Maeva Halgrain

Nelly Bernardet

Marine Crepeau

Nathalie Même

Agnès Narcy

Maxwell Hincke # †

Sophie Réhault-Godbert

Eggshell decalcification and skeletal mineralization during chicken embryonic development: defining candidate genes in the chorioallantoic membrane

Keywords: chicken, eggshell, embryo, mineral, chorioallantoic membrane

During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 days of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC).

Our results confirmed that eggshell weight, thickness and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation.

In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected.

This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization.

INTRODUCTION

The nutrient reserve of the fertile egg consists of three distinct compartments that are progressively mobilized to support embryonic development: the yolk, the albumen and the shell [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF][START_REF] Romanoff | Biochemistry of the avian embryo: a quantitative analysis of prenatal development[END_REF][START_REF] Bellairs | The atlas of chick development[END_REF]. During incubation, these nutrient reservoirs operate dynamically and serve at different stages of growth to meet embryo requirements for lipids, amino acids, carbohydrates, and minerals. When focusing on mineral ions, the shell is the main source of Ca, Mg, and Sr; the albumen is the major source of K and Na; and the yolk provides Cu, Fe, Mn, P, and Zn [START_REF] Richards | Trace mineral metabolism in the avian embryo[END_REF][START_REF] Schaafsma | Mineral, amino acid, and hormonal composition of chicken eggshell powder and the evaluation of its use in human nutrition[END_REF][START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF][START_REF] Hopcroft | Changes to mineral levels in the yolk of meat chicken embryos during incubation[END_REF]. Many studies indicate the importance of egg mineral ions for the development of the embryo but also for the skeletal health of chick and adult birds. In fact, a mineral deficiency has adverse repercussions on skeletal, immune and cardiovascular systems, reduces hatchability and increases mortality [START_REF] Richards | Trace mineral metabolism in the avian embryo[END_REF][START_REF] Kidd | A treatise on chicken dam nutrition that impacts progeny[END_REF][START_REF] Angel | Metabolic disorders: Limitations to growth of and mineral deposition into the broiler skeleton after hatch and potential implications for leg problems[END_REF][START_REF] Dibner | Metabolic challenges and early bone development[END_REF]. The transfer of minerals from the yolk during the first half of incubation, and from the eggshell during the second half of incubation to the embryos [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF], intrinsically depends on the functionality of extraembryonic structures, namely the yolk sac (YS) for the yolk and the chorioallantoic membrane (CAM) for the eggshell.

During embryonic development, nutrients are transferred from the yolk contents to the embryo through the yolk sac membrane (YSM) and its surrounding vascular system [START_REF] Uni | Nutritional limitations during poultry embryonic development[END_REF].

From embryonic day (ED) 19, the YS begins to be internalized into the abdominal cavity of the embryo and residual yolk provides critical nutrients until hatched chicks have access to food [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF]. It has been shown that the YSM expresses digestive enzymes and nutrient transporters similarly to the intestine [START_REF] Speake | Transport and transformations of yolk lipids during development of the avian embryo[END_REF][START_REF] Yadgary | The chick embryo yolk sac membrane expresses nutrient transporter and digestive enzyme genes[END_REF][START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF][START_REF] Speier | Gene expression of nutrient transporters and digestive enzymes in the yolk sac membrane and small intestine of the developing embryonic chick[END_REF][START_REF] Bauer | The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of its endodermal epithelial cells[END_REF][START_REF] Yadgary | Temporal transcriptome analysis of the chicken embryo yolk sac[END_REF]. [START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF] observed that the total calcium content of the yolk (30 mg at D0) decreases during the first half of incubation to reach a plateau from ED11 (20 mg) until hatch. During the second half of incubation, the eggshell becomes a major contributor of calcium for supporting skeletal mineralization of the embryo [START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF]. The solubilization of eggshell minerals and the transfer of eggshell calcium to the embryo is ensured by the CAM, which is a highly vascularized structure that lines the inner eggshell and develops from ED5 onwards [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF]. The CAM is complete by ED10-11, grows rapidly from ED11 to ED15-16, and starts to degrade from day 19 onwards [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF][START_REF] Leeson | The chorio-allantois of the chick. Light and electron microscopic observations at various times of incubation[END_REF][START_REF] Narbaitz | The differentiation of the chick chorionic epithelium: an experimental study[END_REF][START_REF] Makanya | Dynamics of the developing chick chorioallantoic membrane assessed by stereology, allometry, immunohistochemistry and molecular analysis[END_REF]. The CAM is fully differentiated at ED15-16 and is composed of three distinct cellular structures, namely the chorionic epithelium, the mesoderm and the allantoic epithelium [START_REF] Makanya | Dynamics of the developing chick chorioallantoic membrane assessed by stereology, allometry, immunohistochemistry and molecular analysis[END_REF], all of which are assumed to play different but complementary roles. The chorionic epithelium participates in acid-base balance of the embryo and mineral solubilization and transport from the eggshell [START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF].

The mesoderm is the site of early development of the extraembryonic vascular system, which serves the CAM epithelia and forms the chorionic capillary plexus to facilitate gaseous exchange [START_REF] Melkonian | Capillary plexus development in the day five to day six chick chorioallantoic membrane is inhibited by cytochalasin D and suramin[END_REF]. The allantoic epithelium is involved in ion and H2O reabsorption from the allantoic fluid and maintains the acid-base balance of this fluid (proton secretion and bicarbonate reabsorption) [START_REF] Stewart | Transport functions of the chick chorio-allantoic membrane. I. Normal histology and evidence for active electrolyte transport from the allantoic fluid, in vivo[END_REF][START_REF] Narbaitz | Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H + -ATPase in two specialised cell types in the chick embryo chorioallantoic membrane[END_REF]. Hence, the CAM is involved in the dissolution and transport of calcium from the eggshell to the embryo, gaseous exchange, maintenance of acid-base balance, water and electrolyte reabsorption from the allantoic cavity and innate immunity [START_REF] Romanoff | The Avian Embryo. Structural and functional development[END_REF]Coleman and Terepka, 1972a, b;[START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF][START_REF] Hincke | Dynamics of structural barriers and innate immune components during incubation of the avian egg: Critical interplay between autonomous embryonic development and maternal anticipation[END_REF]. Only a few candidate proteins in the CAM have been proposed to participate in eggshell solubilization and mineral ion transport to date: a calbindin-like protein, an anion exchanger (AE1), a H + -ATPase, and soluble and membrane-bound isoforms of carbonic anhydrase [START_REF] Tuan | Functional Involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane[END_REF][START_REF] Rieder | Autoradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane[END_REF][START_REF] Anderson | Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunochemistry[END_REF][START_REF] Narbaitz | Carbonic anhydrase activity in the chick embryo chorioallantois: regional distribution and vitamin D regulation[END_REF][START_REF] Tuan | Carbonic anhydrase and calcium transport function of the chick embryonic chorioallantoic membrane[END_REF]Tuan et al., 1986;[START_REF] Narbaitz | Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H + -ATPase in two specialised cell types in the chick embryo chorioallantoic membrane[END_REF][START_REF] Gabrielli | Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis[END_REF][START_REF] Gabrielli | Carbonic Anhydrases in chick extra-embryonic structures: A role for CA in bicarbonate reabsorption through the chorioallantoic membrane[END_REF][START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF]. Moreover, the identity of the associated genes remains ambiguous and depends on the annotated chicken genome assemblies used as a reference [Galgal4 (GCA_000002315.2) in 2013, Gallus_gallus-5.0 (GCA_000002315.3) in 2016, GRCg6a (GCA_000002315.5) in 2018) [START_REF] Peona | How complete are "complete" genome assemblies? An avian perspective[END_REF]].

In this work, we studied the expression of 10 candidate genes in the CAM and YS at ED12 (CAM is developed but not fully mature), and at ED16 (corresponding to a fully differentiated stage). The demineralization of the eggshell as well as the calcification of the embryo skeleton have been assessed in parallel to further appreciate the interrelationship between these two physiological processes. The data obtained in this article revisit some of the statements from the literature and show for the first time that the molecular components involved in mineral mobilization from the yolk and the eggshell are different.

MATERIALS AND METHODS

Incubation Procedures and Sample Collection

Sixty fertilized eggs were obtained from 29-wk old laying hens (Rhode Island Red, Novogen, France) and handled in the Poultry Experimental Facility (PEAT) UE1295 (INRAE, Nouzilly, France, DOI: 10.15454/1.5572326250887292E12). Eggs were incubated under standard conditions (45% RH, 37.8°C, automatic turning every hour; Bekoto B64-S, Pont-Saint-Martin, France), after a 3-day storage at 16°C, 85% RH to favor synchronization of developmental stages. For each embryonic day studied (ED12 and ED16), 30 eggs (64.1±1.8g) containing viable embryos were selected. At each stage, egg weight and eggshell strength were measured prior to tissue sampling (Digital Egg Tester 6000, Nabel, Kyoto, Japan). Eggs were opened at the air chamber end and the egg contents were poured into a Petri dish. Embryos were sacrificed by decapitation, and placed in a sterile flask containing 75 ml of 90% ethanol prior to staining (see below). The yolk sac and the chorioallantoic membrane were removed, washed several times with sterile saline solution (NaCl 0.9%), immersed in liquid nitrogen and stored at -80°C. Eggshell thickness was measured using a small piece of eggshell (Digital Egg Tester 6000, Nabel, Kyoto, Japan) and eggshell weight was obtained after drying for 2 hours at 110°C.

Resulting eggshells were further stored at 4°C prior to analysis of mineral content. These experiments performed on embryos at day 12 and day 16 of development were conducted in compliance with the European legislation on the "Protection of Animals Used for Experimental and Other Scientific Purposes" (2010/63/UE) and under the supervision of an authorized scientist (S. Réhault-Godbert, Authorization no. 37-144). These experiments meet the guidelines approved by the institutional animal care and use committee (IACUC).

Alizarin Red S (Bone) and Alcian Blue (Cartilage) Staining of Chicken Embryos

Chicken embryo bone and cartilage staining was performed as described for mouse embryos (Scientific Protocols, 2014) with some small adjustments. After sampling, embryo bodies were fixed in 90% ethanol for 11 days at 4°C with renewal of the solution every 4 days. Skin, viscera, liver, kidney and gut were removed. Embryos were placed in an Alcian blue solution (0.1g/L) (Sigma, Saint-Louis, USA, ref. A5268) for 3 days, followed by rehydration with several baths of decreasing percentages of ethanol (from 70% to 0% in demineralized water; 70% during 2 hours/ 40% during one night/ 15% during 2 hours and finally, demineralized water for 4 hours).

The clearing of embryos was achieved with 1% KOH solution for 2 days and the staining of mineralized structures was performed during 3 days in Alizarin red / KOH solution at 0.01 g/L (Sigma, Saint-Louis, USA, ref. A5533). Stained embryos were rinsed in 1% KOH, followed by increasing solutions of glycerol (20 to 80%, over 5 days) / 1% KOH, before storage at 4°C in 100% glycerol. Stained ED12 and ED16 embryos were analyzed visually and photographed (Nikon apparatus D5100, Itteville, France). Identification of bones was based on the Atlas of Chick Development (plates 230 and 231, [START_REF] Bellairs | The atlas of chick development[END_REF].

Eggshell Mineral Content

Eggshell mineral quantification was performed according to [START_REF] Park | The influence of hen aging on eggshell ultrastructure and shell mineral components[END_REF], with small adjustments. Eggshell fragments were washed, dried, weighed and then grinded using a Cryomill ball mill (Retsch, Haan, Germany). Eggshell powder (300 mg) was dissolved in 10 mL of 65% nitric acid, and then heated / digested in a microwave for 15 min at 200°C, 1800 W (Ethos Up, Milestone, Sorisole, Italy). Total P, Mg, K, Na and Ca were determined using an inductive coupled plasma atomic emission spectrometer (ICP OES ThermoscientificTM iCAPTM 7200; method 990.08; AOAC International, 2006). Standard solutions (P, Mg, K, Na and Ca) were prepared from a 1,000 mg/mL stock solution (Certipur® Merck, Darmstadt, Allemagne). All assays were performed in duplicates.

mRNA extraction and real-time quantitative PCR

All tissue samples (n=18 per stage, ED12 and Ed16) were homogenized in liquid nitrogen with a mechanical crusher A11 Basic (IKA, Staufen im Breisgau, Germany). For CAM samples, total RNA was extracted using the Nucleospin RNA kit according to the manufacturer's recommendations (Macherey-Nagel, Düren, Germany). For yolk sac samples, total RNA was extracted using RNeasy® Lipid Tissue Mini Kit, according to the manufacturer's recommendations (Qiagen, Hilden, Germany).

To remove traces of genomic DNA, a second treatment with DNAse was performed on all samples (kit Turbo DNA-freeTM, Life Technologies, Carlsbad, USA). Concentration and quality of the extracted RNA were assessed by spectrophotometry (Nanodrop 1000 spectrophotometer Nanodrop Technology, Wilmington, USA) and by migration of total RNA on a 1% agarose gel. Total RNA samples (1 µg) were reverse transcribed using RNase H-MMLV reverse transcriptase (Superscript TM II RT, Invitrogen, Cergy Pontoise, France). Gene quantification was achieved by SYBR Green incorporation, using LightCycler 480 (Roche, Mannheim, Germany).

Candidate genes to be analyzed were selected according to the literature [START_REF] Gabrielli | Carbonic Anhydrases in chick extra-embryonic structures: A role for CA in bicarbonate reabsorption through the chorioallantoic membrane[END_REF][START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF]. Other studies related to eggshell calcification or intestinal calcium transporters in chickens provided additional candidates [START_REF] Jonchère | Identification of uterine ion transporters for mineralisation precursors of the avian eggshell[END_REF][START_REF] Gloux | Candidate genes of the transcellular and paracellular calcium absorption pathways in the small intestine of laying hens[END_REF][START_REF] Gautron | Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit[END_REF]. The list of candidate genes is presented in Table 1.

Calculation of Relative Gene Expression

The relative normalized expression (R) of a candidate gene was calculated, based on the Efficiency (E) and the cycle threshold (Ct) deviation of cDNA samples (CAM or YS at ED12 and ED16 from individual embryos) vs. a calibrator. The calibrator corresponds to the pool of cDNA from all CAM samples (CAM analysis) or a pool of cDNA from all YS samples (YS analysis). Data were expressed relative to the normalization factor of housekeeping genes calculated by GeNorm (version 3.5) [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data[END_REF]. Briefly, normalized quantities were calculated using the following formula: gene efficiency^(Ctcalibrator -Ctsample) / normalization factor of each sample (calculated based on geometric mean of housekeeping genes).

Five stable housekeeping genes could be utilized for CAM samples, while for the yolk sac, only one gene (ACTB) was shown to be invariant between stages and thus was selected for calculation of relative expression. The selected housekeeping genes were: ACTB (Gene ID : 396526, Actin, Beta; Forward CTGGCACCTAGCACAATGA; Reverse CTGCTTGCTGATCCACATCT); PPIA (Gene ID : 776282, Peptidylpropyl isomerase A; Forward : CGCTGACAAGGTGCCCATAA; Reverse : GTCACCACCCTGACACATGA); STAG2 (Gene ID : 422360, Stromal antigen 2; Forward: GCACACACCAGTCATGATGC; Reverse: TGGTGTTCAGGCTGCATAGG) ; TBP (Gene ID : 395995, TATA-box binding protein;

Forward: GCGTTTTGCTGCTGTTATTATGAG; Reverse:

TCCTTGCTGCCAGTCTGGAC); YWHAZ (Gene ID : 425619, Tyrosine 3monooxygenase/tryptophan 5-monooxygenase activation protein zeta; Forward:

TGCTGCTGGAGATGACAAGA, Reverse: AGGCCTTCTCTGGGGAATTG).

Statistical Analyses

All statistical analyses were performed using R Software, version 4.0.2 (R Core Team, 2017, Vienna, Austria). Because the samples were not normally distributed (Shapiro test), statistical analyses were performed using a Wilcoxon test (P<5%).

RESULTS

Eggshell Physical Characteristics and Mineral Content

The effect of incubation on the eggshell quality parameters and mineral ion content (phosphorus (P), magnesium (Mg), potassium (K), sodium (Na) and calcium (Ca)) are shown in Table 2.

The following parameters decreased the egg and eggshell weights, breaking strength and thickness all decrease significantly from ED12 to ED16 (P=0.034, P=0.0133, P<0.0001, P<0.0001, respectively). Concomitant to the decrease in eggshell weight, we observed a significant decrease in the total content of Mg, Na and Ca between ED12 and ED16 (P=0.0112, P=0.0307 and P<0.01, respectively).

Kinetic of skeleton Mineralization

The typical staining pattern of embryo skeletons with Alcian blue (a cationic dye that binds glycosaminoglycans and sulfated glycoproteins in cartilage) and Alizarin red (an anionic dye that binds cationic calcium and calcium deposits) at ED12 (A) and ED16 (B) is presented in Figure 1. The wing and leg bones are already partially mineralized at ED12 (humerus, radius, ulna, and femur, tibiotarsus, respectively), while calcification of the ribs is initiated at ED12 and complete around ED16. Some skeletal regions corresponding to the cervical vertebra, the ribs, the pelvic bones (ilium, ischium, pubis, caudal vertebra) and the digits of the legs exhibit a visually apparent increase in mineralization between these stages (Figure 1).

Relative Expression of Candidate Genes in the Yolk Sac and the CAM

The mRNA expression of eight candidate genes in both the CAM and the yolk sac is presented in Figures 2A and3A. Three genes that are specifically expressed in the CAM but not the YS (TRPV6), and in the yolk sac but not in the CAM (CALB1 and GC), are presented in Figure 2B and 3B, respectively.

In the CAM, the expression of CA2, SGK1 and TRPV6 are higher at ED16 (about 2 fold, P<0.001) while the expression of PKD2 and ATP6V1B2 decreases (about 1.4 fold) between ED12 and ED16 (P<0.01 and P<0.05, respectively). The incubation stage has no effect on the mRNA expression of carbonic anhydrase 9 and 4 (CA9 and CA4), SLC4A1 or SCGN.

In the yolk sac, stage of development does not affect PKD2 expression (Figure 3A) while the expression of CA2 and CALB1 increases when comparing ED16 to ED12 (15-20 fold, P<0.001) (Figure 3A and 3B, respectively). The relative expression of other candidate genes (CA9, CA4, SLC4A1, ATP6V1B2, SGK1, SCGN, and GC) decreases during incubation (up to a 20-fold decrease depending on the gene, P<0.001).

DISCUSSION

The Alteration in Eggshell Integrity Reflects Mineral Release

In birds, the egg contains all the protective and nutritive elements to ensure the development of the embryo until hatching. The eggshell is a physical barrier that protects the embryo from environmental changes and microbes. It also regulates gaseous exchange through its pores while limiting water loss, and provides most of the calcium that is necessary for mineralization of the embryonic skeleton [START_REF] Nys | Structure, propriétés et minéralisation de la coquille de l'oeuf : rôle de la matrice organique dans le contrôle de sa fabrication[END_REF]. The yolk contains about 30 mg of calcium while up to 800 mg of eggshell calcium are resorbed from the day of lay to the day of hatch [START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF]. Such amounts may slightly differ depending on initial egg weight. The decrease in eggshell weight is essentially observed during the second half of incubation when the embryo skeleton needs to be reinforced to support a 5-fold increase of the embryo body weight (from 5 g at ED11 to 25 g at ED18) [START_REF] Makanya | Dynamics of the developing chick chorioallantoic membrane assessed by stereology, allometry, immunohistochemistry and molecular analysis[END_REF]. In our experiment, between ED12 and ED16, eggshell loss is about 300 mg, corresponding to 120 mg of calcium (Table 2). The eggshell ultrastructure is complex, and is characterized (from inside to outside) by the mammillary layer (where biomineralization is initiated), the palisade layer (responsible for most of the eggshell thickness and resistance to fracture), the vertical crystal layer and the cuticle. It has been reported that eggshell resorption mainly occurs from the calcium reserve body in the shell mammillary region [START_REF] Tyler | Studies on egg shells. XII. Some changes in the shell during incubation[END_REF][START_REF] Simons | Ultrastructure of the hen eggshell and its physiological interpretation[END_REF][START_REF] Bond | A comparative study of changes in the fine structure of avian eggshells during incubation[END_REF], and progressively induces the detachment of eggshell membranes together with erosion of the mammillary knobs [START_REF] Simons | Ultrastructure of the hen eggshell and its physiological interpretation[END_REF][START_REF] Bond | A comparative study of changes in the fine structure of avian eggshells during incubation[END_REF][START_REF] Chien | Ultrastructure of avian eggshell during resorption following egg fertilization[END_REF]. The loss of eggshell mineral and the weakening of the underlying support for the thick palisade layer likely explains the decrease in eggshell thickness and associated strength, as observed in Table 2. Such eggshell thinning may result in an increased susceptibility to penetration by environmental microbes, but concomitantly facilitates chick emergence [START_REF] Hincke | Dynamics of structural barriers and innate immune components during incubation of the avian egg: Critical interplay between autonomous embryonic development and maternal anticipation[END_REF]. Previous publications have reported that although the eggshell is 96% calcium carbonate, the distribution of minor mineral ions is heterogeneous: the Mg concentration is higher in the mammillary layer and at the outer palisade layer, while phosphorus (as inorganic phosphate or associated with phosphoproteins)

is mainly incorporated during the eggshell termination process and is found in the outer palisade layer and cuticle [START_REF] Cusack | Magnesium and phosphorus distribution in the avian eggshell[END_REF][START_REF] Shen | The role of magnesium and calcium in eggshell formation in Tsaiya ducks and Leghorn hens[END_REF][START_REF] Hincke | The eggshell: structure, composition and mineralization[END_REF]. Calcium carbonate is deposited constantly throughout the process of eggshell formation (Waddel et al., 1989;Waddel et al., 1991;[START_REF] Shen | The role of magnesium and calcium in eggshell formation in Tsaiya ducks and Leghorn hens[END_REF][START_REF] Gautron | Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit[END_REF]. Regulation of this process by the organic matrix results in the distinctive ultrastructure and microstructure of the eggshell [START_REF] Dennis | Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)[END_REF][START_REF] Hincke | The eggshell: structure, composition and mineralization[END_REF]Rodriguez-Navarro et al., 2015;[START_REF] Gautron | Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit[END_REF]. The innermost layer that is in contact with the eggshell membranes is named the mammillary layer; each mammillary cone consists of a base plate that is the calcified foundation of the eggshell, a calcium reserve body, a cover and a crown. The calcium reserve body is described as the main source of calcium which is mobilized for skeletal mineralization during embryonic development [START_REF] Dennis | Microstructure of matrix and mineral components of eggshells from White Leghorn chickens (Gallus gallus)[END_REF][START_REF] Chien | Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro[END_REF]. A positive correlation is observed between the number of mammillary tips and calcium removal from the eggshell [START_REF] Karlsson | Eggshell structure, mode of development and growth rate in birds[END_REF]. In line with previously published data [START_REF] Schaafsma | Mineral, amino acid, and hormonal composition of chicken eggshell powder and the evaluation of its use in human nutrition[END_REF], our results show that eggshell mineral (approximately 96% calcium carbonate) is composed of calcium (364 mg/g of eggshell) and magnesium (3.11 mg/g of eggshell) as its main cations, but also phosphate (1.08 mg/g of eggshell) (Table 2). In our experiment, the decrease in eggshell weight is associated with the decrease in calcium, magnesium and sodium but not in potassium and phosphorus (Table 2). These data suggest that potassium and phosphorus are concentrated in the outermost and intermediate layers of the eggshell that are not resorbed during incubation.

Indeed, phosphate was described previously to regulate the termination of eggshell formation, which is consistent with its outer localization [START_REF] Gautron | Precursor matrix proteins in the uterine fluid change with stages of eggshell formation in hens[END_REF][START_REF] Cusack | Magnesium and phosphorus distribution in the avian eggshell[END_REF]. These findings underline that eggshell phosphate is not required for bone mineralization, which supports the general statement that the phosphate reservoir for the embryo is the yolk, with about 180 mg at ED0 [START_REF] Romanoff | Biochemistry of the avian embryo: a quantitative analysis of prenatal development[END_REF], and not the eggshell [START_REF] Tuan | Regulation of extraembryonic calcium mobilization by the developing chick embryo[END_REF][START_REF] Chien | Ultrastructure of avian eggshell during resorption following egg fertilization[END_REF].

The staining of the embryos collected at ED12 and ED16 with Alcian blue and Alizarin red (Figure 1) showed that long bones are already partially mineralized at ED12, which corroborates previous studies: the tibial calcium content begins to increase from 12 days of incubation to reach maximum values around ED19 [START_REF] Kubota | Vitamin D metabolism and its possible role in the developing chick embryo[END_REF][START_REF] Torres | Influences of trace mineral nutrition and maternal flock age on broiler embryo bone development[END_REF]. This increase in bone calcification occurs in parallel with bone citrate decarboxylation and alkaline phosphatase activities, both reflecting osteoblast activity. These activities start to increase around ED10-12, reach a peak at ED19, and then decrease, and are strongly correlated with calcium-binding activity in the chorioallantoic membrane [START_REF] Kubota | Vitamin D metabolism and its possible role in the developing chick embryo[END_REF]. From embryonic stages ED14 to ED19, chicken long bones roughly double their length, thickness and total amount of bone mineral [START_REF] Yair | Bone characteristics of late-term embryonic and hatchling broilers: Bone development under extreme growth rate[END_REF][START_REF] Bellairs | The atlas of chick development[END_REF], in order to support the rapid growth of the embryo. These structural modifications require massive transport of calcium from the eggshell and phosphorous from the yolk [START_REF] Yair | Content and uptake of minerals in the yolk of broiler embryos during incubation and effect of nutrient enrichment[END_REF], through the substantial vasculature of the CAM and of the YS. [START_REF] Kerschnitzki | Transport of membrane-bound mineral particles in blood vessels during chicken embryonic bone development[END_REF] reported the presence of membrane-bound mineral particles (calcium and phosphorus) in blood vessels during long bone development of chicken embryos, and it is generally observed that osteogenesis is coupled with angiogenesis during this process [START_REF] Kusumbe | Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[END_REF]. Between ED12 and ED16, a constant decrease in blood Ca 2+ (measured via the allantoic vein) is accompanied by an increase in tibial mineral calcium, which collectively corroborates the rapid assimilation of circulating Ca 2+ for bone mineralization [START_REF] Everaert | Changes in acid-base balance and related physiological responses as a result of external hypercapnia during the second half of incubation in the chicken embryo[END_REF].

The transfer of calcium from the eggshell to the embryo in mediated by a three-layer structure, namely the chorioallantoic membrane. The chorionic epithelium of the CAM lines the eggshell membranes and is involved both in the solubilization of calcium from the inner eggshell, and in the transfer of solubilized ions to the embryo via its capillary network.

The CAM expresses carbonic anhydrases and ion-binding proteins but not calbindin (CALB1) nor vitamin-D binding protein (GC)

Distinct and specialized cell types characterize the mature chorionic epithelium: the villus cavity (VC) cells and the capillary covering (CC) cells (Figure 4A). Previous publications have shown that the major molecular components for extracellular acidification adjacent to the eggshell are localized within the VC cells (Figure 4A). These are the AE1 anion exchanger (AE1, possibly corresponding to SLC4A1), a cytoplasmic carbonic anhydrase 2 (CA2), a membrane-bound carbonic anhydrase, and a H+ ATPase [START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF]; however, except for CA2, the identity (i.e. gene ID) of most these major proteins remains undetermined.

In this context and based on information available in the literature, we investigated the expression of 10 candidate genes in the CAM. Our results showed that the expression of CA2 increases over time as previously published [START_REF] Tuan | Functional Involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane[END_REF]), but we also showed for the first time that CA4 and CA9 are constantly expressed between ED12 and ED16 (Figure 2).

Indeed, carbonic anhydrases have been reported to play a major role in proton secretion for solubilization of the eggshell mineral calcite via VC cells [START_REF] Tuan | Functional Involvement of carbonic anhydrase in calcium transport of the chick chorioallantoic membrane[END_REF][START_REF] Rieder | Autoradiographic localization of carbonic anhydrase in the developing chorioallantoic membrane[END_REF][START_REF] Anderson | Ultrastructural localization of carbonic anhydrase in the chorioallantoic membrane by immunochemistry[END_REF][START_REF] Narbaitz | Carbonic anhydrase activity in the chick embryo chorioallantois: regional distribution and vitamin D regulation[END_REF][START_REF] Tuan | Carbonic anhydrase and calcium transport function of the chick embryonic chorioallantoic membrane[END_REF]Tuan et al., 1986;[START_REF] Narbaitz | Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H + -ATPase in two specialised cell types in the chick embryo chorioallantoic membrane[END_REF][START_REF] Gabrielli | Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis[END_REF][START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF]. Noticeably, CA2 was also previously identified in mitochondria-rich cells (MRC) that are highly concentrated in the allantoic epithelium [START_REF] Narbaitz | Ultrastructural and immunocytochemical evidence for the presence of polarised plasma membrane H + -ATPase in two specialised cell types in the chick embryo chorioallantoic membrane[END_REF][START_REF] Gabrielli | Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis[END_REF]. This protein is assumed to participate in maintaining acid-base homeostasis in the allantoic fluid during embryonic development. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to produce protons and bicarbonate ions and, in parallel, are thought to regulate several bicarbonate transporter activities. SLC4A1 (AE1) is continuously expressed between the two stages and may transport bicarbonate ions into cells, to maintain an extracellular acidic environment, but also into erythrocytes, where bicarbonate ions accumulate between ED10 and ED16 of incubation [START_REF] Everaert | Changes in acid-base balance and related physiological responses as a result of external hypercapnia during the second half of incubation in the chicken embryo[END_REF]. Besides VC cells, both CA2 and SLC4A1 were reported to be expressed in erythrocytes to contribute to blood homeostasis. A vacuolar H+ ATPase was also previously described as participating in the extracellular proton flux. In our experiment, the subunit ATP6V1B2 was shown to be expressed in the CAM but its expression decreased modestly over time, which is not in accordance with the increased concentration of intracellular protons. However, the profile of expression of ATP6V1B2 between ED12 and ED16 resembles the activity profile of a proton pump that was described to be calciumdependent [START_REF] Tuan | Calcium-activated ATPase of the chick embryonic chorioallantoic membrane. Identification, developmental expression, and topographic relationship with calcium-binding protein[END_REF]. The activity of this protein followed a bimodal pattern with a decrease from day ED12 to ED16 followed by an increase essentially after ED16 of incubation to reach a maximum value around hatch (Tuan and Knowles, 1978).

We have shown above that ED16 is characterized by the loss of calcium, sodium and magnesium from the eggshell (Table 1). Ion transfers and exchanges are believed to be mediated by the so-called capillary covering cells (CC cells, Figure 4A). Although we did not explore magnesium-binding proteins and transporters, we identified SCGN whose expression remains stable over time, and TRPV6 that is slightly overexpressed at ED16, as potential calcium-binding proteins. Surprisingly, when looking at calcium-binding proteins, we found that SCGN expression level remains stable between ED12 and ED16, while TRPV6 expression is low and no expression of CALB1 could be detected. Altogether, these data suggest that other not yet identified calcium-binding proteins are responsible for ion movements in the CAM. An alternative hypothesis is that the calcium flux in the cells and subsequently in the blood, does not involve calcium-binding proteins at this stage but maybe later during the time course of development. Indeed, calcium-binding activity has been shown to be maximal at day 18 (6-fold increase between ED15 and ED18) [START_REF] Torres | Influences of trace mineral nutrition and maternal flock age on broiler embryo bone development[END_REF]. The resulting high intracellular calcium concentration is likely to downregulate PKD2 expression. Our hypothesis is that the lower expression of PKD2 (a regulator of intracellular calcium signaling) may reflect the necessity of CC cells to concentrate high intracellular calcium without triggering cellular pathological signaling through an undesired PKD2 activation. The regulation of sodium flux may involve SGK1, which is localized in the nucleus, but also in mitochondria and the plasma membrane, and that is an important regulator of ion channels, including sodium channels [START_REF] Lang | Regulation of ion channels by the serum-and glucocorticoidinducible kinase SGK1[END_REF]. This gene was shown to be overexpressed in the CAM during incubation and may potentially be localized in CC cells but also in erythrocytes, as previously reported [START_REF] Maizels | Cation transport in chicken erythrocytes[END_REF][START_REF] Clarkson | Sodium transfer in human and chicken erythrocytes[END_REF]. A schematic representation of the expression pattern of candidate genes in the chorionic epithelium of the CAM is illustrated in Figure 4A.

Noticeably, the allantoic epithelium is also likely to be involved in the assimilation of calcium and phosphate that accumulate in the allantoic fluid owing to the large increase in embryo metabolism during the second half of incubation [START_REF] Everaert | Changes in acid-base balance and related physiological responses as a result of external hypercapnia during the second half of incubation in the chicken embryo[END_REF]. The absence of GC (vitamin D transport protein) expression in the CAM at these two stages is consistent with the fact that calcium transport and uptake by the CAM were not regulated by vitamin D [START_REF] Packard | The effect of calcium-regulating hormones on transport of calcium across the chorioallantoic membrane of the chicken embryo[END_REF].

The YS expresses CA2, calbindin (CALB1) and vitamin-D binding protein (GC)

In the YS, a variety of expression patterns were observed for the genes under study. Most of the candidate genes demonstrated a decrease in expression between ED12 and ED16; however, CA2 expression increased similarly to the CAM during incubation, PKD2 did not exhibit any difference in expression between the two stages, and TRPV6 was not expressed at either stage (Figure 4B). Carbonic anhydrases were demonstrated to be important in the formation of subembryonic fluid in early Japanese quail and turkey embryos and are localized in the endoderm of yolk sac during the early stages of incubation [START_REF] Babiker | The role of ion transport in the formation of subembryonic fluid by the embryo of the Japanese quail[END_REF][START_REF] Bakst | Impact of egg storage on carbonic anhydrase activity during early embryogenesis in the turkey[END_REF]. To our knowledge, the present study is the first to demonstrate the expression of carbonic anhydrase isoforms in the yolk sac. Calcium binding protein (CALB1) was reported to be essentially upregulated during the later stages of incubation, which we have corroborated, as seen in Figure 3 [START_REF] Sechman | Tissue-specific expression of calbindin-D28K gene during ontogeny of the chicken[END_REF][START_REF] Yadgary | Temporal transcriptome analysis of the chicken embryo yolk sac[END_REF]. In addition, we observed a decrease in expression of GC between ED12 and ED16. These findings suggest that vitamin D uptake and calcium transport are mechanistically uncoupled. The vitamin D status of embryonic blood remains low up to hatching but may be concentrated in the bones [START_REF] Nys | Ontogeny and oestradiol dependence of vitamin D-binding protein blood levels in chickens[END_REF].

Indeed, vitamin D and a calcium-binding protein were shown to co-localize in dividing chondrocytes around hatch [START_REF] Zhou | Bone vitamin D-dependent calcium-binding protein is localized in chondrocytes of growth-plate cartilage[END_REF]. The uptake of vitamin D from the yolk during the first half of incubation and of eggshell calcium during the second half of incubation may correspond to highly orchestrated mechanisms that ultimately assist bone mineralization. In the yolk sac, we failed to detect TRPV6, a calcium selective channel that mediates Ca 2+ uptake in various tissues, including intestine and uterus of laying hens [START_REF] Yang | Localisation and expression of TRPV6 in all intestinal segments and kidney of laying hens[END_REF][START_REF] Yang | Expression of TRPV6 and CaBP-D28k in the egg shell gland (uterus) during the oviposition cycle of the laying hen[END_REF]. TRPV6 was reported to decrease from ED11 to ED13 followed by an increase up to ED19 in the yolk sac [START_REF] Yadgary | The chick embryo yolk sac membrane expresses nutrient transporter and digestive enzyme genes[END_REF][START_REF] Wong | Centennial Review: The chicken yolk sac is a multifunctional organ[END_REF], but remarkably, its expression remains low between 10 and 15 days of incubation, and variability in later stages is surprisingly high [START_REF] Yadgary | The chick embryo yolk sac membrane expresses nutrient transporter and digestive enzyme genes[END_REF]. In view of these results, the physiological role of this calcium channel in calcium uptake from the eggshell and the yolk requires further study.

To conclude, the role of the yolk sac and the chorioallantoic membrane in transferring and transporting ions from yolk (phosphorus) and eggshell (calcium, magnesium and sodium), respectively, are complementary and involve distinct molecular components. These processes are associated with specialized cell types and their expression is temporally regulated in a coordinated manner. Both previous work and the novel results presented here highlight the need to consider the entire period of embryonic development, in order to have a more comprehensive picture of the relative functional roles of the CAM and the YS. None of the candidate genes encoding calcium binding proteins were shown to be significantly regulated during incubation:

SCGN expression remains stable between the two developmental stages, CALB1 is not expressed in the CAM and TRPV6 expression is barely detectable. Thus, to date, the CAM proteins involved in the binding and transport of calcium from the eggshell to the embryo are not known. This example demonstrates the limitations of a candidate-gene approach. We are currently conducting a more systematic approach (RNA-Seq transcriptomics), which, combined with the localization of highly expressed genes within the CAM, will help to decipher the exact role of this multifunctional structure. We believe that the physiological and functional characterization of the CAM needs to be revisited using modern high-throughput techniques, similar to the strategy that has been used to explore the physiology of the yolk sac [START_REF] Yadgary | Temporal transcriptome analysis of the chicken embryo yolk sac[END_REF].

In addition to its interest in understanding the physiology of the extraembryonic structures which support embryonic development, this field of research may also have positive outputs for the poultry industry. There is increasing evidence that intensive genetic selection of broiler breeders for meat production and layer hens for egg quality has precipitated the development of metabolic disorders including skeletal abnormalities [START_REF] Thorp | Skeletal disorders in the fowl: A review[END_REF]Buzala et al., 2015;[START_REF] Eusemann | The role of egg production in the etiology of keel bone damage in Laying hens[END_REF]. It is well known that skeletal integrity in chickens is affected by many factors including rapid growth rate, nutrition and genetics [START_REF] Thorp | Skeletal disorders in the fowl: A review[END_REF]. Fast growing broiler chicks exhibit impaired bone mechanical properties compared with slow-growing broiler chicks [START_REF] Williams | Skeletal development in the meat-type chicken[END_REF][START_REF] Shim | The effects of growth rate on leg morphology and tibia breaking strength, mineral density, mineral content, and bone ash in broilers[END_REF][START_REF] Yair | Maternal and genetic effects on broiler bone properties during incubation period[END_REF], while there is a very high prevalence of keel bone fractures in layer hens, regardless of the production system [START_REF] Eusemann | The role of egg production in the etiology of keel bone damage in Laying hens[END_REF][START_REF] Thøfner | Keel bone fractures in Danish laying hens: Prevalence and risk factors[END_REF]. Such bone pathologies compromise bird welfare and result in substantial economic losses for the poultry industry. The genetic determinants of bone and mineral metabolism are complex and involve multiple genetic loci [START_REF] Chantry-Darmon | Genetic determinism of bone and mineral metabolism in meat-type chickens: A QTL mapping study[END_REF]. The characterization of CAM and YS functions in skeletal mineralization of the embryo might help to identify in ovo markers as predictors of adult chicken bone health in modern poultry lines and lead to new selection tools.

Figure legends Figure 1 :

 legends1 Figure legends

Figure 2 .

 2 Figure 2. RT-qPCR (n=18 per stage) of candidate genes in the CAM. A. Carbonic anhydrases

Figure 3 .

 3 Figure 3. RT-qPCR (n=18 per stage) of candidate genes in the YS. A. Carbonic anhydrases

Figure 4 .

 4 Figure 4. Hypothetical representation of the role of candidate genes in the chorionic epithelium

Table 1 :

 1 Information related to candidate genes. Primer sequences from Jonchère et al., 2012. Fw, forward; Rev, reverse 667

	666
	Symbol	Gene ID	Gene Name	Function	Subcellular location	Primer Sequence 5'-3'	Amplicon size (bp)
	CA2*		Carbonic anhydrase 2	Bone resorption and osteoclast differentiation. Hydration of carbon dioxide and intracellular pH regulation	Cytosol/plasma membrane	Fw :ATCGTCAACAACGGGCACTCCTTC Rev :TGCACCAACCTGTAGACTCCATCC	101
				Hydration of carbon dioxide,		Fw :GGAAGCAAACAGTCACCCATC
	CA4		Carbonic anhydrase 4	stimulation of the sodium/bicarbonate transporter activity of SLC4A4 (pH	Plasma membrane	Rev :GACTCCCCAGTGCAGATGAAA	225
				homeostasis)
	CA9		Carbonic anhydrase 9	Hydration of carbon dioxide, pH regulation	Plasma membrane	Fw :CCTGACAACCTGCACCTCTA Rev :GAGGTGGTTGTCGTCTGTCT	159
	PKD2		Polycistin 2, transient receptor potential cation channel	Component of a heteromeric calcium-permeable ion channel formed by PKD1 and PKD2	Plasma membrane	Fw :ACCTGAGAAGTGTTTTGCGG Rev :GAGCTGCGACATAACCCTCG	122
	SLC4A1		Solute carrier family 4 member 1	Mediation of chloride-bicarbonate exchange in the kidney, urine acidification	Plasma membrane	Fw :TGAGACCTTCGCCAAACTCG Rev :TTCAGCTTCTGCGTGTAGGT	291
	ATP6V1B2		ATPase H+ transporting V1 subunit B2	Acidification of intracellular compartments-organelles	Cytosol	Fw :CCCCACAATGAGATTGCAGC Rev :CATGGACCCATTTTCCTCAAAGTC	171
	SGK1		Serum/glucocorticoid regulated kinase 1	Regulation of various ion channels, renal Na+/K+ and intestinal Na+/H+ exchange and nutrient transport	Nucleus	Fw :GCCCAGTCCATCACAACAGA Rev :ATGCCGTGCAAGAAGAACCT	124
	TRPV6		Transient receptor potential cation channel, subfamily V, member 6	Mediation of Ca2+ uptake in various tissues, Ca2+ ion homeostasis (body and bones)	Plasma membrane	Fw :CACTCCTTCAAGCTGCCAAG Rev :CTGGTTCACTGCTGCAATGT	242
	CALB1*		Calbindin-1	Intracellular Ca2+ binding protein	Cytosol	Fw :CAGGGTGTCAAAATGTGTGC Rev :GCCAGTTCTGCTCGGTAAAG	215
	GC		GC vitamin D binding protein	Vitamin D transport and storage	Secreted	Fw :TAGCAACTCACGCCGAACAC Rev :CATGGCTCGGAAGTCATCCTT	95
	SCGN		Secretagogin, EF-hand calcium binding protein	Regulation of calcium ion concentration	Cytosol	Fw :GATGGACGTCTGGACCTGAA Rev :CCACTGATGCTGGGCTTGAC	221

*

Table 2 :

 2 Eggshell physical characteristics and mineral content for two stages of embryo development (ED12 vs ED16) (n=30 per stage). P-values lesser than 0.05 were considered as significant (in bold type) with arrows to describe the evolution between both stages.

				ED12	ED16	p-value
			Initial egg weight (g)	64.46 ± 1.85	63.95 ± 1.99	0.3669
			Egg weight at sampling (g)	60.4 ± 1.81	58.82 ± 2.09	0.0034 ↓
	Eggshell physical	characteristics	Strength (N) Eggshell weight (g) Thickness (mm)	39.79 ± 4.69 6.31 ± 0.47 0.47 ± 00.3	34.01 ± 4.92 6.01 ± 0.44 0.43 ± 0.03	<.0001 ↓ 0.0133 ↓ <.0001 ↓
	Eggshell mineral content	(mg)*	P Mg K Na Ca	6.78 ± 0.93 19.59 ± 3.39 2.65 ± 0.35 6.43 ± 0.70 2298.49 ± 191.01 2171.66 ± 182.39 6.49 ± 0.88 17.86 ± 2.90 2.54 ± 0.29 6.03 ± 0.65	0.1872 0.0112 ↓ 0.1124 0.0307 ↓ <0.01 ↓

* For the eggshell mineral content, values correspond to those of the total shell

ACKNOWLEDGEMENTS

The authors are grateful to Sabine Alves (INRAE, IFC, PRC, Centre Val de Loire) for providing Rhodes Island eggs, to Joel Delaveau and Christophe Rat (INRAE, PEAT, Centre Val de Loire, DOI : 10.15454/1.5572326250887292E12) for egg handling and incubation and Anaïs Vitorino Carvalho for assistance with statistical analysis. The authors also thank "LE STUDIUM Institute for advanced studies -Loire Valley" for supporting the residency of M. Hincke at DOVE, BOA, INRAE Centre Val de Loire, and the Region Centre Val de Loire and the PHASE Department (INRAE) for the financing of M. Halgrain's PhD. Hincke's participation was additionally supported by the Canadian Natural Sciences and Engineering Research Council (NSERC, Discovery program RGPIN-2016-04410).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest. discussion section) and the expression data obtained in the present study. A. The CAM is composed of three distinct layers (A) where the chorionic epithelium is assumed to be involved in the transepithelial ion transport [START_REF] Gabrielli | The chick chorioallantoic membrane: A model of molecular, structural, and functional adaptation to transepithelial ion transport and barrier function during embryonic development[END_REF] from the blood (red rectangle) and the eggshell (grey rectangle). In this scheme inspired by Gabrielli and Accili, 2010, VC cells are specialized chorionic cells, which, via a vacuolar-type H+-ATPase present at the apical pole (ATP6V1V2), pump protons generated by cytoplasmic carbonic anhydrases (CA2, CA4 and CA9) towards the eggshell (step ❶). Proton secretion results in a local acidification (step ❷), thereby causing solubilization of the calcite mineral (step ❸). HCO 3-is proposed to be reabsorbed through VC cells via the anion exchanger SLC4A1, to maintain acid-base-balance within the CAM. Ca 2+ and other ions including HCO 3-, Mg 2+ and Na + become available to be reabsorbed via by CC cells, for transport via the vasculature to the embryo. Our results suggest that ions transporters such as SCGN and PKD2 participate in Ca 2+ binding and transport; however, CALB1expression was not detected at ED12 or ED16. The very low expression of TRPV6 in the CAM brings into question the role of this candidate gene in calcium uptake.

SGK1 may be involved in Na + transport while the transporter for Mg 2+ is not yet known. As expected, since the eggshell does not contain vitamin D, the GC gene is not expressed in the CAM. B. In the yolk sac, carbonic anhydrases (CA2, CA4, CA9) and a proton-pumping ATPase (ATP6V1V2) contribute to the acid-base balance of YS cells and the yolk (step ❶). Minerals and vitamin D are absorbed from the yolk (yellow rectangle) to the blood (red arrows), via the transporters expressed by the YS (CALB1, SCGN, PKD2, SGK1, GC -step ❷). TRPV6 in not expressed in YS at ED12 or ED16 (TRPV6). In these proposed mechanisms (A and B), erythrocytes may also express CA2, SLC4A1 and SGK1. Schematic representation of the CAM and the YS (left part) is inspired by [START_REF] Hincke | Dynamics of structural barriers and innate immune components during incubation of the avian egg: Critical interplay between autonomous embryonic development and maternal anticipation[END_REF] and [START_REF] Bauer | The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of its endodermal epithelial cells[END_REF]. Some elements were obtained from Servier Medical Art (https://smart.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License. CC, capillary covering cells; VC, villous cavity cells. Grey arrows illustrate the transport from the blood to the eggshell via the CAM or the yolk via the yolk sac, and red arrows illustrate transport into the blood vessels.

It must be emphasized that the intact yolk sac and CAM tissues were analyzed: endoderm, mesoderm, and ectoderm for the yolk sac, and allantoic epithelium, mesoderm and chorionic epithelium for the CAM. Each candidate gene in this study may be differentially expressed in the various layers or cell types (Discussion section). Hence, the specific localization of all candidate genes and proteins in YS and CAM will require further experimental study.

Figure 1


N Labchir

A Hannour

A Ait Hssi

D Vincent

A Ihlal

Self-biased coplanar circulator based on electrochemically grown ferrimagnetic nanowires

Keywords: porous alumina membrane, CoFe2O4 nanowires, self-biased circulator, HFSS, circulation phenomenon, S parameters

This paper is about a self-biased coplanar circulator. The design approach is based on the use of magnetic composite substrates self-polarized made from ferrimagnetic nanowires. It consists of integrating and orienting cobalt ferrite (CoFe2O4) nanocrystallites in porous alumina membrane using the magnetoelectrodeposition technique. XRD, SEM and EDX analysis confirmed the formation of the spinel phase of cobalt ferrite with a good distribution of Co, Fe and O elements in the unit cell of CoFe2O4 nanowires. The performed SQUID-VSM measurements have revealed that the nano-composite electrodeposited and cooled under a magnetic field of 0.6 T has the best magnetic properties, i.e. a large anisotropy field ( ), moderate coercive field ( ), and partial squareness ratio ( / ). This nano-composite has been selected as an interesting candidate for the fabrication of a miniaturized self-biased circulator. The study has started with the implementation of an analytical dimensioning process based on Bosma's theory. Based on interesting HFSS simulation results, a first prototype was fabricated in the clean room and characterized at high frequencies. The measurements of the S parameters have highlighted a circulation phenomenon of 3 dB.

Introduction

The microwave devices are currently used for applications in many industries, from mobile telephony to aerospace sectors and radar systems. In recent years, their development has rapidly progressed due to increasing demand, has led engineers to enlarge the frequency bands usually and has looked for advanced miniaturization of the devices, through circuit integration among other the ability to master the magnetic properties of the materials making up these devices. In this respect, the electromagnetic metamaterials, defined as effective structures with properties generally not available using conventional materials, and designed from elements whose size is much smaller than the guided wavelength [START_REF] Koschny | Metamaterials in microwaves, optics, mechanics, thermodynamics, and transport[END_REF], form a promising class of materials for the telecommunications sector. Several works have been published on innovative research on metamaterials [START_REF] Vendik | Metamaterials and their application in microwaves: A review[END_REF][START_REF] Cui | Microwave metamaterials[END_REF][START_REF] Kaipa | Characterization of negative refraction with multilayered mushroom-type metamaterials at microwaves[END_REF][START_REF] Saib | Transmission lines on periodic bandgap metamaterials: from microwaves to optics applications[END_REF][START_REF] Maystre | Metamaterials: from microwaves to the visible region[END_REF], but very few magnetic materials had been involved in the design of metamaterials at microwave frequencies. However, metamaterials can produce non-reciprocal effect without adding active devices [START_REF] Marques | Metamaterials with negative parameters: Theory, Design and Microwave Applications[END_REF]. In other hand the magnetic materials exhibit a high magnetic activity at microwave frequencies [START_REF] Gurevich | Magnetization Oscillations and Waves[END_REF]. Depending on the nature of the material and the experimental conditions, a magnetic material subjected to a static and dynamic magnetic field can exhibit a gyromagnetic resonance response at high frequencies.

Several microwave devices based on magnetic materials, such as circulators, make use of the gyromagnetic resonance of magnetic materials, by operating out of to the resonance frequency band. This type of device is often used in the systems with antennas [START_REF] Chou | Ferrite Circulator Integrated Phased-Array Antenna Module for Dual-Link Beamforming at Millimeter Frequencies[END_REF][START_REF] Mung | The challenge of active circulators: design and optimization in future wireless communication[END_REF], The circulator is a device non-reciprocal, typically having three ports. Basically, a device is non-reciprocal if the transmission of the wave differs according to the directions of propagation of the device. The conventional circulator operates with ferrite substrates polarized by an external magnetic field to ensure the non-reciprocity of the electromagnetic signals propagation.

However, the presence of bulky and heavy permanent magnets that ensures the polarization of the ferrite remains a major obstacle to miniaturize and to integrate this circulator in microelectronic circuit. However, their integration into planar devices requires overcoming some technological challenges [START_REF] Harris | Recent advances in processing and applications of microwave ferrites[END_REF]. The nanowires technology offers an interesting alternative for massive and bulky ferrites for the fabrication of self-polarized planar devices. Several prototypes of microwave devices based on ferromagnetic nanowires have been demonstrated. Microstrip circulators have been constructed by Saib et al, [START_REF] Saib | An unbiased integrated microstrip circulator based on magnetic nanowired substrate[END_REF] Allaeys et al, [START_REF] Allaeys | Numerical modelling of unbiased microstrip circulators based on magnetic nanowired substrate : Use of a ferrite-equivalent model[END_REF] and more recently by Darques et al. [START_REF] Darques | Microwave circulator based on ferromagnetic nanowires in an alumina template[END_REF] Circulators asymmetrically charged with nanowires have been designed in microstrip configuration by Spiegel et al. [START_REF] Spiegel | Isolator concept based on ferromagnetic nanowired substrate[END_REF] and in coplanar guide configuration by Kuanr et al. [16] Carignan et al. reported a circulator in isolator mode at double-band displacement, in microstrip line configuration, using the double resonance of ferromagnetic nanowires [START_REF] Carignan | Ferromagnetic nanowire metamaterials : Theory and applications[END_REF]. De La Torre Medina et al. manufactured a circulator in phase shifter mode based on nanowires [START_REF] La Torre Medina | Differential phase shift in nonreciprocal microstrip lines on magnetic nanowired substrates[END_REF]. Stripline circulators have been reported by Spiegel et al. [START_REF] Spiegel | Ferromagnetic inductors on commercial nanoporous anodic alumina[END_REF] and Hsu et al. [START_REF] Hsu | The inductance enhancement study of spiral inductor using Ni AAO nano-composite core[END_REF] In 2011, Boyajian et al. [START_REF] Boyajian | Coplanar circulator made from composite magnetic material[END_REF] produced a prototype of a coplanar circulator. The magnetic substrate that has been used consists of magnetic particles of barium hexaferrite mixed in SU-8 resin matrix deposited by technical spin coating on an alumina substrate. During the magnetic substrate growth process, a permanent magnet was placed under the alumina substrate in order to orient the magnetic moments of the particles. The measurements showed low reciprocity caused by wrong orientation particles and lower concentration. In the same context, Ardaches et al. [START_REF] Tchangoulian | Realization of Unbiased Ferrite Cobalt Nano-Composite for Non-Reciprocal Microwave Components[END_REF] tried to develop a selfbiased coplanar circulator. The approach chosen is based on the synthesis of nano-ferrimagnetic composite substrates. It consists of making a deposit by dip-coating of CoFe2O4 nanoparticles into porous alumina membranes. He manufactured and characterized his coplanar circulator at high frequencies. The measurement has shown a circulation phenomenon, which remains very low due to the small concentration of the magnetic nanoparticles in the composite. This type of circulator is still under study because it should allow to avoid using permanent magnets and to reduce the costs with simple and collective manufacturing techniques. Since the performance of circulators based on magnetic materials is based on the ability to control the magnetic response of the magnetic material, the new approach is to fabricate a self-polarized ferrimagnetic nanowire substrate. Among the techniques for synthesizing magnetic nanowires, we can cite RF sputtering, plasma vapor deposition, laser pulse deposition, etc [START_REF] Jilani | Advance deposition techniques for thin film and coating[END_REF]. These techniques, which are still under development, are very expensive, require sophisticated equipment [START_REF] Riveros | Crystallographically oriented singlecrystalline copper nanowire arrays electrochemically grown into nanoporous anodic alumina templates[END_REF]. Many research centers are interested in processes to obtain low-cost self-polarized materials.

The electrochemical deposition technique is considered to be a very promising, simple and cheaper alternative for the manufacture of self-polarized nanowires. This paper is devoted to the magnetoelectrochemical growth of CoFe2O4 ferrimagnetic nanowires, due to its high magneto-crystalline anisotropy and magnetization saturation and its physico-chemical and microwave characterization. We focus on the modelling and fabrication of a coplanar circulator based on the synthesized auto-polarized ferrimagnetic nanowires. Firstly, the permeability tensor model of the nanowires will be presented.

Afterwards a first circulator sizing based on the Bosma's theory is obtained, followed by a numerical modelling of the coplanar circulator on the HFSS software. Then, several structures will be simulated in order to determine which one gives the best performances. Finally, the manufacturing steps of such component on the nanowires surface will be discussed. Afterwards, a microwave characterization will be performed for the fabricated circulator.

Experimental

Synthesis procedure

Analytical grade iron sulfate and cobalt sulfate purchased from Sigma Aldrich company were used as starting precursors. The electrochemical growth tests were carried out using a Teflon conventional cell of three electrodes connected to a Potentiostat VoltaLab 40 PGZ 301 controlled by a computer via VoltaMaster 4 software. Anodized aluminum oxide (AAO) membranes of 20 nm in pore diameter, 50 µm in thickness,

10

/ in porosity, 1 cm 2 of area and metallized by 3 µm of copper layer were used as working electrodes (Fig. 1). A Platinum rod and a silver/silver-chloride (Ag/Ag-Cl) were used as the counter electrode and as the reference electrode, respectively. During the electrodeposition experiments, a perpendicular applied magnetic field, generated by a commercial NdFeB magnet (30 mm in diameter and 15 mm in height) is placed under the AAO membrane substrate in order to enhance the electrochemical reaction of the ions reduction.

The CoFe2O4 nanowires were electrodeposited from an electrolyte that containing 0.06 M of Fe(SO4).7H2O, 0.04 M of Co(SO4).7H2O. In order to adjust the pH value at 3, 0.04 M of sulfuric acid (H2SO4) was added to the electrolyte. Before starting the electrochemical deposition, the AAO membranes substrates were ultrasonically cleaned with acetone for 10 min each. Next, the Co and Fe ions were reduced by applying a voltage of -1.2 V vs the working electrode. Thus, the as-prepared nanowires were annealed in air at 500 o C for 24 h and then magneto-cooled at 0.6 T in order to complete the crystallographic formation process and obtain a pure spinel phase of CoFe2O4.

2.2.Characterization

The chronoamperometry technique was exploited to study the electrochemical deposition evolution of the Co and Fe elements. The microstructure of the synthetized nanowires was investigated by recording the Xray diffraction (XRD) spectrum using a PAnalytical X'Pert PRO X-ray diffractometer that operating with Cu-beam (λ = 0.154 nm). Then, the morphology and the elemental composition of CoFe2O4 nanowires were studied using Quanta 200-FEI scanning electron microscope (SEM) that operated at 40 kV and equipped with energy dispersive X-ray detector (EDX). Moreover, the surface topography and roughness have been estimated by a Bruker Dektak XT profilometer. In addition, the hysteresis loop was evaluated by a superconducting quantum interference device (SQUID) magnetometer. Finally, the numerical and experimental S-parameters, of the etched coplanar circulator on the nanowires surface, were investigated using high frequency structure simulator (HFSS) and RS ZVA67 vector network analyzer (VNA).

Results and Discussion

Microstructural, morphological, and magnetic properties

The chronoamperometric study offers an idea of the mechanism of nucleation and growth of CoFe2O4 inside the AAO membrane pores. From this study, it is possible to determine the required time to completely fill the pores of the nanoporous membranes and also to determine the critical overflow time of the deposit. Finally, the current density continues to negatively increase and the deposit continues to accumulate on the surface of the nanoporous membrane. From this result, it is assumed that the deposition time is fixed at 38 min in order to prevent the presence of a top layer that can influence the measurement and fabrication processes that will be presented in the next sections.

To examine the crystal structure of the obtained nanowires after being annealed at 500 o C for 24 hours and magneto-cooled at 0.6 T, X-ray diffraction spectrum was recorded. Fig. 3 shows the X-ray diffraction reflections of the grown nanowires in an AAO membrane at room temperature. As observed, the XRD peaks correspond to the CoFe2O4 spinel structure (JCPDS n. 22-1086) [START_REF] Labchir | Controlled electrochemical growth and magnetic properties of CoFe2O4 nanowires with high internal magnetic field[END_REF]. We note the absence of any kind of secondary impurities. The Al2O3 peaks originate from the alumina substrate. The lattice parameter a of CoFe2O4 which is crystallized in face-centered cubes (fcc), was determined by an average on all intense peaks. The calculation provided a value of 8.38 Å. This value is in accordance with bulk material CoFe2O4 [START_REF] Labchir | Controlled electrochemical growth and magnetic properties of CoFe2O4 nanowires with high internal magnetic field[END_REF]. the elaborated nanowires array present a ferrimagnetic behavior [START_REF] Labchir | Controlled electrochemical growth and magnetic properties of CoFe2O4 nanowires with high internal magnetic field[END_REF]. From the recorded loop, it is noted that the nanowires possess a squareness ratio ( / ) of 0.6, a coercive field ( ) of 140 kA/m and an anisotropy field of 1.5 T. This result confirms the elaboration of partially hard ferrimagnetic substrate with high shape anisotropy and magnetic internal magnetic field of CoFe2O4 crystallites. According to the magnetic response of CoFe2O4 nanowires, a coplanar circulator based on these auto-polarized nanowires has been designed.

Application of CoFe2O4 nanowires

After examining the magnetic properties of CoFe2O4 nano-composite synthesized by the magnetoelectrodeposition technique, an analytical modeling and electromagnetic simulation of the Y-junction circulator has to be made. A theoretical model used to predict the permeability tensor of CoFe2O4 nanocomposite in non-saturated state (remanence state) has been defined. Tests will be performed using different squareness ratios (m) for CoFe2O4 nano-composite. Afterwards, we will discuss more concretely the phenomena involved in the circulator. We note that the circulator has been primarily studied in its stripline configuration. In our work, a first approach uses the theoretical study of the stripline circulator based on Bosma's formulas to find the approximate circulator dimensions. Then, the calculation of the S-parameters, taking into account the different loss factors, will make it possible to evaluate the performance of the circulator and to transpose its dimensions towards the coplanar topology. On the other hand, a protocol of integration for the theoretical model of the magnetic permeability in the Ansoft HFSS commercial software will be followed in order to numerically simulate the structure previously studied in an analytical approach.

Indeed, the 3D electromagnetic simulation enables the ferrite thickness to be taken into account, which is not the case in the first analytical modeling.

Finally, best resulting design will be used for the manufacturing of a typical prototype.

Magnetic permeability tensor of CoFe2O4 nano-composite

Electromagnetic models of unsaturated ferromagnetic nanowires leading to the permeability tensor are often complex. A permeability model on Ni, CoFeB and Ni/Cu nanowires has been developed by Carignan et al. [START_REF] Carignan | Ferromagnetic nanowire metamaterials : Theory and applications[END_REF][START_REF] Labchir | Microwave response of coplanar waveguide based on electrodeposited CoFe2O4 nanowires[END_REF], but it is applied on metallic ferromagnetic nanowires and it should be modified for application on ferrite nanowires. The tensor permeability model must take into account the response of the nanowired ferrimagnetic composite with their magnetic characteristics such as , / , and geometrical ones such as the porosity p and the filling rate ℎ of the AAO membrane pores. In this first approach the interactions between the nanowires and the interactions between the domains into each nanowire are neglected. Owing to these simplifications, the modified Irashagi model permeability tensor model of the nano-composite has been selected [START_REF] Hamoir | Control of microwave circulation using unbiased ferromagnetic nanowires arrays[END_REF][START_REF] Spiegel | Isolator concept based on ferromagnetic nanowired substrates[END_REF].

̂ = -! 0 + ! 0 0 0 # $ (1)
Where As shown in the obtained results, by increasing the orientation of the nanocrystallites from 30 % to 100 %, the two elements and ! increase at the resonance while for the element # the real part tends towards 1 and the imaginary part becomes zero for an orientation of 100 % magnetic material. As a result, the adopted model converges well towards the Polder model in the saturated case. The resonance frequency is practically unchanged.

Stripline circulator based on CoFe2O4 nano-composite

The Y-junction stripline circulator designed by Bosma is the most widely used in theoretical studies of circulators. As illustrated in Figure 8, the core of this circulator of stripline technology consists of three access lines oriented at 120 o to each other and connected to a central metal disk [START_REF] Bosma | On stripline Y-circulation at UHF[END_REF]. This conductor is inserted between two ferrite cylinders with the same shape as the central disk, in order to ensure the homogeneity of the transverse structure. The stripline structure is sandwiched by two ground planes above and below the ferrite disks. Two permanent magnets located on each side of the structure create a static magnetic polarization field that magnetizes the ferrimagnetic material along the axis of the magnetic cylinders. As a first approach, we determined the effective permeability of the nanowired CoFe2O4 composite. In our study, we considered that we have CoFe2O4 nanowires oriented along the wire axis. Thus, the magnetic substrate has a saturation magnetization of 0.18 T and an internal static magnetic field of 1.52 T (it is the sample electrodeposited and cooled under a magnetic field of 0.6 T).

The evolution of the effective permeability of the CoFe2O4 nanowires as a function of frequency was modelled using the following formula:

<< = -! (5)
Here and ! are the calculated tensors by taking = 0.6, which is the best squareness ratio value determined for our CoFe2O4 nano-composite. As shown in Fig. 10, the resonance phenomenon of the effective permeability, when the internal field is fixed at 1.52 T, appears at a frequency of 42 GHz. Our circulator should operate around 40 GHz. However, this component does not operate at resonance (high losses); but either below or above resonance. The operation of the circulator known as above and below resonance are understood to be in field, i.e. when the internal field is respectively higher or lower than the field required for the resonance. The geometrical parameters of the central conductor of the circulator have been approximately determined using the Bosma's theory which depend on the effective permeability of the CoFe2O4 nanowires. The estimations provide a radius R of the central conductor disk of 475 µm, and an access line width W of 72 µm.

Once the effective ferrite permeability has been evaluated, the values of the S-parameters between two consecutive ports of the circulator, as a function of frequency, can be analytically determined from the theoretical analysis performed previously. The conductor material is the copper. The dielectric material is the alumina surrounding the ferrite disks, which has a relative permittivity of (> ? = 10). The permittivity value of the cobalt ferrite is around 13. A dielectric loss tangent (tan C = 10 -) has been taken into account in the analytical calculation. As can be shown in Figure 10, the modelled circulator using Matlab (Bosma) software operates at frequency of 35.8 GHz. According to the evolution of the S parameters as a function of frequency, the reflection is 20.45 dB, the isolation is 20.64 dB and the insertion loss is 0.98 dB. This is the best result obtained with the parameters given previously.

As the structure concerned in our work is the coplanar topology, it does not correspond exactly to the one studied by Bosma, even though the operation principle remains the same. Therefore, the optimized geometrical parameters (R,W) will be maintained in the coplanar structure for the next analysis.

Integration of CoFe2O4 permeability tensor in HFSS

The anisotropic nature of the nanowires can be modeled using HFSS, either by using Polder formulations or by setting a user-defined permeability tensor. Conventionally, the Ansys HFSS uses the Polder model to calculate the components of the permeability tensor. However, the Polder model is only valid for the saturated state of the magnetic material [START_REF] Zerrougui | High frequency characterization of the thin film ferrites BaM and YIG[END_REF].

When the applied field is along z direction, the permeability tensor can be expressed as,

̂= D E -EE -(! E -! EE ) 0 + (! E -! EE ) E -EE 0 0 0 # E -# EE F ( 6
)
The last versions of this software allow to define the nine components of the permeability tensor as frequency-dependent functions. This makes it possible to integrate our adopted model for the permeability of the CoFe2O4 nano-composite into HFSS, in order to accurately predict the microwave response of the targeted component.

Our approach is based on the integration of the data points (calculated from the theoretical model in Matlab software) and on the proceeding of their linear interpolation into frequency dependent functions. The real and imaginary parts of the permeability tensor components are imported as separate datasets into HFSS.

Each dataset is interpolated into frequency-dependent functions using an intrinsic piecewise linear interpolation function.

Simulation of a coplanar circulator based on CoFe2O4 nano-composite

The circulator uses coplanar topology with a bottom ground plane as shown in Fig. 12a. The coplanar structure, as the name suggests, is a topology having its conductor planes in the same level separated by a small distance G (Fig. 12b). It should be noted that the access line width W and the radius R of the central disk have been approximatively determined in the previous section. Moreover, the radius of the bottom ground plane disc is defined as the sum of the width of the central radius and the spacing gap G in order to improve the phenomenon of the electric field displacement at the central part of the circulator. So, keeping the value of W and R, the only remaining task is the optimization of G. To obtain the right G-value and to evaluate the performance of our ferrimagnetic nano-composite, simulations of the CPW, which ensure the input and output of the EM signal to the circulator, were carried out.

The nanowired ferrimagnetic substrate employed in our work is an alumina membrane, electromagnetically charged by CoFe2O4 nanocrystallites, that forming 60 µm in thickness. Before proceeding to the HFSS simulation, we have used a simple law of proportionality in order to approximate the effective permittivity > << of the nano-composite, determined as a function of the nanocrystallites concentration and the AAO membrane pores filling coefficient [START_REF] Darques | Microwave circulator based on ferromagnetic nanowires in an alumina template[END_REF]:

> << = (1 -)> GGH + I J)K L H M N> J)K L H M (7)
where is the porosity of the AAO membrane, β the filling coefficient of the AAO membrane pores by CoFe2O4 nanocrystallites, I J)K L H M the concentration of CoFe2O4 nanocrystallites contained in the pores, > GGH the permittivity of alumina, and > J)K L H M the permittivity of bulky CoFe2O4.

The typical characteristic properties of the ferrimagnetic nano-composite are listed in Table 1. We must keep in mind that we used CoFe2O4 nanowires in order to benefit from its strong magnetocrystalline anisotropy to prevent the application of an external field. Subsequently, the excitation field of the component is limited in our case to the anisotropy field of the CoFe2O4 nanowires.

Table 1: Characteristics of CoFe2O4 nano-composite. N (%) (%) > GGH > << ) (4) ∆ (P ) tan C , (4)
The coplanar circulator has been dimensioned according to the previously optimized values and then simulated on HFSS (Fig. 13). The circulator was modeled and adapted to have the input impedance of the ports equal to 50 Ω. Fig. 14 shows the simulation results of the designed circulator. As shown in the simulation finding, one can observe a remarkable operation of the circulator around 35.7

GHz for a bandwidth of 1 GHz. In this band, the insertion losses are -2.57 dB, the insulation varies from 6 to 10.33 dB and the reflection coefficient is around -20.2 dB. A non-reciprocal effect of the circulator based on CoFe2O4 nano-composite can be observed. This requires the fabrication of the circulator as well as its microwave characterization.

Fabrication and microwave characterization of the coplanar circulator

After designing and dimensioning the coplanar circulator through various analytical and numerical studies, prototypes were fabricated and characterized. The fabrication process uses the different technological steps.

The prototypes are then characterized using a microwave measurement bench consisting of a vector network analyzer (VNA) coupled with a probe tester. The measured S-parameters enable to conclude about the performance of the prototypes. No external magnetic field is applied during these experimental measurements. This makes it possible to check that the concept of self-biased circulator is achievable with our ferrimagnetic nano-composite.

Fabrication protocol of the circulator

The realization of the structures modeled under HFSS, requires many technological processes. Fig. 15 illustrates the different steps required to fabricate a coplanar circulator based on the ferrimagnetic nanocomposite. The preparation of the ferrimagnetic nano-composite involves the deposition of copper, the sticking and the grinding processes. A glass slide serves as a mechanical carrier for the ferrimagnetic nano-composite on which a 5 µm layer of copper is deposited. This first layer of copper acts as a bottom ground plane of the circulator. The copper is deposited by the TSD350 rf sputtering equipment. After, the ferrimagnetic nanocomposite is bonded on the glass slide using the "Geo-fix" glue. Once glued, the ferrimagnetic substrate is polished with a very fine optical paper in order to obtain a roughness close to 100 nm which will favors a good adhesion of the copper that will be deposited to make the conductor and the top ground planes.

After the substrate cleaning with acetone and distilled water, the next step of the photolithography is initiated in order to transfer the coplanar pattern from the mask to the ferrimagnetic substrate. Then, a thin layer of SPR 505 resin is deposited via the spin coating device. When the resin deposition is finished, the substrate is heated at 90 o C for 90 s. This annealing step aims to evaporate quickly the solvents. The resincoated substrate is placed in a UV contact exposure machine. The substrate-mask contact must be perfect to avoid any diffraction phenomena. It is insulated by the UV light for 3 seconds with a power of 270 Watts.

To activate the polymerization of the resin, a second annealing is carried out at 110 o C for 90 s. The UVexposed resin is dissolved in the MF-319 developer and reveals the non-insulated resin. Once revealed, the substrate is slowly rinsed with distilled water and carefully dried with nitrogen before proceeding to a final annealing at 110 o C for 4 min. This last heat treatment makes the resin mechanically more resistant. The resin development step is followed by the chemical etching phase which consists on removing, using the iron perchloride, the copper that has not been protected by the resin. For this purpose, the substrate is placed in a diluted iron perchloride solution until the copper unprotected by the resin is removed. After, the substrate is rinsed with the distilled water in order to stop the reaction of the iron perchloride and make the conductive lines appear. Finally, in order to protect the copper and the etched circuit from oxidation and corrosion, it is necessary to tin it. The tinning process is chemically done with a cold aqueous Tin (Sn) solution.

Fig. 16 shows multiple coplanar circulators printed on the nano-composite with the theoretically and numerically optimized dimensions.

Microwave characterization

The microwave characterization has been performed to evaluate the performance of the fabricated prototypes. A two-ports vector network analyzer (VNA) operating in the 40 MHz -67 GHz band and equipped with three coplanar GSG (Ground-Signal-Ground) probes and its holders has been used. A microscope is also associated with the measurement bench for positioning the probes on the millimeter sized components as shown in Fig. 17. Concerning the Y-junction circulator that has three access ports, it is necessary to have three points oriented at 120 o . One of the third coplanar probes, used in the measurement, is connected to a load from 50

Ω because even though the circulator is a 3-port component, it is possible to use only 2-port measurements with our VNA. It should be noted that in our measurements the resistance of 50 Ω is sufficient to absorb practically the entire power of the incoming signal, so it can be qualitatively stated that the measured signal corresponds to the intrinsic response of the circulator. Before starting the measurements, an OSTL (Open-Short-Through-Load) calibration must first be performed for each port with a substrate suitable for the coplanar probes (calibration kit). This calibration will enable the analyzer to correct any systematic measurement errors, such as cable and connector errors. Afterwards, we can measure the Sij parameters to visualize the circulation phenomenon for the coplanar circulator. To check the repeatability during the microwave measurement, the Load was connected to every port. The same microwave response was observed, this enables to draw a clear conclusion about the presence of the non-reciprocity phenomenon. Although this first encouraging result, the high losses (5.57 dB) are mainly due metallic losses according to the low substrate thickness, and ferrite nanowires exhibiting more losses in the non-statured state and worked near the gyromagnetic resonance. The magnetic state of the CoFe2O4 nanowire are certainly multidomain. Owing to its atomic structure, the cobalt ferrite has an easy plane magneto-crystalline anisotropy, and the effective anisotropy of the magnetic composite substrate is rather a shape anisotropy according to the nanowire shape.

In this regard, it can be generally pointed out that there is a difference between the simulation and the experimental results for the S-parameters, but at the resonance frequency (35 GHz) there is a close agreement between HFSS predictions and VNA measurements. The observed difference for the S-parameters could be mainly explained by the incomplete Irashagi model used in this work, since our nano-composite is partially magnetized. This model considers the geometrical parameters of the nanowires and the magnetization ratio of the ferrimagnetic nano-composite. A magneto-dynamic model, that will take into account the shape, magnetocrystalline, and surface anisotropies and the dipolar, spin-orbit interactions, will be required in a future work in order to accurately simulate the microwave response of such coplanar topology of the circulator [START_REF] Carignan | Ferromagnetic nanowire metamaterials : Theory and applications[END_REF].

From this first analysis and experimental study on circulator based on ferrimagnetic nanowires, other studies are under consideration to improve the nano-composite properties. Using thicker alumina membranes could facilitate all the fabrication steps in the clean room (annealing, cooper deposition, surface etching, etc).

Moreover, the increase in thickness will increase the quantity of cobalt ferrite crystallites, which will be a bonus to improve the magnetic properties responsible for the enhancement of the microwave response.

Conclusion

In this study, we have electromagnetically embedded CoFe2O4 nanowires that exhibit a strong internal magnetic field. This nanowired substrate is selected for the fabrication of a microwave circulator. First, we have presented the theoretical model used to determine the permeability tensor of the ferrimagnetic nanocomposite. The general functioning and analytical modeling of the circulator in a stripline topology was demonstrated. This modeling leads to a preliminary rapid evaluation of the component performance and its dimensioning before switching to the coplanar topology. To complete the dimensioning study of the coplanar ciculator, several simulation tasks were carried out after the integration of the permeability tensor of the nanocomposite on HFSS software. Moreover, a characterization of the prototype component was performed using a vector network analyzer. A non-reciprocal effect of 3 dB was observed for the constructed coplanar circulator which demonstrates the feasibility of achieving a non-reciprocal effect using our ferrimagnetic substrate. Moreover, our realized experiments in the synthesis of nano-composite and fabrication in a clean room have enabled us to identify the technological challenges and foresee new possibilities to develop magnetic nano-composites applied to the design of self-biased circulator.

Fig. 1 .

 1 Fig. 1. Top and cross sectional view SEM images of AAO membrane.

Fig. 2

 2 Fig.2shows the chronoamperogram recorded during the electrochemical growth of CoFe2O4 within 20 nm-AAO membranes at an applied potential of -1.2 V. It can be observed from the recorded curve that in the early stages of growth when the diffusion layer is formed, the pores start to be filled by CoFe2O4 nanocrystallites. This is translated by practically quasi-static current density. In addition, the deposition is maintained for a duration of up to 40 minutes, in which the porous nanowires become completely filled.

Fig. 2 .

 2 Fig. 2. The chronoamperogram of the magnetoelectrochemical growth of CoFe2O4 into nanoporous AAO membrane at room temperature.

Fig. 3 .Fig. 4 ,

 34 Fig. 3. XRD spectra of elaborated CoFe2O4 nanowires.

Fig. 4 .

 4 Fig. 4. SEM image of CoFe2O4 nanowires and its elemental composition.

= 1 + 2 ( 4 )

 124 ) * ℎ (+ + +,)((+ + +,) -+ ) -. ( 2( ) * ℎ (+ + +,)((+ + +,) -+ ) -. (1 -) (By way of illustration, consider the following conditions: 1 = 0.18 4, + = 42 5 6,, = 0.017, = 50 % and ℎ = 100 %. Some results of this model with an orientation of magnetic crystallite moments varying from 30 % to 100 % have been studied. Figs 6, 7 and 8 show the variations of the components of , ! and # corresponding to the permeability tensor as a function of the average of the moments orientation ( = / ) of the CoFe2O4 nanocrystallites.

Fig. 6 .Fig. 7 .

 67 Fig. 6. Evolution of μ components as a function of CoFe2O4 nanocrystallites orientation.

Fig. 8 .

 8 Fig. 8. Evolution of # components as a function of CoFe2O4 nanocrystallites orientation.

Fig. 9 .

 9 Fig. 9. Structure of the Y-junction stripline circulator.

Fig. 10 .

 10 Fig. 10. Evolution of the effective permeability for CoFe2O4 nano-composite as a function of frequency.

Fig. 11 .

 11 Fig. 11. S-parameters magnitudes for the stripline circulator; analytical calculation.

Fig. 12 .

 12 Fig. 12. Coplanar topology; (a) coplanar circulator with a bottom ground plane, (b) top view of the coplanar circulator, (c) cross sectional view of the CPW of the circulator, (d) A-B cross-sectional view of the circulator with ferrimagnetic substrate, (e) schematic distribution of the RF fields for a CPW line, (f) field displacement in the center of the circulator with coplanar junction.

Fig. 13 .

 13 Fig. 13. Simulated coplanar circulator on HFSS.

Fig. 14 .

 14 Fig. 14. S parameters magnitudes of the simulated coplanar circulator.

Fig. 15 .

 15 Fig. 15. Technological steps for the fabrication of the circulator in the clean room.

Fig. 16 .

 16 Fig. 16. Printed coplanar circulators on the nano-composite surface.

Fig. 17 .

 17 Fig. 17. Microwave characterization bench; measurement equipments and probes configuration.

Fig. 18 .

 18 Fig. 18. Measured S parameters magnitudes of the fabricated coplanar circulator.

Fig. 18

 18 Fig.18represents the S parameters, measured on a frequency band from 15 to 65 GHz of the coplanar circulator made of the ferrimagnetic substrate, which has been separated into two components: reflection and transmission parameters. It should be noted that no magnetic field was applied during this phase. As shown in Fig.18, the reflection curves at port 1 (Q .. ) and port 2 (Q ) show a good matching in the measurement frequency band.Moreover, the ferrimagnetic nano-composite confers to the circulator its non-reciprocity. The reflection level is -19.3 dB at 35 GHz. The transmission levels are -5.57 and -8.59 dB for the insertion (Q . ) and isolation (Q . ) loss, respectively. Furthermore, it is observed that the non-reciprocal phenomenon is present almost over the range of 25-45 GHz, but the frequency that shows the lowest reflection is 35 GHz as predicted by HFSS. This information can be exploited as an additional tool to design multi-band circulators. For the present fabricated prototype. The non-reciprocal phenomenon is about 3 dB and the circulation phenomenon is observable around 35.5 GHz. The observed non-reciprocal effect (Q . ≠ Q . )) appears to be small but is a first indication of the possible circulation function.

Acknowledgment

The authors acknowledge the financial support of PHC-Toubkal under the project TBK/85/17, Campus France: 38983UH.