The initial group weight at the beginning of feeding the experimental diets, on day 16 of the experiment, was similar for all treatments, at 9,604 g/pen (standard deviation = 15 g/pen, P = 0.183). Mortality during the experimental period was low (0.6% of all animals) and not related to any treatment (six cases in six different treatments).
Growth performance
The average daily gain (ADG) and average daily feed intake (ADFI) were lower compared to other treatments when the Ca level was high and no phytase was supplemented (P < 0.001, Figure 1). 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
In the crop, 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). 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.
Microbial community
In 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, 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 was higher for Ca-formate when phytase was supplemented (P ≤ 0.050). The abundance of OTU1 increased for Ca-formate at the high Ca level. OTU8 and OTU12 (both L. vaginalis) were more abundant in the CaCO3 treatment compared to the CaCO3+formic acid and Ca-formate treatments (P < 0.001). CaCO3 increased the abundance of the OTUs closely related to S. alactolyticus (OTU5, P = 0.006 and OTU10, P = 0.008), L. reuteri (OTU9, P = 0.001; OTU11, P = 0.003; OTU15, P < 0.001 and OTU19, P < 0.001), and Gallibacterium sp. (OTU23, P < 0.001). The high Ca level led to an increased abundance of OTU2, assigned to L. gallinarum (P = 0.007), and 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 (assigned to L. reuteri), and decreased the abundance of OTU2 (L. gallinarum). Crop pH was negatively correlated with the abundance of two out of three OTUs assigned to L. gallinarum (P ≤ 0.008) and positively correlated with seven out of nine OTUs assigned to L. reuteri (P ≤ 0.027, Table S5, 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) and L. vaginalis (OTU8, P < 0.001) were decreased for Ca-formate and the abundance of OTU19 and OTU10 (L. reuteri and S. alactolyticus, respectively, P ≤ 0.049) were increased for CaCO3. The abundances of OTU7, OTU8, and OTU20 were significantly affected by the interaction between Ca level and phytase supplementation (P < 0.049). Unclassified Firmicutes (OTU7) and unclassified Bacterium (OTU20) were less abundant in the ileum of chicken at the high Ca level without phytase supplementation. Supplementation of phytase leveled this effect. Abundance of OTU8 (L. vaginalis) increased upon an increase in Ca level, but not when phytase was supplemented.
Six significant correlations (P ≤ 0.038) with OTUs were determined for InsP6 concentration in the ileum, pc P digestibility, and pc InsP6 disappearance (OTU1, OTU2, OTU4, OTU9, OTU11, and OTU13) (Table S5, Figure S3). Correlations with OTUs were significant in five cases for pH in the ileum (OTU7, OTU9, OTU10, OTU11, and OTU23), for concentrations of myo-inositol (OTU1, OTU2, OTU9, OTU11, and OTU13), and for Ins(1,2,4,5,6)P5 (OTU2, OTU4, OTU8, OTU9, and OTU10) in the ileum.
Functional prediction
The broad classification hierarchy of KEGG pathways of functions resulted in a similar distribution for crop and ileum. For both sections, the most abundant pathways were assigned to the metabolism category (74–75%), followed by environmental information processing (8–9%), and genetic information processing (8–9 %).
The same P-related pathways were significantly influenced in the crop and the ileum (Figure 5). 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 significantly influenced by Ca level. Phytase supplementation had no significant effect on InsP metabolism 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 had a significant influence on the abundance of genes related to the phosphatidylinositol signaling system and phosphonate and phosphinate metabolism in the crop and in some treatments in the ileum, as indicated by a significant three-way interaction. The other pathways were significantly influenced by acidification, Ca level, or the two-way interaction between acidification and Ca level.
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). 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 significantly influenced by the treatments used in this study.
InsP6 disappearance and prececal digestibility of P and Ca
In the crop, phytase supplementation increased InsP6 disappearance in the CaCO3 treatments with and without formic acid by 20 and 37 percentage points (pp), respectively (P < 0.001), but not in the Ca-formate treatment (P = 0.090, Figure 7). Without phytase supplementation, Ca level did not affect InsP6 disappearance in the crop (P = 0.536). At the high Ca level, the effects of phytase supplementation on InsP6 disappearance in the crop increased by 6 pp to 37 % (P = 0.025).
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 significant 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 S9). 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 significantly affected.
In the gizzard, InsP6 concentrations were lower in diets with supplemented phytase compared to those without (P < 0.001, Table S10). 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 significant effect on myo-inositol concentrations.
In the ileum, treatment effects on InsP6 concentration were inverse to those on pc InsP6 disappearance (Table S11). Phytase supplementation had no significant effect on Ins(1,2,4,5,6)P5 concentration, but significantly 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).