band2 was significantly related to the amount of net absorbed P with higher expression of Band1 at higher amounts of net absorbed P. In addition, a second Pi transporter, PiT1, was detected in which ileal expression decreased (P < 0.05) in response to higher phytase supplementation. The expression of the calcium channel TRPV6 was increased in BD+ groups. A trend for an interaction between MCP and phytase supplementation on mucosal phytase activity was observed (P = 0.079) with a decrease in activity when BD+ with 12,500 FTU/kg phytase was fed. Chicken intestinal epithelial cells responded to dietary supplemented phytase and MCP by changing the Pi transporter expression in apical membranes. In conclusion, availability of Pi is most likely the key modulator of transporter protein expression. However, a contribution of lower inositol phosphates generated by phytases and other phosphatases may also be relevant.