4.1. Strain Effects
Strain effects were observed for several traits driving Ca and P metabolism along the digestive tract (Figure 1
). In the gizzard, LB hens had higher concentrations of InsP6
than did LSL hens. This might indicate a slower degradation of InsP6
in the anterior digestive tract by LB hens or a different passage rate of small particles through the gizzard between the strains. The significantly lower MI concentration in LSL hens fed Ca+ diets, which was not observed in LB hens, indicated that the LSL strain reacted more sensitively to different dietary Ca levels. A very high Ca level (23.3 g/kg) compared to a standard level (10.7 g Ca/kg) significantly increased the pH of crop and ileum content of broiler chickens [21
]. Thus, the contrasting reaction to the higher Ca level between the hen strains might be due to the pH changes affecting the strains differently or indicating a strain-specific Ca requirement in the diet.
Strain effects were also observed in the jejunum, with LB hens having higher concentrations of InsP6
, and Ins(1,2,3,4,5)P5
than LSL hens. This confirms the assumption of differences in InsP6
degradation between the two strains, as has already been shown by Abudabos [11
] with two other hen strains. Additionally, LB hens revealed significantly higher expression rates of two sodium/phosphate co-transporters (SLC20A1, SLC34A2) in the ileum than LSL hens. SLC34A2 plays an important role in the transcellular P transport mechanism along the intestine in rodents and human [22
] while its RNA expression was low in pigs [24
]. In laying hens, SLC34A2 showed a remarkable mRNA copy number in the ileum, suggesting that it is a relevant transcellular P transporter in the intestine of poultry. Correspondingly, SLC20A1 has been reported to be expressed in the mid and posterior intestine, including the ileum, underlining its contribution to P absorption in this segment [25
]. In view of the strain-specific intestinal capacity of phytate degradation in LB and LSL laying hens, the results of the ileal mineral P transporters suggest compensatory mechanisms in LB hens to balance P absorption.
Significantly higher concentrations of InsP6
, and Ins(1,2,3,4)P4
and lower concentrations of MI in the ceca of LB hens compared to that in LSL hens seem to confirm the assumption of Sommerfeld et al. [13
] that these strains might differ in their intestinal morphology, leading to different degrees of InsP degradation. What both strains had in common is that the InsP pattern became more diverse from the jejunum to the ileum (Figure 2
), and the relative proportion of MI and other InsP isomers in the sum of all InsPs changed along the digestive tract.
In the crop, one of the InsP5
isomers was affected by strain and the other by strain × P. However, the detected isomers likely originated from the feed (Table 1
) and the numerical differences were negligibly small. The concentrations of InsP6
and MI in the crop were not affected by the treatments, which is another indication for negligible phytase activity in the crop of laying hens, which has already been proposed by Marounek et al. [26
]. Phytase activity in the crop contents of the 38- or 47-week old laying hens was lower than that in the small intestine when measured as activity per segment [26
]. However, phytase activity in the crop content was identical to that of the small intestine of laying hens aged 47 weeks when measured per gram of digesta [26
]. Possibly, the relatively short retention time in the crop when feed is offered for ad libitum consumption [28
] leads to an insignificant degradation of InsP6
In this study, we also analyzed the concentrations of the indigestible marker TiO2
along the digestive tract and its total excretion. The TiO2
concentrations in the gizzard, jejunum, terminal ileum, and ceca were significantly higher in LB hens than in LSL hens, but the TiO2
recovery in the excreta was not (Supplementary Table S2
). This indicates that the passage rate of different constituents of the feed differed between the two strains. The recovery ranged from 88% to 95% in all treatments. In the study by Peddie et al. [30
], the daily recovery of TiO2
ranged between 76% and 145% in the excreta of individual laying hens over several days. It was speculated that these variations derived from methodological inaccuracies in sampling feed and excreta for individual birds over a short time. In the present study, the feed being in mash form possibly made it harder to collect than pellets when spilled and, coupled with the relatively low amount of excreta, led to some inaccuracies and the recovery deviating from 100%.
Not only differences between the strains were observed, but also remarkable differences between individuals within different treatments. Similar observations, given by the high standard deviations for all measured traits among individual hens, were found by Marounek et al. [26
4.2. Effects of Dietary Ca and P
The MI concentrations in the jejunum as well as in the ileum were lower when Ca+ diets were fed, and more Ca was present in the digesta. Possibly, Ca had a diminishing effect on endogenous phosphatases promoting the degradation of lower InsP to MI. Consistent with this assumption, a decreased activity of alkaline phosphatase and phytase in duodenal mucosa of broiler chickens with increased dietary Ca was found [31
]. In the present study, the MI concentration in the jejunum and the MI content in egg yolk were positively related (r = 0.872; p
= 0.005). However, no relationship between MI content in the egg yolk and MI concentration in the ileum was found. This is in line with the results of Sommerfeld et al. [13
], who found a similar relationship, which might indicate the jejunum being the main absorption site of MI in the digestive tract [33
With more Ca in the diet, hens ingested and excreted more Ca. A greater difference between Ca− and Ca+ in excretion than intake resulted in a lower percentage Ca utilization with Ca+. A higher Ca utilization at a lower dietary Ca concentration has already been shown by Rodehutscord et al. [34
]. It seems that the absorption and use efficiency of dietary Ca increases when less Ca is provided [35
]. Accordingly, the ileal Ca transporter CALB1 tended to be higher expressed with lower dietary Ca levels. Studies on chicken with reduced dietary Ca concentration showed a significant increase in duodenal CALB1 mRNA levels [8
] suggesting that CALB1 is part of the compensatory response to variable dietary Ca levels.
A higher dietary P level led to a higher P intake and a higher P excretion. The utilization of P, however, was not influenced by the dietary P level. Nonetheless, due to the complex regulatory mechanisms in mineral metabolism, including in the intestine, bone, and kidney [37
], the elevated dietary Ca contents decreased both P intake and P utilization, as additionally reflected in the numerically lower mRNA copy numbers of SLC34A2 between laying hens fed high and low dietary Ca contents. The lower P intake could be explained by the general lower feed intake due to the higher dietary Ca concentration. The P excretion was not affected by Ca, which means that, although less P reached the small intestine, the same amount was excreted. This could indicate that the excess Ca in the Ca+ treatments formed complexes with P that were then not absorbable and thus excreted, leading to a lower P utilization. Supporting evidence for a Ca−P−complex formation in the small intestine is provided by the P concentrations in the jejunum and ileum, which were not, as expected, lower in the P− treatments, but instead did not differ among treatments. The observation that P utilization was only influenced by dietary Ca level and not by P level is consistent with a previous study [34
] and might be an indication for Ca not only altering the P intake level, but also the absorption process of P.
Although effects of the diets on the utilization of Ca and P were observed in this study, there was no effect on the plasma Ca and P concentrations. No effects of increasing P levels (from 3.8 to 6.1 g P/kg feed) on plasma mineral concentrations were found by Boorman and Gunaratne [38
] feeding wheat-soybean meal-based diets to 37-week-old ISA brown hens. However, the plasma P was significantly increased with excess dietary P (13.9 g P/kg feed) and dependent of the time of oviposition and whether an egg was laid or not on the day of blood sampling. Jing et al. [39
] measured lower plasma P concentrations when Lohmann LSL-classic hens were fed corn-soybean meal-based diets containing 0.38% P compared to 0.63% P or higher. Plasma Ca concentrations did not differ among these treatments. As a proportion of the total Ca measured in the blood is bound and not available for eggshell formation [40
], it might be recommended to analyze ionized Ca in future experiments. One supportive previous finding is that of Frost and Roland [40
], who observed a dietary P effect on ionized Ca in the plasma, but not on total Ca.