The initial group weight at the beginning of feeding the experimental diets, on day 16 of life, was similar for all treatments, at 9,604 g/pen (standard deviation = 15 g/pen, P = 0.183). The experimental period ended on day 21 and day 22 post-hatch for half of the replicate pens of each treatment. Mortality was low (0.6% of all animals) and not related to any treatment (six cases in six different treatments).
The average daily gain (ADG) and average daily feed intake (ADFI) during the experimental period were lower compared to other treatments when the Ca level was high and no phytase was supplemented (P < 0.001; Figure 1, Table S1). The ADFI was significantly lower for Ca-formate compared to CaCO3 and CaCO3+formic acid (P = 0.002). Phytase supplementation increased the gain to feed ratio (G:F) by 0.04 g/g (P < 0.001). Further effects on growth performance were not significant.
pH in the digestive tract
The contents of crop, gizzard, and posterior small intestine were obtained immediately after slaughter of the birds. In the crop content, the highest pH of 5.5 was observed for CaCO3. Crop pH was decreased by 0.6 and 0.3 units for CaCO3+formic acid and Ca-formate, respectively, compared to CaCO3 (P < 0.001; Figure 2, Table S2). With a 0.1-unit increase, crop pH was marginally but significantly higher for the high compared to the low Ca level. In the gizzard, phytase supplementation increased pH by 0.2 units (P < 0.001) and decreased pH by 0.1 units (P = 0.005) at the low and high Ca level, respectively. In the ileum, phytase supplementation increased pH by 0.8 units at the high Ca level (P < 0.001) but had no significant effect at the low Ca level.
In content of both the crop and the ileum, mainly Lactobacillus species, including L. johnsonii, L. crispatus, L. reuteri, L. gallinarum, and L. vaginalis, were identified (Figure 3 and Figure 4). Streptococcus alactolyticus was highly abundant in the crop and the ileum for diets with CaCO3 at the low Ca level, irrespective of phytase supplementation. This was reflected by a high similarity between CaCO3 treatments at the low Ca level and a separation of these treatments from the other diets in a cluster analysis (Figure S1). Permutational multivariate analysis of variance (PERMANOVA) analyses (Table S3) revealed that the microbial communities in the crop and the ileum were significantly affected by all the main effects (P ≤ 0.034), with no interaction being significant. The microbial community in birds receiving CaCO3 differed significantly in both sections of the digestive tract compared to CaCO3+formic acid or Ca-formate (P ≤ 0.005).
In the crop content, between 34% and 53% of the detected relative abundance of microorganisms corresponded to operative taxonomic unit (OTU) 1 (L. johnsonii) and between 11% and 29% to OTU2 (L. gallinarum). We conducted analyses of variance (ANOVA) to get information on treatment effects on single OTUs being aware that explanatory power is partly impinged by physiological interrelationships between microorganisms. Abundance of OTU1 (L. johnsonii) was higher for Ca-formate when phytase was supplemented (P ≤ 0.050) (Table S4). The abundance of OTU1 increased for Ca-formate at the high Ca level. CaCO3 increased the abundance of the OTUs assigned to L. reuteri (OTU9, OTU11, OTU15, and OTU19, P ≤ 0.001). The high Ca level led to an increased abundance of OTU4 (P = 0.005), OTU9 (P = 0.036), and OTU11 (P < 0.001), which were assigned to L. reuteri. Phytase supplementation increased the abundance of OTU9, OTU11, and OTU13 (L. reuteri). Further significant influences on OTUs assigned to L. vaginalis, L. gallinarum, S. alactolyticus, Gallibacterium sp., Unc. Bacterium, and Unc. Firmicutes are shown in Table S4 and Table S5. Crop pH was positively correlated with seven out of nine OTUs assigned to L. reuteri (P ≤ 0.027, Table S6, Figure S2). Similar to the crop, the most abundant OTUs in the ileum were OTU1 (L. johnsonii, 24–46%) and OTU2 (L. gallinarum, 15–41%). Phytase supplementation increased the abundance of OTU1 (P = 0.040) and OTU2 (P = 0.015). The abundance of OTU2 was increased in the ileum of chicken fed with the high Ca level diets (P = 0.009). The abundance of OTUs assigned to L. reuteri (OTU6, OTU15, and OTU18, P ≤ 0.049) were decreased for Ca-formate and the abundance of OTU19 (L. reuteri, P = 0.049) was increased for CaCO3.
Six significant correlations (P ≤ 0.038) with OTUs assigned to L. johnsonii, L. gallinarum, and L. reuteri were determined for InsP6 concentration in the ileum, pc P digestibility, and pc InsP6 disappearance (OTU1, OTU2, OTU4, OTU9, OTU11, and OTU13) (Table S7, Figure S3). Correlations with OTUs assigned to Unc. Firmicutes, L. reuteri, S. alactolyticus, and Gallibacterium sp. were significant in five cases for pH in the ileum (OTU7, OTU9, OTU10, OTU11, and OTU23). Correlations with concentrations of myo-inositol in the ileum were significant for OTUs assigned to L. johnsonii, L. gallinarum, and L. reuteri (OTU1, OTU2, OTU9, OTU11, and OTU13).
The broad classification hierarchy of KEGG pathways of functions showed that same P-related pathways were significantly influenced in the crop and the ileum (Figure 5, Table S8). No interaction related to genes connected to InsP metabolism was significant in the crop and the ileum. In the crop, genes encoding for InsP metabolism were higher in the CaCO3 than in the CaCO3+formic acid and Ca-formate treatments (P ≤ 0.001) and more abundant at the low than at the high Ca level (P = 0.007). In the ileum, the abundance of genes related to InsP metabolism was lower in the Ca-formate treatment than in the CaCO3 and CaCO3+formic acid treatments (P ≤ 0.011) and not influenced by Ca level. Phytase supplementation had no effect on InsP metabolism pathways in the crop and the ileum. Other significantly influenced pathways were mineral absorption, the phosphotransferase system, the phosphatidylinositol signaling system, and phosphonate and phosphinate metabolism in the crop and the ileum. Phytase supplementation decreased the abundances of genes related to the phosphonate and phosphinate metabolism (P = 0.048) in the crop and increased the abundances of genes related to the phosphatidylinositol signaling system in the ileum (P = 0.031). The other pathways were influenced by acidification, Ca level, or the two-way interaction between acidification and Ca level with no apparent pattern in changes.
Within the category of InsP metabolism, increasing the Ca level reduced abundance of genes coding for myo-inositol-1(or 4)-monophosphatase in the CaCO3 and Ca-formate treatments (P ≤ 0.018), but not in the CaCO3+formic acid treatment (P = 0.520) in the crop (Figure 6, Table S9). In the ileum, myo-inositol-1(or 4)-monophosphatase was increased by phytase supplementation (P = 0.040). Increasing dietary Ca had no effect on myo-inositol-1(or 4)-monophosphatase in the CaCO3 and CaCO3+formic acid treatments (P ≥ 0.269) and decreased myo-inositol-1(or 4)-monophosphatase in the Ca-formate treatment (P = 0.008). Myo-inositol-1-phosphate synthase coding genes were lower in the CaCO3+formic acid and Ca-formate treatments than in the CaCO3 treatment in the ileum (P ≤ 0.001). Other genes annotated to InsP and myo-inositol degradation in the KEGG database were not influenced by the treatments used in this study.
InsP6 disappearance and prececal digestibility of P and Ca
Contents of crop, gizzard, and ileum were analyzed for InsP6 and degradation products, P, Ca, and titanium dioxide. InsP6 disappearance and mineral digestibility were calculated using titanium dioxide as the undigestible reference. In the crop, Ca level did not affect InsP6 disappearance (P = 0.536) when no phytase was supplemented (Figure 7; Table S10). At the high Ca level, the effects of phytase supplementation on InsP6 disappearance in the crop increased by 6 percentage points (pp) to 37 % (P = 0.025). Phytase supplementation increased InsP6 disappearance in the CaCO3 treatments with and without formic acid by 20 and 37 pp, respectively (P < 0.001), but not in the Ca-formate treatment (P = 0.090).
Phytase supplementation increased pc InsP6 disappearance (P < 0.001) to approximately 80 %, irrespective of the dietary Ca level. Without phytase supplementation, pc InsP6 disappearance was 9 pp higher for the low compared to the high Ca level (P < 0.001). Ca level had no effect on pc InsP6 disappearance for CaCO3+formic acid, but the high Ca level decreased pc InsP6 disappearance by 4 pp for CaCO3 (P = 0.047) and by 10 pp for Ca-formate (P = 0.016). Increasing dietary Ca decreased pc P digestibility. This effect was more pronounced for Ca-formate, with 12 pp (P < 0.001), than for CaCO3 and CaCO3+formic acid, with 7 pp each (P < 0.001). The three-way interaction was significant for pc Ca digestibility (P = 0.012, Figure 8). The pc Ca digestibility in CaCO3 and CaCO3-formate was increased by phytase supplementation at the low Ca level (P ≤ 0.012) but was not affected at the high Ca level (P ≥ 0.160). Phytase supplementation increased and decreased pc Ca digestibility for CaCO3+formic acid at the low and high Ca level, respectively (P ≤ 0.002).
Inositol phosphate isomer and myo-inositol concentrations
Significant interactions between acidification and phytase supplementation were detected for InsP6 and two InsP5 isomers in the crop (P < 0.001; Table S11). Decrease in concentrations of InsP6 and InsP5 isomers upon phytase supplementation was most pronounced in the CaCO3+formic acid treatment and less in the CaCO3 and the Ca-formate treatments. Myo-inositol concentrations in the crop were not affected.
In the gizzard, InsP6 concentrations were lower in diets with supplemented phytase compared to those without (P < 0.001, Table S12). Highest InsP6 concentrations were found in the Ca-formate treatment without phytase supplementation. Phytase supplementation decreased concentrations of InsP5 isomers below level of detection, while making some InsP4 and InsP3 isomers detectable. Phytase supplementation increased myo-inositol concentrations to a greater extent at the low Ca level compared to the high Ca level (P = 0.022). Acidification had no effect on myo-inositol concentrations.
In the ileum, treatment effects on InsP6 concentration were inverse to those on pc InsP6 disappearance (Table S13). Phytase supplementation had no effect on Ins(1,2,4,5,6)P5 concentration, but increased Ins(1,2,3,4,5)P5 concentrations and decreased Ins(1,2,3,4,6)P5 concentrations in most cases. Concentrations of InsP4 and InsP3x were increased upon phytase supplementation (P < 0.001). Ins(1,2,3,4,5)P5 concentrations were highest at the high Ca level for CaCO3+formic acid and both Ca levels for Ca-formate when phytase was supplemented. Concentrations of Ins(1,2,3,4)P4 and InsP3x were high when phytase was supplemented to CaCO3+formic acid and Ca-formate at the high Ca level. An increase in myo-inositol concentrations upon phytase supplementation was more pronounced in the ileum at the low compared to the high Ca level. The highest myo-inositol concentration was determined for CaCO3+formic acid (P ≤ 0.003) with no difference between CaCO3 and Ca-formate (P = 0.150).