Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

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.

Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit.

BACKGROUND: The avian eggshell is a natural protective envelope that relies on the phenomenon of biomineralization for its formation. The shell is made of calcium carbonate in the form of calcite, which contains hundreds of proteins that interact with the mineral phase controlling its formation and structural organization, and thus determine the mechanical properties of the mature biomaterial. We describe its mineralogy, structure and the regulatory interactions that integrate the mineral and organic constituents during eggshell biomineralization. Main Body. We underline recent evidence for vesicular transfer of amorphous calcium carbonate (ACC), as a new pathway to ensure the active and continuous supply of the ions necessary for shell mineralization. Currently more than 900 proteins and thousands of upregulated transcripts have been identified during chicken eggshell formation. Bioinformatic predictions address their functionality during the biomineralization process. In addition, we describe matrix protein quantification to understand their role during the key spatially- and temporally- regulated events of shell mineralization. Finally, we propose an updated scheme with a global scenario encompassing the mechanisms of avian eggshell mineralization. CONCLUSION: With this large dataset at hand, it should now be possible to determine specific motifs, domains or proteins and peptide sequences that perform a critical function during avian eggshell biomineralization. The integration of this insight with genomic data (non-synonymous single nucleotide polymorphisms) and precise phenotyping (shell biomechanical parameters) on pure selected lines will lead to consistently better-quality eggshell characteristics for improved food safety. This information will also address the question of how the evolutionary-optimized chicken eggshell matrix proteins affect and regulate calcium carbonate mineralization as a good example of biomimetic and bio-inspired material design.

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.

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.

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.

Investigating proteins and proteases composing amniotic and allantoic fluids during chicken embryonic development.

In amniotes, the amniotic fluid is a significant contributor to fetal development and health. While numerous studies have been conducted in mammalian amniotic fluid, the composition of amniotic and other extraembryonic fluids in avian egg along with their physiological functions remain largely unexplored. In such a context, our objective was to characterize the chicken amniotic fluid (AmF) and allantoic fluid (AlF) properties, protein composition, and some associated functions from day 8 to day 16 of incubation. SDS-PAGE combined to mass spectrometry analysis revealed common and specific proteins to each fluid, suggesting distinct properties and functions. Indeed, major AlF proteins are mostly "egg yolk" proteins involved in lipid, vitamin metabolisms, and metal ion transport, while major AmF proteins resemble those of albumen. Drastic changes in the AmF protein profiles were observed during incubation, when the albumen transfers from day 12 onwards, while few changes were detected for the AlF protein profile. The decreases in osmolality (from 231 to 183 mOsm/kg) and pH (from 8.26 to 7.26) observed in the AlF during incubation are associated with water and electrolytes reallocation for the embryo needs. In contrast, AmF pH value remained stable (≈7.5). Active proteolytic enzymes have been identified in the 2 fluids using gelatin zymography, followed by mass spectrometry analysis for protease identification. A total of 12 proteases was detected in the AlF, compared to 5 in the AmF. We have shown that AlF concentrates proteolytic enzymes assumed to participate in digestive processes: aminopeptidase N, dipeptidyl peptidase-4, meprin A, and 72 kDa type IV collagenase preproprotein. The other proteases identified in both fluids also could have a role in morphogenesis (hepatocyte growth factor activator, suppressor of tumorigenicity 14, astacin-like metalloendopeptidase) and hemostasis (prothrombin and coagulation factor X). Altogether, these data suggest that the roles of chicken AlF and AmF are not merely associated with protection of the embryo and regulation of metabolic disposable wastes, but also they could have more sophisticated roles during embryonic development.

Characterization of egg white antibacterial properties during the first half of incubation: A comparative study between embryonated and unfertilized eggs.

Egg white is an important contributor to the protection of eggs against bacterial contaminations during the first half of incubation (day zero to 12), prior to the egg white transfer into the amniotic fluid to be orally absorbed by the embryo. This protective system relies on an arsenal of antimicrobial proteins and on intrinsic physicochemical properties that are generally unfavorable for bacterial multiplication and dissemination. Some changes in these parameters can be observed in egg white during egg storage and incubation. The aim of this work was to characterize changes in the antibacterial potential of egg white in embryonated eggs (FE) during the first half of incubation using unfertilized eggs (UF) as controls. Egg white samples were collected at day zero, 4, 8, and 12 and analyzed for pH, protein concentration, and protein profile. Antibacterial properties of egg white proteins were evaluated against Listeria monocytogenes, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, and Salmonella Enteritidis. During incubation, differential variations of egg white pH and protein concentrations were observed between UF and FE. At equal protein concentrations, similar activities against L. monocytogenes and S. uberis were observed for FE and UF egg white proteins. A progressive decline in these activities, however, was observed over incubation time, regardless of the egg group (UF or FE). SDS-PAGE analysis of egg white proteins during incubation revealed discrete changes in the profile of major proteins, whereas the stability of some less abundant antimicrobial proteins seemed more affected. To conclude, the antibacterial activity of egg white proteins progressively decreased during the first half of egg incubation, possibly resulting from the alteration of specific antimicrobial proteins. This apparent decline may be partly counterbalanced in embryonated eggs by the increase in egg white protein concentration. The antibacterial potential of egg white is very effective during early stages of embryonic development but its alteration during incubation suggests that extra-embryonic structures could then progressively ensure protective functions.

Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?

In the past 50 years, selection starting initially at the breed level and then using quantitative genetics coupled with a sophisticated breeding pyramid, has resulted in a very productive hybrid for a variety of traits associated with egg production. One major trait currently being developed further is persistency of lay and the concept of the "long life" layer. Persistency in lay however cannot be achieved without due consideration of how to sustain egg quality and the health and welfare of the birds in longer laying cycles. These multiple goals require knowledge and consideration of the bird's physiology, nutritional requirements, which vary depending on age and management system, reproductive status and choice of the selection criteria applied. The recent advent of molecular genetics offers considerable hope that these multiple elements can be balanced for the good of all in the industry including the hens. The "long life" layer, which will be capable of producing 500 eggs in a laying cycle of 100 weeks, is therefore on the horizon, bringing with it the benefits of a more efficient utilisation of diminishing resources, including land, water, raw materials for feed as well as a reduction in waste, and an overall reduced carbon footprint.

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.

Sex- and tissue-specific expression of "similar to nothepsin" and cathepsin D in relation to egg yolk formation in Gallus gallus.

Egg yolk constitutes the main storage compartment of the avian egg and the first nutritional source that supports embryonic growth. Most egg yolk components are synthesized by the liver of laying hens at sexual maturity and are secreted into the blood to be further transferred into the ovarian oocyte (yolky follicle) by receptor-mediated endocytosis. Egg yolk proteins are secreted as precursors and must undergo proteolytic processing to be bioactive. It is assumed that chicken cathepsin D, an aspartic protease, is a key enzyme in this process. Very recently, a novel aspartic protease, namely "similar to nothepsin," has been identified in the egg yolk. Previous experiments conducted in Antarctic fish have shown that the expression of nothepsin is tissue- and sex-specific. To gain insight into the specificities of expression of both cathepsin D and "similar to nothepsin" in Gallus gallus, we compared their distribution in various tissues, in male and females. Cathepsin D is ubiquitously expressed in all tissues examined, including liver of both male and female adults, and its expression is stable during sexual maturation. In contrast, "similar to nothepsin" expression is unique to the liver of adult females and is sex steroid-dependent as it increases gradually in the liver of hens during sexual maturation. The sexual dimorphic expression of the "similar to nothepsin" gene suggests that the activity of this protein is regulated by the steroid environment of laying hens and is specifically adapted for inclusion in the yolk. Further studies are needed to assess whether "similar to nothepsin" assists cathepsin D in the proteolytic processing of egg yolk proteins during follicular growth.

Is sequential feeding of whole wheat more efficient than ground wheat in laying hens?

The impact of sequential feeding of whole or ground wheat on the performance of layer hen was investigated using ISABROWN hens from 19 to 42 weeks of age. In addition, the effect of reduced dietary energy content of a complete diet was also investigated. Four treatments were tested. Whole wheat was alternated with a protein-mineral concentrate (balancer diet) in a treatment (sequential whole wheat: SWW), while another treatment alternated ground wheat (sequential ground wheat: SGW) with the same balancer diet. The control (C) was fed a complete layer diet conventionally. Another treatment (low energy: LE) was fed a complete diet conventionally. The diet contained lower energy (10.7 v. 11.6 MJ/kg) compared to the C. Each treatment was allocated 16 cages and each cage contained five birds. Light was provided 16 h daily (0400 to 2000 h). Feed offered was controlled (121 g/bird per day) and distributed twice (2 × 60.5 g) at 4 and 11 h after lights on. In the sequential treatment, only wheat (whole or ground) was fed during the first distribution and the balancer diet during the second distribution. Left over feed was always removed before the next distribution. The total feed intake was not different between SWW and SGW, but the two were lower than C (P < 0.05). Wheat intake was however, lowered with SGW compared to SWW (P < 0.05). Egg production and egg mass (EM) were not different between treatments. Egg weight was lower with SGW than with SWW (P < 0.05), but the two were similar to C. Body weight (BW) was lowered (P < 0.01) with SGW relative to SWW and C, SWW BW being also lower than the C one. The efficiency of egg production was increased (P < 0.01) with the SWW and SGW relative to the control. Birds fed LE had higher feed intake (P < 0.05) but they had similar egg production and EM compared to the two sequential treatments. The efficiency of feed utilization was also reduced (P < 0.01) with LE compared to SWW and SGW. It was concluded that sequential feeding is more efficient than conventional feeding. In addition, whole wheat appeared more efficient than ground wheat in terms of egg and BW.

Adaptation of wheat and protein-mineral concentrate intakes by individual hens fed ad libitum in sequential or in loose-mix systems.

1. Feed intake and performance of birds given sequential or loose-mix feeding was investigated from 19 to 42 weeks of age. A complete diet was fed as control (C). A balancer diet (50) was fed either sequentially (S50) or in a loose-mix (L50) with wheat. This diet was formulated to provide a similar nutritive value as C assuming a 50 : 50 diet and wheat intake. Another balancer diet (25) was fed sequentially (S25) or in a loose-mix (L25) with wheat. The diet was to provide a similar nutritive value as C assuming 75 : 25 diet and wheat intakes. In sequential feeding, only wheat was fed in the morning (4 h after lights-on) and the balancer diet in the late afternoon (4 h before lights-off). In the loose-mix treatment, a mixture of the two diets was fed throughout the 16-h daily light. Each treatment was given ad libitum to 25 birds in individual cages. 2. Birds fed on L25 had lower total feed intakes than those receiving C, S50 or S25. Protein intake was reduced with L25 compared to C, S50, S25 and L50. Metabolisable energy (ME) intake was, however, similar among all treatments. Egg production and weight were reduced with L25 compared to S50 and S25. Body weight (BW) was lowered with L25. However, there was high individual variation in all variables. 3. Feeding system (sequential vs loose-mix) had no effect on ME intake. However, the loose-mix treatment reduced feed and protein intake due to lower balancer diet intake. It also resulted in low egg production, egg and BWs compared to sequential feeding. The weights of pancreas and gizzard were heavier with sequential and loose-mix compared to the control. 4. The loose-mix treatment reduced egg-laying performance. Sequential feeding resulted in similar egg-laying performance to conventional feeding and thus could be used to advantage in situations where it is applicable.

Sequential feeding using whole wheat and a separate protein-mineral concentrate improved feed efficiency in laying hens.

The effect of feeding nutritionally different diets in sequential or loose-mix systems on the performance of laying hen was investigated from 16 to 46 wk of age. Equal proportions of whole wheat grain and protein-mineral concentrate (balancer diet) were fed either alternatively (sequential) or together (loose-mix) to ISA Brown hens. The control was fed a complete layer diet conventionally. Each treatment was allocated 16 cages and each cage contained 5 birds. Light was provided 16 h daily (0400 to 2000 h). Feed offered was controlled (121 g/bird per d) and distributed twice (4 and 11 h after lights-on). In the sequential treatment, only wheat was fed at first distribution, followed by balancer diet at the second distribution. In loose-mix, the 2 rations were mixed and fed together during the 2 distributions. Leftover feed was always removed before the next distribution. Sequential feeding reduced total feed intake when compared with loose-mix and control. It had lower wheat (-9 g/bird per d) but higher balancer (+1.7 g/bird per d) intakes than loose-mix. Egg production, egg mass, and egg weight were similar among treatments. This led to an improvement in efficiency of feed utilization in sequential compared with loose-mix and control (10 and 5%, respectively). Birds fed sequentially had lower calculated ME (kcal/bird per d) intake than those fed in loose-mix and control. Calculated CP (g/bird per d) intake was reduced in sequential compared with loose-mix and control. Sequentially fed hens were lighter in BW. However, they had heavier gizzard, pancreas, and liver. Similar liver lipid was observed among treatments. Liver glycogen was higher in loose-mix than the 2 other treatments. It was concluded that feeding whole wheat and balancer diet, sequentially or loosely mixed, had no negative effect on performance in laying hens. Thus, the 2 systems are alternative to conventional feeding. The increased efficiency of feed utilization in sequential feeding is an added advantage compared with loose-mix and thus could be employed in situations where it is practicable.

Zinc availability and digestive zinc solubility in piglets and broilers fed diets varying in their phytate contents, phytase activity and supplemented zinc source.

The study was conducted to evaluate the effects of dietary zinc addition (0 or 15 mg/kg of Zn as inorganic or organic zinc) to three maize-soybean meal basal diets varying in their native Zn, phytic P contents and phytase activity (expressed in kg of feed: P- with 25 mg Zn and 1.3 g phytic P, P+ with 38 mg Zn and 2.3 g phytic P or P+/ENZ being P+ including 500 units (FTU) of microbial phytase per kg) in two monogastric species (piglets, broilers). Measured parameters were growth performance, zinc status (plasma, and bone zinc) and soluble zinc in digesta (stomach, gizzard and intestine). The nine experimental diets were fed for 20 days either to weaned piglets (six replicates per treatment) or to 1-day-old broilers (10 replicates per treatment). Animal performance was not affected by dietary treatments (P > 0.05) except that all P- diets improved body weight gain and feed conversion ratio in piglets (P < 0.05). Piglets fed P- diets had a better Zn status than those fed P+ diets (P < 0.05). In both species, Zn status was improved with supplemental Zn (P < 0.05), irrespective of Zn source. Phytase supplementation improved piglet Zn status to a higher extent than adding dietary Zn, whereas in broilers, phytase was less efficient than supplemental Zn. Digestive Zn concentrations reflected the quantity of ingested Zn. Soluble Zn (mg/kg dry matter) and Zn solubility (% of total Zn content) were highest in gizzard contents, which also presented lower pH values than stomach or intestines. The intestinal Zn solubility was higher in piglet fed organic Zn than those fed inorganic Zn (P < 0.01). Phytase increased soluble Zn in piglet stomach (P < 0.001) and intestine (P = 0.1), but not in broiler gizzard and intestinal contents. These results demonstrate (i) that dietary zinc was used more efficiently by broilers than by piglets, most probably due to the lower gizzard pH and its related higher zinc solubility; (ii) that zinc supplementation, irrespective of zinc source, was successful in improving animal's zinc status; and (iii) suggest that supplemented Zn availability was independent from the diet formulation. Finally, the present data confirm that phytase was efficient in increasing digestive soluble Zn and improving zinc status in piglets. However, the magnitude of these effects was lower in broilers probably due to the naturally higher Zn availability in poultry than in swine.

Meta-analysis of phosphorus utilisation by broilers receiving corn-soyabean meal diets: influence of dietary calcium and microbial phytase.

Pollution relative to phosphorus excretion in poultry manure as well as the soaring prices of phosphate, a non-renewable resource, remain of major importance. Thus, a good understanding of bird response regarding dietary phosphorus (P) is a prerequisite to optimise the utilisation of this essential element in broiler diets. A database built from 15 experiments with 203 treatments was used to predict the response of 21-day-old broilers to dietary non-phytate P (NPP), taking into account the main factors of variation, calcium (Ca) and microbial phytase derived from Aspergillus niger, in terms of average daily feed intake (ADFI), average daily gain (ADG), gain to feed (G:F) and tibia ash concentration. All criteria evolve linearly (P < 0.001) and quadratically (P < 0.001) with dietary NPP concentration. Dietary Ca affected the intercept and linear component for ADG (P < 0.01), G:F (P < 0.05) and tibia ash concentration (P < 0.001), whereas for ADFI, it affected only the intercept (P < 0.01). Microbial phytase addition impacted on the intercept, the linear and the quadratic coefficient for ADFI (P < 0.01), ADG (P < 0.001) and G:F (P < 0.05), and on the intercept and the linear component (P < 0.001) for tibia ash concentration. An evaluation of these models was then performed on a database built from 28 experiments and 255 treatments that were not used to perform the models. Results showed that ADFI, ADG and Tibia ash concentration were predicted fairly well (slope and intercept did not deviate from 0 to 1, respectively), whereas this was not the case for G:F. The increase in dietary Ca concentration aggravated P deficiency for all criteria while phytase addition had a positive effect. The more P deficiency was marked, the more the bird response to ADFI, ADG, G:F and tibia ash concentration was exacerbated. It must also be considered that even if the decrease in dietary Ca may improve P utilisation, it could in turn become limiting for bone mineralisation. In conclusion, this meta-analysis provides ways to reduce dietary P in broiler diets without impairing performance, taking into account dietary Ca and microbial phytase.

Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens.

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116, osteopontin (SPP1), ovocalyxin-32 (RARRES1), ovotransferrin (LTF), ovalbumin and ovocalyxin-36, as well as key genes in the maintenance and function of the shell gland [estrogen receptor (ESR1) and carbonic anhydrase II (CAII)]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.

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.

Characterisation of European varieties of triticale with special emphasis on the ability of plant phytase to improve phytate phosphorus availability to chickens.

1. A total of 30 varieties and selection lines of triticale grown under similar conditions were characterised. Thousand grain weight, specific weight, Hagberg falling number and N were 50.2 +/- 5.0 g, 72.4 +/- 2.1 kg/hl, 96 +/- 48 s and 16.1 +/- 0.11 g/kg, respectively. 2. Mean phosphorus (P) concentration was 2.86 +/- 0.31 g/kg, of which 77% was of phytic origin. Mean phytase activity was 1018 +/- 319 phytase units (PU)/kg. A genotypic effect on phytase activity was detected amongst 5 varieties studied out of 30. Potential and real applied viscosities were positively correlated and mean values were 3.53 +/- 0.66 and 2.15 +/- 0.31 ml/g, respectively. 3. The efficacy of plant phytase in improving P availability was assessed in chickens up to 3 weeks of age. Growth performance and bone ash concentration were compared in birds given either a maize (450 g/kg) and soybean meal (230 g/kg) phosphorus deficient diet containing 3.5 g P/kg, this basal diet supplemented with 1 or 2 g P/kg as monocalcium phosphate (MCP) or triticale (450 g/kg) and soybean meal (230 g/kg) diets containing 3.2 to 3.8 g P/kg with no MCP. To achieve graded levels of phytase activity, 4 varieties of triticale, intact or in which phytase was denaturated by heat treatment, were used. Estimated metabolisable energy, protein, amino acids and calcium concentrations were similar in all diets. 4. Phytase activity in the triticale-based diets ranged between 135 and 1390 PU/kg. Growth performance and bone ash were responsive to plant phytase and to MCP. Non-linear models of these responses were adjusted with the best fit for bone ash parameters. The values of 250, 500 and 1000 PU of plant phytase were estimated to be equivalent to 0.46, 0.67 and 0.81 g P as MCP, respectively.