Effect of limestone particle size and microbial phytase on phosphorus and calcium digestion kinetics along the gastrointestinal tract in laying hens.

1. This study assessed the effect of limestone particle size and microbial phytase incorporation on the fate of phosphorus (P) and calcium (Ca) along the gastrointestinal tract in 72 laying hens.2. Four experimental diets were formulated according to a 2 × 2 factorial arrangement to evaluate the effect of two coarse limestone (CL) inclusion. This included a mix (MIX) of 75% CL (2 - 4 mm) and 25% fine particles (FL, <0.5 mm) or 100% FL, in two different basal diets formulated without (MIX0 and FL0) or with 300 FTU of microbial phytase/kg (MIX300 and FL300).3. Contents of the crop, gizzard, duodenum, jejunum and ileum were collected to determine the mean retention time (MRT) of dry matter (DM), the recovery rate of Ca and P in each segment of the gastrointestinal tract and the apparent fractional digestibility coefficient (AD) of Ca and P in each intestinal segment.4. In hens fed FL, microbial phytase decreased the MRT of DM along the intestine (p < 0.05). In the crop and the gizzard, Ca recovery increased with MIX incorporation to a greater extent in hens fed without microbial phytase (p < 0.05). The mixed particle size incorporation decreased absorption kinetics of Ca in hens fed microbial phytase. The AD of P and the absorption kinetics of P were significantly decreased in hens receiving FL300, probably due to complex formation between Ca and phytic acid.5. This study showed that coarse limestone particles incorporation improved mineral utilisation along the digestive tract.

Effect of the physicochemical properties of zinc oxide sources on their bioavailability in broilers.

1. The bioavailability of a trace mineral source is related to its intestinal solubility (bioaccessibility), which in turn is determined by its physicochemical properties. It is still not clear which characteristics are more relevant in affecting solubility and bioavailability of mineral sources. Zinc oxide (ZnO) is a common feed additive used to supplement zinc in the diet of monogastric animals. However, different sources have shown variable responses on animal bioavailability.2. This study hypothesised that different sources of feed grade ZnO have various physicochemical features that lead to distinct bioavailability values. Feed grade ZnO samples collected from the feed industry worldwide were characterised for their physicochemical features and tested in broilers to allow bioavailability determined.3. A total of 135 male Cobb broiler chickens were fed a standard starter diet from day 1 after hatching up to d 7. At d 8, animals were allocated in individual cages and fed one of the following dietary treatments during 15 days: a basal diet with 23.5 ppm of zinc and seven test diets with supplemented ZnO or zinc sulphate (ZnSO4) at 6 or 12 ppm.4. Different sources of ZnO showed an effect of solubility in the stomach and supplementation influenced total Zn levels in the ileum. The bioavailability of the different sources varied from 49% to 160% in relation to ZnSO4. Aggregate size of particles seems to explain most of the variability in the bioavailability of the different sources tested in broilers. In conclusion, physicochemical properties of ZnO can partly explain the variability observed in terms of biological value.

Dietary cation-anion difference and day length have an effect on milk calcium content and bone accretion of dairy cows.

Milk and dairy products are an important source of Ca for humans. Recent studies have shown fluctuations in cow milk Ca content during the year in France, with high values in winter and with corn silage diets, and a decrease during May and June and with grass diets. The aim of this study was to identify the reasons for this seasonal decrease in milk Ca content by testing the effect of 2 levels of dietary cation-anion differences (DCAD; 0 mEq/kg of dry matter for DCAD 0 and 400 mEq/kg for DCAD 400) and 2 day lengths (8 h of light/d for short days: SD; and 16 h/d for long days: LD) on the Ca balances of dairy cows. The DCAD treatments were designed to mimic diets based either on corn silage or on herbage. The cows were only illuminated by solarium lights providing UVA and UVB. The trial was conducted according to 2 simultaneous replicates of a 4×4 Latin square design using 8 dairy cows averaging 103±44 d in milk with 4 periods of 14 d. Data were analyzed by ANOVA with a model including treatment, cow, and period effects. No significant interaction was found between day length and DCAD treatments. With DCAD 400 compared with DCAD 0, blood pH increased and plasma ionized Ca content decreased, whereas the plasma total Ca content did not differ between treatments. Milk Ca content, however, increased with DCAD 400 compared with DCAD 0, in relation to a decrease in the amount of Ca excreted in urine. The DCAD had no significant effect on protein and casein contents and DCAD 400 tended to decrease milk yield. This illustrates that the udder did not decrease Ca uptake from the blood at high DCAD even though DCAD 400 decreased the mammary availability of Ca by decreasing the proportion of blood ionized Ca. Milk Ca and casein contents were significantly lower with LD compared with SD, whereas day length had no effect on milk yield after 14 d of treatment. Bone accretion of cows increased when the Ca content of milk increased (i.e., with DCAD 400 compared with DCAD 0 and with SD compared with LD). This work suggests that long and sunny days could explain part of the seasonal decrease in milk Ca content in summer and refutes the hypothesis that low milk Ca contents at grazing could be due to the high DCAD of herbage.

Robustness to chronic heat stress in laying hens: a meta-analysis.

Chronic heat is a major stress factor in laying hens and many studies on the effect of heat stress have been published. It remains difficult, however, to draw general conclusions about the effect of chronic heat stress on performance and its relationship with genetic and environmental factors, as these studies have been done under varying experimental conditions and using various experimental designs. A meta-analysis enabled us to make a quantitative review of the results from 131 published papers. The relative effects of four factors (genotype, age, group size, and amplitude of temperature variation) and their interactions with temperature were analyzed for 13 traits. After pre-correcting the data for a random study effect, the best model for each trait was selected in a stepwise procedure based on its residual sum of squares. Shell strength, daily feed intake, egg mass, and hen-day egg production were found to be more sensitive to heat stress than the other traits as they dropped by 9.0 to 22.6% between thermo-neutrality (15 to 20°C) and heat stress (30 to 35°C) while yolk and albumen proportions or Haugh units showed nearly no variation with temperature (<1.2% between thermo-neutrality and heat stress). Many interactions (17) were found between temperature and one or more factors in the 13 traits studied here, which reinforces the interest of using a meta-analysis to summarize data from the literature. This study highlighted that the impact of heat stress in laying hens depends on the genotype, age, and group size, some of which have rarely been investigated.

Basmisanil, an α5-GABAAR negative allosteric modulator, produces rapid and sustained antidepressant-like responses in male mice.

There is a critical need for safer and better-tolerated alternatives to address the current limitations of antidepressant treatments for major depressive disorder. Recently, drugs targeting the GABA system via α5-containing GABAA receptors (α5-GABAAR) as negative allosteric modulators (α5-NAMs) have shown promise in alleviating stress-related behaviors in preclinical studies, suggesting that α5-NAMs may have translational relevance as novel antidepressant medications. Here, we evaluated the efficacy of Basmisanil, an α5-NAM that has been evaluated in Phase 2 clinical studies as a cognitive enhancer, in a battery of behavioral tests relevant to coping strategies, motivation, and aversion in male mice, along with plasma and brain pharmacokinetic measurements. Our findings reveal that Basmisanil induces dose-dependent rapid antidepressant-like responses in the forced swim test and sucrose splash test without promoting locomotor stimulating effects. Furthermore, Basmisanil elicits sustained behavioral responses in the female urine sniffing test and sucrose splash test, observed 24 h and 48 h post-treatment, respectively. Bioanalysis of plasma and brain samples confirms effective blood-brain barrier penetration by Basmisanil and extrapolation to previously published data suggest that effects were observed at doses (10 and 30 mg/kg i.p.) corresponding to relatively modest levels of α5-GABAAR occupancy (40-65 %). These results suggest that Basmisanil exhibits a combination of rapid and sustained antidepressant-like effects highlighting the potential of α5-NAMs as a novel therapeutic strategy for depression.

Effect of phytase and limestone particle size on mineral digestibility, performance, eggshell quality, and bone mineralization in laying hens.

The effect of microbial phytase and limestone particle size (LmPS) was assessed in Lohmann Tradition laying hens from 31 to 35 wk of age. Seventy-two hens were used in a completely randomized trial according to a 2 × 2 factorial arrangement with 2 levels of phytase/basal available P (aP); 0 FTU/kg with 0.30% aP or 300 FTU/kg with 0.15% aP, and 2 limestone particle sizes; fine particles (FL, <0.5 mm) or a mix (MIX) of 75% coarse limestone (CL, 2-4 mm) and 25% FL. Diets contained equivalent levels of Ca (3.5%), phytic P (PP; 0.18%), and aP (0.30%) considering the P equivalency of phytase. Thus, dietary treatments were FL0 and MIX0 without phytase, and FL300 and MIX300 with 300 FTU/kg phytase. Performance were recorded daily and eggshell quality (eggshell weight proportion, weight, thickness, and breaking strength) was measured weekly. At the end of the trial, bone parameters (tibia breaking strength, elasticity, and ash) and the apparent precaecal digestibility (APCD) of P and Ca were determined. No differences were observed between treatments in feed intake, FCR and bone parameters. Addition of MIX increased the eggshell proportion, weight and thickness in groups receiving no phytase (+6.5, +6.9, and +4.5%, respectively) while no effect was observed in groups receiving phytase (Phytase × LmPS, P < 0.05). In hens receiving FL, the APCD of P was lower in diets supplemented with phytase (-14 percentage points; Phytase × LmPS, P < 0.001). A higher phytate disappearance was observed in hens fed diets with phytase in combination with MIX (Phytase × LmPS, P = 0.005). Phytase and MIX together increased the APCD of Ca by 7.3 percentage points (Phytase × LmPS, P < 0.001). In conclusion, addition of CL could limit the formation of Ca-phytate complex thus improving the response of the birds to phytase compared to FL.

A first model of the fate of dietary calcium and phosphorus in broiler chickens.

To reduce P excretion and increase the sustainability of poultry farms, one needs to understand the mechanisms surrounding P metabolism and its close link with Ca metabolism to precisely predict the fate of dietary P and Ca and related requirements for birds. This study describes and evaluates a model developed to estimate the fate of Ca and P consumed by broilers. The Ca and P model relies on three modules: (1) digestion of Ca and P; (2) dynamics of Ca and P in soft tissue and feathers; and (3) dynamics of body ash. Exogenous phytase affects the availability of Ca and P; thus, to predict the absorption of those minerals, the model also accounts for the effect of phytase on Ca and P digestibility. We used a database to estimate the consequences of dietary Ca, P, and phytase over feed intake response. This study followed a four-step process: (1) Ca and P model development and its coupling with a growth broiler model; (2) model behavior assessment; (3) sensitivity analysis to identify the most influential parameters; and (4) external evaluation based on three databases. The proportion of P in body protein and the Ca to P ratio in bone are the most sensitive parameters of P deposition in soft tissue and bone, representing 91 and 99% of the total variation. The external evaluation results indicated that body water and protein had an overall mean square prediction error (rMSPE) of 7.22 and 12.3%, respectively. The prediction of body ash, Ca, and P had an rMSPE of 7.74, 11.0, and 6.56%, respectively, mostly errors of disturbances (72.5, 51.6, and 90.7%, respectively). The rMSPE for P balance was 13.3, 18.4, and 22.8%, respectively, for P retention, excretion, and retention coefficient, with respective errors due to disturbances of 69.1, 99.9, and 51.3%. We demonstrated a mechanistic model approach to predict the dietary effects of Ca and P on broiler chicken responses with low error, including detailed simulations to show the confidence level expected from the model outputs. Overall, this model predicts broilers' response to dietary Ca and P. The model could aid calculations to minimize P excretion and reduce the impact of broiler production on the environment. A model inversion is ongoing that will enable the calculation of Ca and P dietary quantities for a specific objective. This will simplify the use of the model and the feed formulation process.

Phosphorus utilization in finishing broiler chickens: effects of dietary calcium and microbial phytase.

A decrease in dietary P, especially in finishing broilers (21 to 38 d old), is a crucial issue in poultry production from an environmental and economic point of view. Nevertheless, P must be considered together with other dietary components such as Ca and microbial phytase. Different corn and soybean meal-based diets varying in Ca [low (LCa) 0.37, medium (MCa) 0.57, and high (HCa) 0.77%], and nonphytate P [nPP; low (LnPP) 0.18 and high (HnPP) 0.32%] content were tested with and without microbial phytase [0 or 500 phytase units (FTU)/kg]. Feed intake, BW gain, bone mineralization, and mineral retention were examined in 144 Ross PM3 broilers (22 to 38 d old) reared in individual cages. Growth performance was not significantly affected by the treatments. Nevertheless, a numerical decrease of ADG and ADFI was observed in HCa-LnPP and LCa-HnPP associated with an increase of feed conversion ratio. Decreased dietary Ca reduced tibia ash content (Ca, linear: P < 0.001; quadratic: P = 0.034) and tibia ash weight for the highest level of nPP (Ca × nPP; P = 0.035). In parallel, increasing dietary Ca reduced the flow of retained P (P = 0.022) but also tibia ash weight in LnPP diets (Ca × nPP; P = 0.035). The responses of the animals in terms of tibia ash content and P retention were improved by the addition of microbial phytase especially for the lowest P diets (nPP × phytase, P = 0.021 and P = 0.009; respectively). Phytase increased dry tibia weight, bone breaking strength, and tibia diameter in broilers fed the highest Ca diets (Ca × phytase; P < 0.05). We conclude that is possible to decrease P levels in finishing broilers, if the Ca content is appropriate. Nevertheless, decreasing the dietary P and Ca cannot allow a maximization of bone mineralization, but the optimal threshold remains to be determined.

Effect of limestone particle size on performance, eggshell quality, bone strength, and in vitro/in vivo solubility in laying hens: a meta-analysis approach.

Numerous publications over the past 5 decades have investigated the effect of limestone particle size (LmPS) on production performance, bone mineralization, and limestone solubilization in laying hens. Coarse limestone particles have been shown to improve eggshell quality and bone mineralization. However, there is a large variability of responses in birds to this factor, indicating the need to better quantify the effect of modulating factors related to coarse particles that could explain this variability. The objective of this meta-analysis was to study the impact of LmPS on the digestive and metabolic fate of Ca to optimize its utilization by laying hens. Fifty-eight papers published between 1971 and 2019, including 71 experiments were included in this study. Four categories of dependent variables were identified: Ca solubility, production performance, eggshell quality, and bone strength. Independent variables tested were LmPS and age. Results showed that the in vitro solubilization of limestone linearly decreased (P < 0.001; R² = 0.91) while in vivo solubilization linearly increased with LmPS (P < 0.001; R² = 0.91). Coarse limestone particles were retained longer in the gizzard (P < 0.001; R² = 0.60), inducing higher solubilization by gastric juices than fine limestone. LmPS showed no effect on production performance while all eggshell quality parameters increased with LmPS (P < 0.001; R² > 0.91): increasing specific gravity by 0.8%, eggshell thickness by 1.1%, and eggshell breaking strength by 3% when increasing from 0.15 mm to 1.5 mm. LmPS had an effect on tibia breaking strength dependently of age (Age × LmPS, P < 0.001; R² = 0.89): coarse limestone particles increased tibia breaking strength with aging compared to fine limestone particles. The current study renders it possible to quantify the effects of age and LmPS on eggshell quality and tibia breaking strength. This work showed an interaction between eggshell quality and bone strength and showed that LmPS increases bone strength in older laying hens.

Digestive tract measurements and histological adaptation in broiler lines divergently selected for digestive efficiency.

Two lines of broilers divergently selected for a high (D+) or a low (D-) AME(n) on a wheat-based diet were studied for morphological and histological characteristics of the digestive tract. A total of 630 birds of both lines were slaughtered after a 23-d feeding period. Digestive tract morphology and intestinal histology were investigated on a total of 24 birds to describe the consequences of divergent selection. Birds of the D+ line had 34% heavier gizzards (P < 0.001) and 22% heavier proventriculi than their D- counterparts. In contrast, intestines were 15 to 40% heavier in D- birds, mainly in the jejunum (P < 0.001) and ileum (P < 0.001). Intestinal segments were also longer (between 3 and 6%) in the D- birds. Intestinal villi were larger and longer in D- birds (P < 0.001), mainly in the jejunum (14 to 16%), and crypts were 10 to 15% deeper for the 3 intestinal segments in D- birds (P < 0.001). Muscle layers of the intestine were 17 to 24% thicker (P < 0.001) and goblet cells were 27 to 34% more numerous in the jejunum and ileum of D- birds (P = 0.027). This new characterization of the 2 lines shows that divergent selection based on AME(n) modified the morphology of the proventriculus and gizzard, suggesting greater activity of this compartment in D+ than in D- birds. Intestinal adaptation revealed by visceral organ weight and length and histological modifications in D- birds can be viewed as an attempt to compensate for the low functionality of the gastric area.

Phosphorus and calcium requirements for bone mineralisation of growing pigs predicted by mechanistic modelling.

Phosphorus (P) is an essential nutrient in livestock feed but can pollute waterways. In order for pig production to become less of a threat to the environment, excreta must contain as little P as possible or be efficiently used by plants. This must be achieved without decreasing the livestock performance. Phosphorus and calcium (Ca) deposition in the bones of growing pigs must be optimised without affecting the muscle gain. This requires precision feeding based on cutting-edge techniques of diet formulation throughout the animal growth phase. Modelling and data mining have become important tools in this quest. In this study, a mechanistic model taking into account the distribution of P between bone and soft tissues was compared to the established factorial models (INRA (Jondreville and Dourmad, 2005) and NRC (National Research Council, 2012)) that predict P (apparent total tract digestible, ATTD-P; or standardised total tract digestible, STTD-P) and Ca (total and STTD) requirements as a function of BW and protein deposition. The requirements for different bone mineralisation scenarios, namely, 100% and 85% of the genetic potential, were compared with these two models. Sobol indices were used to estimate the relative impact of growth-related parameters on mineral requirements at 30, 60 and 120 kg of BW. The INRA showed the highest value of ATTD-P requirement between 29 and 103 kg of BW (6%) and lower for lighter and higher BW. Similarly, the model for 85% bone mineralisation showed lower STTD-P requirement than NRC between 29 and 93 kg of BW (7%) and higher for lighter and higher BW. Contrary to other models, the Ca requirement of the proposed model is not fixed in relation to P. It increases from 95 kg of BW while the others decrease. The INRA showed the highest Ca requirements. The model Ca requirements for 100% bone mineralisation are higher than NRC from 20 to 38 kg of BW similar until 70 kg of BW and then higher again. For 85% objective, the model showed lower Ca requirements from 25 to 82 kg of BW and higher for lighter and higher BW. The potential Ca deposition in bones is the most sensitive parameter (84% to 100% of the variance) of both ATTD-P and Ca at 30, 60 and 120 kg. The second most sensitive parameter is the protein deposition, explaining 1% to 15% of the ATTD-P variance. Studies such as this one will help to usher in a new era of sustainable and eco-friendly livestock production.

Effects of dietary electrolyte balance and calcium supply on mineral and acid-base status of piglets fed a diversified diet.

Dietary electrolyte balance (dEB) is known to affect acid-base status and mineral metabolism, but is rarely considered in diet formulation for pigs. Yet, the use of a wide variety of local feedstuffs in Europe contributes to lowering the dEB and increasing the fibre content. Hence, mineral requirements may be modified and skeletal health affected. Therefore, the effects of a lower dEB and a higher dietary Ca level on acid-base balance and mineral status were assessed in young pigs fed a diversified diet. A total of twenty-four weaned pigs were fed a control moderate-dEB diet (C) or a diversified moderate-dEB (D), low-dEB (D-A) or low-dEB supplemented with Ca (D-CA) diet. Growth performance, venous blood gas and chemistry, urine pH, mineral balance and femur characteristics were determined. With an equivalent dEB compared with the C diet, the D diet caused an acidification of the urine and increased the excretion of P as a result of a higher dietary content of S. Low-grade metabolic acidosis occurred in piglets fed the D-A diet with changes at systemic and urine levels. A higher excretion of ammonia and P in urine was observed and some bone characteristics tended to be negatively affected. Ca supplementation partially counteracted the effects of low-grade acidosis. Urine excretion of P and ammonia was alleviated and bone characteristics improved. In conclusion, a higher Ca supply must be considered in more diversified diets to counteract the risk of evolving towards low-grade metabolic acidosis which can negatively affect bone.

Possible roles of parathyroid hormone, 1.25(OH)2D3, and fibroblast growth factor 23 on genes controlling calcium metabolism across different tissues of the laying hen.

This study provides an integrative description of candidate gene expression across tissues involved in calcium (Ca) metabolism during the egg laying cycle, using the well-defined model of Ca supply as fine or coarse particles of calcium carbonate (CaCO3). Plasma and tissue samples were collected from hens at the peak of laying at 0 to 1, 9 to 10, and 18 to 19 h postovulation (PO). After mRNA preparation from the parathyroid gland, medullary bone, liver, kidney, duodenum, and jejunum, gene expressions were quantified using RT-qPCR. The highest levels of parathyroid hormone (PTH) mRNA in the parathyroid gland (P < 0.05), and of the active form of vitamin D3 1.25(OH)2D3 in the plasma (P < 0.01) were observed at 18 to 19 h PO. During this active phase of eggshell formation, bone resorption was attested to high levels of plasma inorganic phosphorus (iP) and the receptor activation of nuclear factor-κB expression in the bone (P < 0.001 and P < 0.05, respectively). At this stage, 5 genes of the transcellular and the paracellular Ca absorption pathways in the intestine (P < 0.05) and the Ca channel transient receptor potential cation channel subfamily V member 5 (P < 0.05), involved in its reabsorption in the kidney, were overexpressed. At 0 to 1 h PO during the subsequent daylight period, 2 candidates of the transcellular and the paracellular Ca pathways (P < 0.05) remained at high levels in the intestine, while calbindin D 28K expression was the highest in the kidney (P < 0.05). As PTH mRNA and 1.25(OH)2D3 were low, bone accretion was likely active at this stage. The phosphaturic hormone fibroblast growth factor 23 (FGF23) was overexpressed at 18 to 19 h PO (P < 0.05) in the bone when plasma iP was high, which suggested a role in the subsequent reduction of P reabsorption in the kidney, as attested to the decreased expression of P cotransporters, leading to iP clearance from the plasma at 0 to 1 h PO (P < 0.05). The low levels of 1.25(OH)2D3 at this stage coincided with increased expression of the 24-hydroxylase gene in the kidney (P < 0.05). In hens fed fine particles of CaCO3, higher plasma levels of 1,25(OH)2D3 and higher expression of several genes involved in bone turnover reflected a stronger challenge to Ca homeostasis. Altogether, these data support the hypothesis that FGF23 could drive vitamin D metabolism in the laying hen, as previously documented in other species and explain the tight link between P and Ca metabolisms.

Early-life conditioning strategies to reduce dietary phosphorus in broilers: underlying mechanisms.

Chickens adapt to P and Ca restriction during the very first days of life by improving P utilisation efficiency. The present study was built to identify the mechanisms underlying this adaptive capacity, and to identify the optimal window of application of the restriction (depletion). A total of 1600 Cobb 500TM male broilers were used. During each phase (from age 0 to 4 d, 5 to 8 d, 9 to 18 d and 19 to 33 d), the animals received either a control diet (H) or a restricted diet (L) with reduced levels of non-phytate P (nPP) and Ca (between -14 and -25 % for both) with four dietary sequences: HHHH, HLHL, LHHL and LLHL. None of the feeding strategies affected growth. Tibia ash content at day 4 and 8 was impaired when the L diet was fed from 0 to 4 and 5 to 8 d, respectively (P = 0⋅038 and P = 0⋅005). Whatever the early restriction period or length between 0 and 8 d of age, the mineralisation delay was compensated by day 18. This was accompanied by an increased mRNA expression of the Ca transporter, CALB1, and an increased apparent ileal digestibility of Ca at day 8 (P < 0⋅001). This adaptation was limited to the starter phase in restricted birds. No effect was seen on P transporters mRNA or protein expression. In conclusion, birds adapted to mineral restriction by increasing Ca and nPP utilisation efficiencies. Depletion-repletion strategies are promising in improving the sustainability of broiler production but need to be validated in phytase-supplemented diets.

Increased expression of fibroblast growth factor 23 is the signature of a deteriorated Ca/P balance in ageing laying hens.

The present study concerned the effect of ageing in laying hens, from 23 to 90 weeks of age, on the regulation of Ca metabolism related to the requirement for eggshell mineralization. Samples were collected from parathyroid gland (PG), liver, jejunum, medullary bone (MB) and kidney for a quantitative study of candidate gene expression. Although parathyroid hormone (PTH) gene expression in the PG did not vary with age, a stronger challenge to Ca homeostasis was suggested in aged hens. Indeed gene expression of Ca transporters , Vitamin D Receptor (VDR) in the jejunum, and that of transient receptor potential channel subfamily V member 5 (TRPV5) in the kidney decreased. This could exacerbate bone resorption and impair bone accretion, as attested by a higher expression of the Carbonic Anhydrase 2 (CA2) gene and a lower expression of collagen type I alpha 1 chain (COL1A1) in the MB. The increased expression of Fibroblast Growth Factor 23 (FGF23) in the MB likely contributed to the decreased plasma levels of 1.25(OH)2D3 and the altered expression of target genes under its regulation. Our data highlights the molecular mechanisms underlying the osteoporotic syndrome previously documented in aged laying hens, thus providing new perspectives for future interventions.

Candidate genes of the transcellular and paracellular calcium absorption pathways in the small intestine of laying hens.

To meet the high calcium (Ca) demand during eggshell biomineralization (2 g of Ca per egg), laying hens develop specific metabolic regulations to maintain Ca homeostasis. The intake of Ca, its solubilization, and absorption capacity are enhanced at sexual maturity (SM). A better knowledge of the intestinal Ca transporters involved in their variations at this stage could indicate new nutritional strategies to enhance Ca digestive utilization. Transcellular Ca absorption pathway and its major player calbindin-D 28 K (CALB1) mediate a saturable transport, which has been extensively described in this model. Conversely, a contribution by the paracellular pathway involving non-saturable Ca transport through intercellular tight junction has also been suggested. The aim of the present study was to identify candidate genes of these two pathways and their patterns of expression, in immature pullets (12, 15, and 17 wk old) and mature laying hens (23 wk old) in the duodenum, jejunum, and ileum. Using RT-qPCR, this study identifies 3 new candidate genes for transcellular, and 9 for paracellular Ca transport. A total of 5 candidates of the transcellular pathway, transient receptor potential cation channels subfamily C member 1 (TRPC1) and M member 7 (TRPM7); CALB1 and ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) and ATPase plasma membrane Ca2+ transporting 2 (ATP2B2) were enhanced with age or after SM in the duodenum, the jejunum or all 3 segments. A total of 4 candidates of the paracellular pathway Claudin 2 (CLDN2) and tight junction proteins 1, 2, and 3 (TJP1, TJP2 and TJP3) increased in the small intestine after SM. Additionally, CALB1, ATP2B2, and CLDN2 were overexpressed in the duodenum or the jejunum or both segments after SM. The enhanced expression of candidate genes of the paracellular Ca pathway after SM, supports that the non-saturable transport could be a mechanism of great importance when high concentrations of soluble Ca are observed in the intestinal content during eggshell formation. Both pathways may work cooperatively in the duodenum and jejunum, the main sites of Ca absorption in laying hens.

Digestion dynamics in broilers fed rapeseed meal.

Rapeseed proteins are described to be poorly digestible in chickens. To further identify some molecular locks that may limit their use in poultry nutrition, we conducted a proteomic study on the various chicken digestive contents and proposed an integrative view of the proteins recruited in the crop, proventriculus/gizzard, duodenum, jejunum, and ileum for digestion of rapeseed by-products. Twenty-seven distinct rapeseed proteins were identified in the hydrosoluble fraction of the feed prior ingestion. The number of rapeseed proteins identified in digestive contents decreases throughout the digestion process while some are progressively solubilized in the most distal digestive segment, likely due to a combined effect of pH and activity of specific hydrolytic enzymes. Fifteen chicken proteins were identified in the hydrosoluble proventriculus/gizzard content, including chymotrypsin-like elastase and pepsin. Interestingly, on the 69 distinct proteins identified in duodenum, only 9 were proteolytic enzymes, whereas the others were associated with homeostasis, and carbohydrate, lipid, vitamin and hormone metabolisms. In contrast, chicken proteins identified in jejunal and ileal contents were mostly proteases and peptidases. The present work highlights the relevance of using integrative proteomics applied to the entire digestive tract to better appreciate the protein profile and functions of each digestive segment.

Effects of reduced dietary calcium and phytase supplementation on calcium and phosphorus utilisation in broilers with modified mineral status.

1. The impact of modified mineral status and dietary Ca:P ratio on Ca and P utilisation was measured in chicks with or without phytase supplementation. 2. In a preliminary study, 4 diets were given to chicks from 3 to 15 d of age: D1 (6.5 g P/kg and Ca:P = 1.5) and D2, D3 and D4 (6.0, 5.4 and 5.0 g P/kg, respectively, and Ca:P = 1.2). Growth performance was similar across diets. Tibia ash was similar in chicks given D1 and D2, but was gradually depressed from D2 to D4 (-22%). 3. In the depletion period, two groups of chicks, with similar performance, but with different mineral status were achieved by feeding them, from 5 to 15 d of age, diets with a similar Ca:P ratio of 1.2, but containing 6.3 or 5.2 g P/kg. 4. During the subsequent 11 d of the repletion period, chicks from each of the two previous groups were given one of the 4 diets containing 5.7 g P/kg, but differing in their Ca (8.3 and 5.3 g Ca/kg) and microbial phytase (0 or 1000 FTU, Natuphos levels in a 2 x 2 x 2 factorial arrangement. 5. At the end of the repletion period, the initially depleted chicks could not be differentiated from the non-depleted chicks, indicating the capacity of chicks to compensate for their initial depleted mineral status. 6. Interaction between dietary Ca and phytase levels was not significant. Phytase improved growth performance and bone characteristics. Reduced dietary Ca enhanced feed intake and growth rate, but depressed bone dry matter and ash weight. 7. At the end, diets supplemented with phytase maximised bone ash weight when chicks were fed with a Ca:P ratio of 1.5 but elicited the highest growth rate when chicks were fed with a Ca:P ratio of 0.9.

Consequences of dietary calcium and phosphorus depletion and repletion feeding sequences on growth performance and body composition of growing pigs.

The effect of a calcium (Ca) and phosphorus (P) depletion and repletion strategy was studied in four consecutive feeding phases of 28 days each. In all, 60 castrated male pigs (14±1.6 kg initial BW) received 60% (low (L) diet; depletion) or 100% (control (C) diet; repletion) of their Ca and digestible P requirements according to six feeding sequences (CCCC, CCCL, CLCC, CCLC, LCLC and LLLL; subsequent letters indicate the diet received in phases 1, 2, 3 and 4, respectively). Pigs bone mineral content in whole-body (BMCb) and lumbar vertebrae L2 to L4 (BMCv) was measured in every feeding phase by dual-energy X-ray absorptiometry. Growth performance was slightly (<10%) affected by depletion, however, dietary treatments did not affect overall growth. Compared with control pigs, depletion reduced BMCb (34%, 38%, 33% and 22%) and BMCv (46%, 54%, 38% and 26%) in phases 1 to 4, respectively. Depletion increased however digestible P retention efficiency from the second to the fourth phases allowing LLLL pigs to present no differences in BMCb and BMCv gain compared with CCCC pigs in phase 4. Growth performance in repleted compared with control pigs was lower in phase 2, was no different in phase 3 and was lower in CLCC pigs in phase 4. Repletion increased body P and Ca retention efficiency when compared with control pigs (respectively, 8% and 10% for LC v. CC, P<0.01; 8% and 10% for CLC v. CCC, P<0.10; 18% and 25% for CLCC, CCLC, LCLC v. CCCC, P<0.001). Moreover, BMCv gain was higher in CLC pigs (P<0.001) and gains of body P, Ca, BMCb and BMCv in phase 4 were also higher in repleted than in CCCC pigs (respectively, 14%, 20%, 20% and 52%; P⩽0.02). Repletion reduced body P, Ca, BMCb and BMCv masses in phase 2 but no differences were found in phase 4 compared with control pigs. Lumbar vertebrae L2 to L4 bone mineral content was more sensitive to depletion and repletion sequences than BMCb especially in the first phase probably due to a higher proportion of metabolically active trabecular bone in vertebrae than in the whole skeleton. Dietary Ca was, however, oversupply in L compared with C diets (3.1 v. 2.5 Ca:digestible P ratio, respectively) suggesting that P has probably driven the regulations. Phosphorus and Ca depletion and repletion increases dietary P utilization efficiency and can help to reduce dietary P supply, but the underlying mechanisms need elucidation before its practical application.

Phytase supplementation in diets rich in fiber from rapeseed enhances phosphorus and calcium digestibility but not retention in broiler chickens.

Two experiments were conducted on broilers to assess the effect of dietary fiber from 00-rapeseed meal (RSM) on phosphorus (P) and calcium (Ca) apparent ileal digestibility (AID) and retention (AR) during the growing (Exp1: 10 to 21 d) or finishing period (Exp2: 21 to 31 d) in diets supplemented or not with microbial phytase. Each experiment involved 144 male Cobb 500 fed one of 8 diets. Fiber content was modulated by incorporating whole RSM, RSM from dehulled rapeseeds, either raw or supplemented with 2 levels of defatted rapeseed hulls. Diets were supplemented or not with 750 phytase units of microbial phytase per kg. Excreta were collected from d 14 to d 17 (Exp1) and from d 27 to d 30 (Exp2) to measure AR. At the end of experiments, digestive tracts were sampled and weighed. The distal ileum and tibias were collected to measure AID and bone mineralization, respectively. Age did not significantly alter the response of birds to the addition of dietary fiber. Inclusion of hulls decreased growth performance (P < 0.05). The weight of the proventriculus-gizzard (PG) increased with the dietary fiber content in Exp1: The decreased weight observed using dehulled RSM was reversed following the inclusion of hulls. In both trials, while the presence of phytase increased the AID of P (P < 0.001) but not Ca, the inclusion of hulls with phytase improved the AID of P and Ca [linear (Lin), P < 0.05]. This effect could depend on the effect of fiber on PG development and physiology. Hulls decreased the moisture content of excreta (P < 0.01), suggesting higher water retention or lower water consumption with fiber. The AR of P was lower than AID of P with hulls, contrary to Ca, suggesting a metabolic imbalance. The decrease of AR together with the decrease of bone characteristics indicates a lack of Ca in diets with hulls and suggests that P and Ca provision should be adapted to the level and the origin of fiber inclusion.