Endocrine and molecular regulation of oviposition sequence and eggshell biomineralization in commercial poultry: mechanisms, genetic control, and nutritional interventions: A review

Introduction

Commercial layers have been subjected to relentless genetic selection for increased egg production, prolonged persistency of lay, and efficient feed conversion over the past few decades. Modern commercial hybrids can now lay more than 300 eggs per year, and long-cycle hybrids targeted to lay approximately 500 eggs over 100 weeks have recently been developed [1][2][3] . The advancements in genetic potential have resulted in increased productivity and profitability of the poultry industry but have also magnified the physiological challenges posed to the reproductive and skeletal system of thelaying hen [4] [5] [4,5] [4] [5] . Thus, the challenge of maintaining eggshell quality in long laying cycles has become one of the most important issues in modern egg production [6][7][8] . Eggshell quality is a primary determinant of biological performance and economic return because defects in the shell increase egg breakage, downgrade rates, microbial contamination, and losses of hatchability in breeder flocks, as well as financial losses through the production chain [7][8][9] . Furthermore, to support uninterrupted eggshell formation, a close to 2.0-2.5 g quantity of calcium carbonate must be deposited per day, which places a significant demand on calcium absorption in the intestine, medullary bone reserves, endocrine regulation, and uterine mineralization mechanisms. With the aging of laying hens, these mechanisms progressively become less efficient; therefore, shell thinness, weakness, and variability in ultrastructure gradually become more pronounced. Sequential oviposition is not just a repetitive biological process but rather the result of a well-coordinated system integrating neuroendocrine function, follicular hierarchy, ovulation, oviductal transport, uterine ion exchange, and controlled biomineralization [10][11][12][13] . Each ovulation under normal physiological conditions should take place within an endocrine window of proper regulation after the oviposition of the previously laid egg to maintain the sequence of clutch and to ensure continuous egg production [14,15] . Even minor perturbations in hormonal synchronization, uterine ion transport, calcium metabolism, or extracellular matrix formation can disrupt oviposition timing, shell mineralization, and hence eggshell quality [16][17][18] . Recent progress in reproductive endocrinology, molecular biology, and high-throughput omics technologies has brought a much better understanding of the mechanism behind oviposition and the formation of the eggshell [19][20][21][22] . Studies on transcription, protein, metabolism, epigenetics, and single-cell sequencing have identified many genes, signaling pathways, and regulatory networks for calcium transport, bicarbonate secretion, organization of the extracellular matrix, nucleation of calcite crystals, response to oxidative stress, regulation of the immune response, and differentiation of uterine epithelial cells [23][24][25][26][27] . These findings have changed the strategy of commercial breeding from the selection based on conventional phenotypes to selection based on genomics and pathways to improve shell quality, persistence of laying, and skeletal health [28][29][30] . Precision nutritional support for eggshell formation over longer production cycles has also come to be of greater importance. At present, split-feeding systems, optimized calcium particle size, evening calcium supplementation, vitamin D metabolites, and trace minerals including probiotics and phytogenic additives, as well as other functional feed ingredients, are being implemented to synchronize nutrient availability with the hen's circadian pattern of shell calcification. All these would improve calcium utilization and reduce skeletal depletion [31][32][33][34][35] . In fact, there have been major advances in single research sectors, but current knowledge, covering endocrinology, reproductive physiology, molecular genetics, biomineralization, and poultry nutrition, remains fragmented [3638] . A comprehensive synthesis is still limited, particularly for the modern high-producing laying strains maintained under extended laying cycles [39] . Therefore, this review concentrates on the crosstalk between endocrine regulation of sequential oviposition and uterine molecular biology, ion transport, biomineralization, and precision nutritional management in the determination of eggshell competence in the modern commercial laying hen. Specific consideration is directed toward the means by which the hormonal signal pathways are linked with molecular mechanisms, recent multi-omics technologies, and realizable measures for improving eggshell quality, laying persistency, and long-term reproductive efficiency.

Endocrine Regulation of Sequential Oviposition 2.1 The Hypothalamic-Pituitary-Gonadal Axis

The egg-laying cycle in commercial laying hens is mainly controlled by the hypothalamic-pituitary-gonadal (HPG) axis. This axis integrates photoperiodic information, circadian rhythms, and metabolic status, as well as ovarian feedback on follicular development and ovulation [1,[6][7][8] . Light stimulation of hypothalamic neurons via retinal and extra-retinal photoreceptors leads to the release of gonadotropin-releasing hormone (GnRH) which in turn stimulates the anterior pituitary to secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) [1] . Follicular recruitment and establishment of the preovulatory hierarchy depend primarily on FSH responsiveness. Terminal maturation of the dominant follicle is increasingly governed by LH-induced steroidogenesis and progesterone secretion [1,7,8] .

Hormonal Control of Ovulation

The hallmark endocrine event that precedes ovulation is the tightly coordinated preovulatory rise in circulating levels of progesterone and LH. Progesterone, which is synthesized by the granulosa cells of the largest preovulatory follicle, was later shown by Scanes [1] to act through positive feedback on the hypothalamus and pituitary to induce the characteristic LH surge in an estrogen-dependent endocrine system. This surge represents the immediate hormonal trigger for ovulation and occurs several hours before rupture of the follicle. Ovulation usually takes place about 30 minutes after the laying of the last egg, and the interval between successive oviposition generally varies from 24 to 27 h in commercial chickens [1,7] . Such precise endocrine coordination accounts for the formation of egg-laying sequences (clutches) [1] [7] where successive oviposition take place on consecutive days until the ovulatory rhythm is interrupted by a pause day [1] .

Endocrine Regulation of Follicular Hierarchy

The follicular hierarchy has to be constantly reestablished for sequential oviposition to occur. Normally, the largest yellow follicle (F1) ovulates first. The smaller follicles will then sequentially advance through the hierarchy to replace the ovulated follicle. At the cellular level, FSH increases granulosacell differentiation by enhancing cyclic AMP (cAMP) signalling, steroidogenic acute regulatory protein (STAR) expression, cholesterol transport, and progesterone biosynthesis [1, 40] . Apart from gonadotropins, several growth factors that act locally regulate the selection of follicles before the formation of hierarchy. The members of the superfamily of transforming growth factor-β (TGF-β), such as anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMP2, BMP4, and BMP6), growth differentiation factor-9 (GDF9), and inhibins, perform the functions of modulation on granulosa-cell proliferation, steroidogenic capacity, differentiation of follicles, and survival of follicles [1,[40][41][42] .

Neuroendocrine and Circadian Regulation

Sequential oviposition is further modulated by the complex neuroendocrine pathways which integrate stimulatory and inhibitory hypothalamic signals. Norepinephrine, vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) stimulate hypothalamic GnRH secretion. Secretion of dopamine and gonadotropin-inhibitory hormone (GnIH) suppresses gonadotropin release and delays ovulation. New evidence also suggests that indirect involvement in avian reproductive neuroendocrine function may be a property of kisspeptin signaling, although its physiological role is far less defined than in mammals. Circadian regulation is another key factor in ovulatory timing. Clock genes such as BMAL1, CLOCK, PER2, and CRY1 have been detected in ovarian follicles and tissues of the oviduct, suggesting that endocrine events are not directly initiated by mechanical events associated with oviposition but are rather synchronized with endogenous biological rhythms [1,43,10] . However, the exact mode of molecular communication between the ovary and the oviduct for synchronization of the next ovulation with completion of the previous laying cycle is not clear [1] .

Hormonal Regulation of Oviposition

The final stage of the reproductive cycle oviposition is initiated by relaxation of the uterovaginal sphincter along with rhythmic contraction of the smooth uterine muscle, actions primarily mediated by arginine vasotonic (AVT), the principal avian neurohypophysial hormone. AVT acts synergistically with locally synthesized prostaglandins, particularly prostaglandin F₂a (PGF₂a) , to stimulate myometrial contractility and facilitate egg expulsion. Oviposition, therefore, is not an isolated mechanical event but rather the terminal phase of an integrated endocrine cycle that simultaneously prepares the ovary for the next ovulation to ensure continuity of clutch formation in commercial laying hens. These facts were elegantly summarized in a review by Perry and co-workers [7] [1] .

Fig 1:

Schematic representation of the neuroendocrine and oviductal events underlying sequential oviposition in commercial laying hens. It depicts photoperiodic and circadian inputs, HPG axis signaling, ovarian follicular steroidogenesis, preovulatory LH surge, ovulation, oviductal transport, uterine biomineralization, and hormonally regulated oviposition, integrated with each other. The schematic draws its basis from the mechanistic framework as described by Scanes [1], Sah and Mishra [2], Yang et al. [6], and Kaspers [7]. Table 1. Principal endocrine regulators involved in sequential oviposition and their physiological functions in commercial laying hens. Induces final follicular maturation, progesterone secretion, ovulation, and maintenance of clutch sequence [1, 7] Progesterone (P4) Granulosa cells of the F1 follicle

Hypothalamus and pituitary

Triggers the preovulatory LH surge through positive feedback and initiates ovulation [1, 9] Estradiol (E2) Ovarian follicles Oviduct, liver, hypothalamus

Stimulates oviduct development, medullary bone formation, vitellogenesis, and progesterone responsiveness [1, 6, 41] Arginine vasotocin (AVT) Neurohypophysis Uterine smooth muscle and uterovaginal junction Induces uterine contraction and facilitates oviposition [7, 11] Prostaglandin F₂α ( PGF₂α ) Shell gland (uterus) Uterine smooth muscle Acts synergistically with AVT to stimulate myometrial contraction during oviposition [7, 12] Neuropeptide Y (NPY) Hypothalamus GnRH neurons Stimulates GnRH secretion and supports reproductive endocrine activity [1, 44] Norepinephrine

Central nervous system

Hypothalamic GnRH neurons

Enhances GnRH release and facilitates gonadotropin secretion [1, 44] Gonadotropininhibitory hormone (GnIH)

Hypothalamus

Pituitary and GnRH neurons

Suppresses LH and FSH secretion and delays ovulation [1, 45] Dopamine Hypothalamus Pituitary Inhibits gonadotropin secretion and modulates reproductive timing [1, 44] Melatonin Pineal gland Hypothalamus and ovary Synchronizes circadian rhythms with photoperiodic regulation of reproduction [6, 43]

Oviductal Progression and the Dominant Role of the Shell Gland

Following ovulation, the ovum enters the oviduct where it undergoes a very wellorganized series of structural and biochemical changes that eventually result in the formation of a true egg [7] [2] . Each anatomical region of the oviduct expresses specialized physiological functions closely coordinated with endocrine regulation and the progressive maturation of the egg. The infundibulum picks up the ovulated ovum; in breeder hens it helps to pick up sperm for fertilization and initiates formation of the vitelline membrane. It is in the magnum where most of the albumin proteins are synthesized and secreted. The isthmus is the site where the inner and outer shell membranes are deposited providing the structural scaffold on which the mineralized eggshell will later develop. The egg finally enters the uterus, which is commonly referred to as the shell gland; this is where extensive deposition of calcium carbonate occurs to form the hard eggshell [13,7,2] . The temporal distribution of egg transit through the oviduct would obviously reflect the dominant physiological importance of the shell gland. Kaspers [7] claims that ovum remains in the infundibulum for about 15 to 30 minutes, in the magnum for about 3 to 4 hours, and in the isthmus for 1 to 2 hours before going to the shell gland where it stays for about 18 to 20 hours. Thus, nearly three-quarters of the entire ovulatory-ovipositional cycle is devoted to uterine residence and eggshell formation [7] . This length of a retention period obviously underscores the extremely complex nature of shell biomineralization continuous transport of calcium, secretion of bicarbonate, assembly of extracellular matrix, control over crystal nucleation, and progressive maturation of the ultrastructure of the shell. Instead of being a passive reservoir for calcium deposition, the shell gland is described as a highly specialized biomineralization organ that is endocrinologically regulated so that epithelial transport mechanisms, extracellular matrix proteins, acid-base balance, and immune defense are integrated for the production of a mechanically competent eggshell. Active transcellular calcium transport by uterine epithelial cells includes calcium channels, ATP-dependent calcium pumps, carbonic anhydrase for bicarbonate generation, and matrix protein secretion controling calcite crystal orientation and shell microarchitecture. Therefore, the quality of the shell will depend not only on how much calcium is deposited but also on how well these molecular events take place at the right time. Thus, the time the egg stays in the uterus is one of the main factors that determine the quality of the eggshell. Changes in the timing of hormones, environmental stress, aging, imbalance in food, or damage to the ways the uterus moves can all make the time an egg is in the shell gland shorter or longer. This will then affect shell thickness, strength, how the crystals are arranged, and how the cuticle is laid down [18] [16] [1] . Even quite small differences in how well ovulation is matched with the time the shell forms can result in eggs with poor shell strength even though egg production seems normal. From a commercial perspective, the shell gland is the primary physiological bottleneck in the quality of the eggshell for modern laying hens. Genetic selection has markedly increased egg number; thus, the capacity of the uterus to sustain efficient biomineralization has become even more critical for shell integrity during extended laying cycles [19] [17] . Therefore, knowledge of the endocrine regulation and molecular physiology of the shell gland will provide the biological basis for the development of genetic, nutritional, and management strategies that will improve eggshell quality and increase the productive lifespan of current poultry strains.

Molecular Basis of Eggshell Biomineralization

The process of forming eggshells is not simply a passive one of carbonate calcium precipitating, but rather a highly coordinated biomineralization event. It is regulated via endocrine signals, uterine epithelial transport, extracellular matrix proteins, and the exact physicochemical conditions [5,6,15] . Specialized organic macromolecules and mineral ions are secreted within the shell gland into the uterine fluid. This provides a cell-free mineralization environment where nucleation of calcite crystals can take place, as well as growth and assembly into the highly organized structure of the eggshell [5] . Brionne et al. [5] described the process of eggshell formation as a model system of biological biomineralization since microstructure, mechanical strength, and fracture resistance of the shell are determined by continuous interactions between inorganic mineral fluxes and the organic matrix. The mineral phase of the eggshell is primarily composed of calcium carbonate (CaCO₃) in the calcite polymorph, which makes up about 95-97% of the weight of the shell; the rest is made up of organic matrix proteins, glycoproteins, proteoglycans, lipids, and water [5,15,20] . Organics influence crystal nucleation and crystal orientation mamillary cone formation palisade layer organization and cuticle deposition, though they represent a small fraction of the total mass of the shell [5,16] . Therefore, shell quality does not only depend on the The schematic shows the egg's movement in order through the infundibulum, magnum, isthmus, uterus (shell gland), and vagina, underscoring the extended uterine stay during the formation of the eggshell and its importance in determining the final quality of the shell. Reference to Scanes [1] [1] , Yang et al. [6] , and Kaspers [7] . Vagina Minutes Egg expulsion (oviposition) and temporary storage before laying Mucus and lubricating secretions [7, 11] quantity of mineral matter deposited but also on molecular regulation of crystal architecture. Uterine calcification requires an uninterrupted supply of calcium and bicarbonate. In the process of shell formation, calcium is supplied mainly from intestinal absorption; however, in cases where the dietary availability of calcium is not able to meet the very high mineral requirement, about 20-40% of the needed calcium may be mobilized from medullary bone [7] . Kaspers [7] estimated about 2.0-2.5 g of calcium to be needed for each eggshell, which depicts the remarkable mineral turnover that occurs daily in modern laying hens. Efficient formation of the eggshell is hence closely related to skeletal metabolism, endocrine regulation, and homeostasis of calcium. At the cellular level, uterine epithelial cells take an active part in the process of calcium transport from the blood into the uterine lumen by systems of transcellular transport coordinated [15, 17] . The major site of entry of calcium into epithelial cells is through apical calcium channels, one of which is transient receptor potential vanilloid 6 (TRPV6); it then binds to intracellular calcium-binding proteins like calbindin-D28k (CALB1), which facilitates cytoplasmic calcium diffusion while it checks any possibility of cytotoxic accumulation [15,21] . Plasma membrane Ca² -ATPases (ATP2B1) and sodiumcalcium exchangers provide the⁺ final step in the extrusion of calcium into the uterine fluid for continuous supply as and when needed for calcite deposition [15,21,22] . At the same time, bicarbonate ions that are needed for the synthesis of calcium carbonate are prepared by hydration of carbon dioxide in uterine epithelial cells via carbonic anhydrase II (CA2) [17,22] . Bicarbonate then gets into the uterine lumen by the action of anion exchangers and bicarbonate transporters, such as some members of the SLC26 family, where it joins with calcium ions to form calcite crystals [17,22] . Proper crystal growth kinetics as well as the prevention of abnormal shell mineralization depend on the strict control of luminal pH and ionic composition.

The organic matrix is believed to provide a molecular template for the initiation of crystal growth and to control the ultrastructure of the shell. The most thoroughly characterized matrix proteins are ovocleidin-116 (OC-116), ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), osteopontin (SPP1), and ovotransferrin, each contributing to different stages in the formation of calcite crystals and their subsequent maturation [5,16,23] . Instead of being structural proteins, these macromolecules determine the morphology of the crystals, the orientation of the crystals, the rate at which mineral is deposited, and the ability to resist crack propagation as an index of mechanical competence of the mature eggshell [5,23,24] .

From recent studies at the transcriptomic and proteomic levels, it has been established that the very early stages of shell calcification play a more important role in determining final shell quality [15,[24][25][26] . A differential expression of genes related to calcium transport, synthesis of the extracellular matrix, oxidative phosphorylation, and ion homeostasis was found to be associated with marked differences in shell strength among commercial laying strains [24][25][26] . In fact, organization of the mammillary layer has appeared as one of the strongest structural predictors of shell mechanical properties; hence, quality of the eggshell is determined at the very early stages of uterine mineralization rather than only during later thickening of the shell [24] [5] . These findings show that eggshell biomineralization is a closely coordinated biological process involving endocrine regulation, epithelial ion transport, assembly of the extracellular matrix, nucleation of crystals, and the metabolism of skeletal calcium.

In other words, these molecular mechanisms are the basis for future genetic improvement as well as precision nutrition and management strategies for enhancing eggshell quality and sustaining reproductive performance of modern commercial laying hens. 3. Principal molecular components involved in eggshell biomineralization and their physiological functions.

Table 3: Principal molecular components involved in eggshell biomineralization and their physiological functions.

Molecular component Primary location Principal function Representative references TRPV6

Uterine epithelium Apical Ca²⁺ uptake [15, 21] CALB1 (Calbindin-D28k) Uterine epithelial cytoplasm Intracellular Ca²⁺ transport and buffering [15, 21] ATP2B1 Basolateral membrane Ca²⁺ extrusion into uterine fluid [15, 22] Carbonic anhydrase II (CA2) Uterine epithelial cells HCO₃⁻ generation [17, 22] SLC26 transporters Uterine epithelium Bicarbonate secretion [17, 22] Ovocleidin-116 (OC-116) Eggshell matrix Crystal nucleation and shell organization [5, 23] Ovocalyxin-32 (OCX-32) Eggshell matrix Matrix stabilization and shell defense [23, 24] Ovocalyxin-36 (OCX-36) Eggshell matrix Shell maturation and antimicrobial activity [23, 24] Osteopontin (SPP1) Eggshell matrix Regulation of calcite crystal growth [5, 23]

Calcium Transport and Intracellular Handling

High rates of calcium carbonate deposition during eggshell formation also point to the need for an effective, tightly regulated transcellular calcium transport system within the uterine epithelium. There is no passive diffusion in the movement of calcium from the maternal circulation to the uterine lumen, but rather, a network of membrane channels, intracellular calcium-binding proteins, ATP-dependent pumps, and ion exchangers, all of which work in a coordinated fashion to achieve continuous calcium delivery while maintaining intracellular calcium homeostasis [3,5,15] . Jonchère et al. [3] provided a comprehensive uterine calcium ion-transport system in which TRPV6 was the primary apical calcium channel for uptake of calcium from the extracellular environment into uterine epithelial cells. Once in the cytoplasm, calcium is immediately bound by calbindin-D28k (CALB1). This facilitates intracellular calcium diffusion while also preventing an excessive rise in free cytosolic Ca² concentration [5] [3] [5] [3] . This⁺ intracellular buffering system allows rapid trafficking of calcium towards the basolateral membrane without inducing cellular toxicity mediated by calcium. Subsequently, calcium efflux to the uterine fluid is mediated by plasma membrane Ca² -⁺ ATPases, primarily ATP2B1, ATP2B2, and ATP2B4 (PMCA isoforms), also known as the isoforms of PMCA, which actively transport calcium against its electrochemical gradient by ATP hydrolysis. In parallel, sodium/calcium exchangers (SLC8A1 and SLC8A3) contribute to calcium efflux and help sustain the high extracellular calcium concentration, over which there is continuous growth of calcite crystals within the uterine lumen [3] . This underlines the importance of intracellular calcium buffering; this is further supported by the very high expression of CALB1 in the uterus during the active phase of shell calcification. Brionne et al. [5] showed a large increase in CALBtranscripts during eggshell formation, which points to its major role in the control of calcium homeostasis in such high mineral flux conditions. In a previous study, Jonchère et al. [3] proposed that calbindin works together with other systems responsible for the storage and release of intracellular calcium such as sarco-endoplasmic reticulum Ca² ATPases (ATP2A2 and ATP2A3) and inositol 1,4,5-⁺ trisphosphate receptors (ITPRs) to control the cycling of intracellular calcium, refill endoplasmic reticulum calcium stores and protect uterine epithelial cells against calcium overload in fast biomineralization.

Together, these molecular transport systems make sure that large amounts of calcium are brought near the mineralization front with maximum efficiency while keeping the cells alive and their metabolism stable. Therefore, eggshell quality depends not only on the total amount of calcium available but also on the ability of uterine epithelial cells to quickly take up, buffer, recycle, and secrete calcium in a highly organized way.

Any disruption to this network may lead to inadequate calcium availability at the mineralization site, thereby reducing shell thickness and strength as well as overall eggshell quality [3,5,15] .

Jonchère et al. [3] identified CAas one of the most highly expressed enzymes in the uterine epithelium during active shell calcification, underlining its major function in sustaining the very high demand for bicarbonate associated with continuous deposition of calcium carbonate. Bicarbonate, once formed, is transported across the epithelial membrane by several anion transport systems, including SLC26A9 and members of the SLC4 bicarbonate transporter family, to enrich uterine fluid with bicarbonate ions which are required for the formation of calcite.

The uterine ionic microenvironment is more than just the availability of bicarbonate. It was argued earlier that since

CaCOcontinuously generates H ions, in the absence of an⁺ effective pathway for removal of these protons, acidification would occur intracellularly and, in the lumen, [15] [3] . The authors went on to show later that systems involved in proton extrusion, like the Na /H exchangers and the vacuolar H ATPases, are of critical importance⁺ ⁺ ⁺ for homeostasis at intracellular pH and for support to mineralization. Thus, production of bicarbonate and removal of protons are tightly coupled physiological processes, not independent mechanisms. Transcriptomic data from recent studies highlight the fact that the regulation of acid-base parameters is important at the initial stage of eggshell formation. When Zhang et al. [4] compared the hens that laid eggs with strong shells to those that laid eggs with weak shells, they observed a significant upregulation of bicarbonate transport genes (SLCA1, SLCA2, and SLCA9) and proton pump subunits (ATP6V0D2, ATP6V1B, ATP6V1C2, and ATP6V1G3) during the initiation phase of calcification in the former group. They proposed that an ionic environment best suited for efficient nucleation of crystals and early formation of the mammillary layer would be one in which bicarbonate is secreted and protons are extruded.

Eggshell quality depends on calcium availability and the physicochemical properties of uterine fluid. Proper regulation of bicarbonate concentration, luminal pH, ionic gradients, and electrochemical balance determines kinetics of crystal growth, matrix-mineral interactions, and eventually ultrastructural organization of mature eggshell. Weak eggshells can be formed even with adequate calcium supply if the ionic microenvironment of the uterus does not support the efficient biomineralization process.

Fig 5:

Bicarbonate generation, proton transport, and regulation of the uterine ionic microenvironment during eggshell biomineralization. This is a schematic which shows intracellular production of bicarbonate by carbonic anhydrase II (CA2), bicarbonate secretion via SLC26A9 and SLC4 transporters, and proton extrusion by Na /H exchangers and members of the ATP6V family of vacuolar H^+-ATPases. This leads⁺ ⁺ to an optimal luminal ionic environment for nucleation of calcium carbonate crystals and eggshell formation.

Matrix Proteins and Crystal Architecture

It is the organic eggshell matrix that provides the molecular framework through which nucleation, growth, orientation, and termination of calcite crystals are to be governed. In biomineralization, crystal formation is thus seen to be guided both spatially and temporally by the eggshell matrix. This matrix was considered merely as the scaffolding structure on which mineral deposition takes place passively; recent work has demonstrated its active participation in regulating crystal shape, kinetics of mineral deposition, ultrastructure of the shell, and mechanical properties of the mature eggshell [5,4,15] . Thus, eggshell quality results not only from the available minerals but also from an organized expression and secretion of matrix proteins over the entire process of calcification. Proteomic studies have shown that active shell formation leads to a very complex mixture of extracellular proteins present in the uterine fluid. Brionne et al. These authors identified 57 proteins secreted into the uterine fluid during the stage of calcification, most of them related to participation in the assembly of extracellular matrix, crystal nucleation, proteoglycan biosynthesis, molecular chaperoning, antimicrobial defense, and regulation of mineral deposition. These proteins are responsible for creating a dynamic molecular environment that controls both the initiation and subsequent maturation of calcite crystals.

The most frequently cited matrix protein in the literature as a major player in eggshell mineralization is ovocleidin-116 (OC-116). It is therefore assumed that ovocleidin-116 is first deposited in the mammillary layer as a structural template that will later facilitate appropriate nucleation of calcite crystals, their orientation, and uniformity of microarchitecture of the shell. High levels of expression of OC-116 have generally been related to good shell organization and high mechanical strength. Osteopontin (SPP1) is mainly a negative regulator of calcium carbonate crystal growth. In fact, osteopontin was shown to specifically bind the surface of the growing calcite crystals in order to limit their overgrowth and reshape the morphology of the crystal by Brionne et al. This work was later confirmed by Zhang et al. [8] SPP1 was reported to be significantly higher in hens with weak-shelled eggs at the onset of calcification, a finding that strongly indicates excessive inhibitory controls during the formation of the mammillary layer, resulting in poorly organized crystals and weak shell [8] .

Other matrix proteins include ovocalyxin-32 (OCX-32), ovocalyxin-36 (OCX-36), ovotransferrin, ovocleidin-17 (OC-17), clusterin, and several proteoglycans; they contribute to crystal stabilization, antimicrobial protection, extracellular matrix organization, and shell maturation [5,[23][24][25] . Even though they have different individual biological functions, these proteins maintain the balance between crystal nucleation, crystal growth, and matrix remodeling; thus, they ensure the formation of a mechanically competent eggshell. From the available evidence, one can therefore argue that the quality of the eggshell depends on a regulatory model wherein there is coordinated interaction between mineral transport and extracellular matrix proteins rather than calcium deposition alone. It is proposed that proper mechanical strength and integrity of the eggshell can only be attained when both crystalpromoting proteins (e.g., OC-116 and OC-17) and crystal-modulating proteins (e.g., SPP1) are precisely temporally expressed to allow for formation of an ordered mammillary layer and maintenance of appropriate crystal orientation [5,4] .

Matrix protein Gene Primary function Effect on crystal architecture Representative references

Ovocleidin-116 OC-116 Crystal nucleation and matrix organization Promotes ordered calcite deposition [5, 6] Ovocleidin-17 OC-17 Initiation of crystal nucleation Directs early calcite crystal formation [5, 23] Osteopontin SPP1 Regulation of crystal growth Inhibits excessive crystal growth; elevated in weak shells [5, 4] Ovocalyxin-32 OCX-32 Matrix stabilization and antimicrobial activity Supports shell maturation [23, 24] Ovocalyxin-36 OCX-36 Matrix remodeling and innate immunity Maintains shell integrity [23, 24] Ovo transferrin TF Iron-binding glycoprotein Matrix stabilization and antimicrobial defense [5, 24] Proteoglycans Multiple genes Extracellular matrix assembly Regulate crystal spacing and orientation [5] Molecular chaperones Multiple genes Protein folding and matrix assembly Maintain proper matrix organization [5]

Why Early Calcification Determines Final Shell Quality

Advances in transcriptomics and the study of eggshell ultrastructure have provided new insight into the biological determinants of shell quality. It is now believed that, rather than being formed progressively over the entire period of calcification, the earliest stages of uterine mineralization have the most significant effect on the structural integrity and mechanical properties of the mature eggshell [4,15,24] . In this context, it is proposed that the initiation phase of calcification should be considered a critical period during which the architecture of the shell is biologically programmed.

In a comparative uterine transcriptomic study, Zhang et al. [4] identified 1,777 differentially expressed genes (DEGs) during the initiation stage of calcification in hens that produce hardshelled and weak-shelled eggs. The authors observed relatively few differences in transcription between the subsequent rapid growth and termination phases of shell formation. This suggests that most molecular mechanisms for quality determination are not primarily activated at the onset of biomineralization but much later during crystal thickening. Ultrastructural analysis further supported these molecular findings. Hens that produce eggs with superior mechanical strength present significantly thinner mammillary layers, a smaller width of mammillary knobs, and a more organized transition between the mammillary and palisade layers. Since mammillary cones represent the primary nucleation sites for calcite crystals, the spatial organization of these structures will determine subsequent crystal orientation, as well as crystal fusion, and vertical column development throughout the palisade layer. Therefore, even minor irregularities established during the initial nucleation process shall be considered to spread throughout the entire shell if not to reduce fracture resistance despite apparently normal shell thickness [4] . Transcriptomic evidence indicates that successful early calcification involves coactivation of multiple biological pathways, including calcium transport, secretion of bicarbonate, assembly of the extracellular matrix, regulation of acid-base, energy metabolism of mitochondria, organization of cytoskeletons, and differentiation of epithelial cells [3,5,4] . It suggests shell quality results from integrated regulation of mineral transport and crystal nucleation-not isolated changes in calcium availability. This was differentially expressed genes for bicarbonate transporters (SLCA1, SLCA2, and SLCA9), ATP6V family proton pumps, TRPV6 and CALB1 calcium transport proteins, and matrix proteins such as OC-116 and SPP1.

These findings substantially change the classic concept of eggshell weakness in the second half of the laying period.

Previously, low dietary calcium or an inability of the intestine to absorb calcium was mostly blamed for poor shell quality. However, molecular evidence at present indicates that shell defects may occur due to disruption of the uterine microenvironment at the very early stage of calcification, including problems with crystal nucleation, extracellular matrix organization, bicarbonate transport, proton extrusion, or epithelial homeostasis. In such a situation, if the molecular machinery of biomineralization is already compromised, an increase in dietary calcium alone may not fully restore the quality of the shell. Thus, from a practical point of view, these results imply that the interventions aiming to sustain the physiological environment during the early stages of calcification, such as uterine ion transport optimization, and maintenance of acid-base balance, synthesis of matrix proteins, and nutritional strategies in precision, and genomic selection for biomineralization-related genes are likely to be more effective than those that focus primarily on calcium intake. Therefore, the initiation phase of eggshell calcification should be considered the main biological target for future genetic, nutritional, and management strategies aimed at improving shell quality and extending laying persistency in modern commercial hens [4,15,24] . 7. Biological characteristics distinguishing early calcification in strong-and weakshelled eggs.

Extensive activation during initiation

Reduced or dysregulated expression

Altered biomineralization pathways [4] Mammillary layer thickness Thinner, well organized Thicker, irregular Lower fracture resistance [4] Mammillary knob width Smaller and uniform Larger and variable Poor crystal fusion [4] Crystal nucleation Efficient and synchronized Delayed or disorganized Reduced shell strength [5, 4]

Matrix protein regulation

Balanced OC-116 and SPP1 expression

Increased inhibitory signaling (SPP1) Abnormal crystal architecture [5, 4] Bicarbonate transport Efficient Reduced Suboptimal ionic microenvironment [3, 4] Final shell quality High mechanical Increased brittleness and Economic losses [4]

Genetics, Modern Layer Strains, and Long-Life Shell Efficiency

The remarkable increase in egg production achieved over the past few decades has been due to genetic selection for egg number, feed efficiency, sexual maturity, and laying persistency. Modern commercial layers can maintain high production rates for over 90-100 weeks, with some breeding programmers aiming at cumulative production approaching 500 eggs per hen housed [6,9,25] . This unprecedented reproductive performance has, however, put increased physiological pressure on calcium metabolism, skeletal integrity, uterine function, and eggshell biomineralization. Long-term shell quality has thus become one of the major constraints in prolonged laying cycles [6, 25] .

Phenotypic selection for shell thickness, shell breaking strength, shell weight, and specific gravity was the primary focus of traditional breeding programmers. Success was achieved in the improvement of shell quality by these methods; however, very little information was attained regarding the intricate molecular mechanisms that control biomineralization. Genomics, transcriptomics, proteomics, metabolomics, and systems biology have redefined poultry breeding by showing that eggshell quality is determined by the coordinated action of biological pathways, rather than by individual genes acting independently [26] . Yang et al. [6] stressed that quality of shell is regulated by interconnected functions involving calcium transport, bicarbonate metabolism, assembly of extracellular matrix, endocrine regulation, immune function, responses to oxidative stress, metabolism of mitochondria, and differentiation of epithelium in the uterus. As a result, the selection of the pathway at the genomic level has become more important than the candidate genes. Some other genes have been identified that play a key role in the regulation of eggshell mineralization. These genes are responsible for calcium transportation (TRPV6, CALB1, and ATP2B1), production of bicarbonates (CA2, SLC26A9, and SLCfamily), and formation of the extracellular matrix (OC-116, OC-17, SPP1, OCX-32, and OCX-36) [3,5,4,6] . These same genes have also been linked with strength, thickness, organization of crystals, and efficiency in uterine calcification. More candidate genes that indirectly affect persistency of laying and quality of shell by regulating the follicular hierarchy, steroidogenesis, hypothalamic-pituitary-gonadal (HPG) [3] [5] [4] signaling, lipid metabolism, mitochondrial function, and innate immunity are also included [6,27] . Genetic studies also reveal that eggshell quality has moderateto-high heritability, though estimates of heritability vary among commercial populations based on breed, age, management system, and environmental condition [28] . The importance of genotype-byenvironment interactions increases during late lay, since, at this stage, age-related deterioration in uterine function and calcium homeostasis may undermine shell quality even when egg production is sustained at high levels. Thus, breeding strategies for extended laying cycles must also consider the preservation of reproductive longevity and skeletal health, as well as the biomineralization capacity of the uterus.

Long-life commercial laying strains are one of the most interesting challenges in contemporary poultry breeding. The ability to support optimal shell quality over production periods approaching 100 weeks reflects sustained physiological coordination among calcium metabolism, bone remodeling, endocrine regulation, ion transport, extracellular matrix synthesis, and mitochondrial energy production [25] . Thus, failure of any one biological system may accelerate shell deterioration during late lay even when egg production remains relatively stable. This has also recently been further shown by the latest advance in genomic prediction, that marker effects may differ much among chicken populations, that it is not possible to apply individual candidate genes universally [6] . Therefore, population-specific genomic evaluation, GWAS, wholegenome sequencing, GEBVs, and multitopic integration are growing in importance for commercial breeding programs to enhance the accuracy of predictions in the selection index [29,28,27,26] . Altogether, the current evidence indicates that future breeding for long-life shell efficiency should give top priority to pathway resilience rather than single-gene effects. In this regard, selection programs should aim at ensuring the coordinated functions of the hypothalamic-pituitary-gonadal axis and uterine ion transport, bicarbonate metabolism, extracellular matrix assembly, bone turnover, mitochondrial metabolism, oxidative stress resistance, and clutch stability over extended laying cycles. System-based breeding strategies of this nature are expected to improve eggshell quality, reproductive longevity, and production efficiency in next-generation commercial laying hens.

Practical Strategies to Improve Eggshell Efficiency in Commercial Layers 7.1 Precision Calcium Supply and Circadian Nutrition

Since eggshell calcification follows a clearly defined circadian rhythm, nutritional interventions are most effective when synchronized with the physiological pattern of shell formation in the hen rather than being uniformly supplied throughout the day [9] [8] [9] [8] . Approximately two-thirds of shell calcification occurs during the dark period, when uterine calcium demand is at its maximum [7] . Therefore, the timing of calcium availability has become more and more important as a determinant of eggshell efficiency in modern commercial laying hens [8] . Akter et al. [8] reported that the afternoon drinking water supplemented calcium and vitamin D as an alternative to the high-calcium feeding program. In a 10-week trial, hens on a basal diet with 4.1% calcium and targeted afternoon supplementation attained laying performance similar to that of birds on a conventional 4.5% calcium diet [8] . This also raised serum vitamin D levels significantly, improved ileal energy digestibility, reduced water intake, and enhanced feather condition-all signs of better nutrient use and general physiological condition [8] . These results imply that it is possible to synchronize calcium delivery according to the period of maximum shell mineralization so that calcium utilization is optimized and undesirable morning calcium intake is avoided [8] . Bicarbonate metabolism CA2, SLC26A9, SLC4A1/2/9 HCO₃⁻ production and transport Maintains optimal calcification environment [3, 4] Matrix proteins OC-116, OC-17, SPP1, OCX-32, OCX-36

Crystal nucleation and shell architecture Determines shell ultrastructure [5, 4, 6] HPG-axis regulation GnRH, FSHR, LHR, ESR1, PGR Endocrine regulation of ovulation Maintains clutch sequence [1, 6] Bone metabolism VDR, RANKL, OPG Calcium mobilization and skeletal homeostasis Sustains calcium availability [6, 25] Mitochondrial metabolism Multiple genes ATP production Supports active ion transport [4, 6] Oxidative stress defense NRF2 pathway, antioxidant enzymes Cellular protection Preserves uterine epithelial function

Split-Feeding Strategies

The split-feeding systems exploit the fact that there are marked changes in nutrient requirements throughout the ovulatoryovipositional cycle. In the morning, hens will have high protein and metabolizable energy requirements to support follicular growth, yolk deposition, and albumen synthesis; calcium requirement will be high in the afternoon and night when uterine shell calcification is most active. Feeding a high-protein, high-energy, lower-calcium diet in the morning, followed by a lower-protein, lower-energy, calciumenriched diet in the afternoon, was also the practice that significantly improved calcium digestibility while keeping eggshell quality, as obtained by Akter et al. [9] . This proposal of feeding synchronizes nutrient supply with physiological demand, thereby enhancing nutrient-use efficiency without raising total dietary calcium intake, as earlier reported by other workers [9] [8] [9] [8] .

Vitamin D Optimization

Efficient eggshell formation is impossible without vitamin D because it controls calcium absorption in the intestines, medullary bone mobilization, renal conservation of calcium, and uterine transport of calcium [8] . Biologically active vitamin D increases the expression of calcium transport proteins including TRPV6, CALB1, and plasma membrane calcium ATPases bringing calcium to the bird's shell gland for shell biomineralization [3] . Therefore, a combination of calcium and vitamin D generally works better than increased dietary calcium alone. This is especially true in old laying hens, where calcium absorption efficiency gradually falls [6,8] .

Gut Health and Probiotic Supplementation

Another approach would be enhancing eggshell quality through improving intestinal health since calcium utilization is directly linked to gastrointestinal function and nutrient absorption. Probiotic supplementation has been reported to improve intestinal morphology as well as stabilizing gut microbiota and increasing mineral digestibility, indirectly supporting shell mineralization.

Onbaşılar et al. [9] stated that drinking water supplemented with probiotics increased the Haugh unit from 84.91 to 87.66, shell thickness from 378.38 to 386.48 μm, and shell breaking strength from 3.60 to 3.87 kg/cm². Since the Haugh unit is mainly related to the quality of the contents of the egg rather than the shell, the simultaneous change of these three measurements indicates that intestinal physiology can be optimized to positively affect more than one characteristic of egg quality [9] .

Practical Implications for Commercial Egg Production

The above studies proved that the deposition of more efficient eggshells does not just involve increasing the concentration of dietary calcium [9] [8] . The most successful nutritional programs will, therefore, avail calcium when there is maximal uterine demand, optimize vitamin D status, improve intestinal nutrient utilization, and synchronize feeding time with the hen's circadian physiology [9,8,7] . Such integrated management strategies are particularly valuable for modern long-life laying strains because they have made the maintenance of shell quality during late lay a major breeding and production objective [6] .

Future Perspectives

It is anticipated that the future of eggshell research will shift from single physiological mechanisms to integrate systems that encompass endocrinology, molecular biology, nutrition, genetics, and precision livestock management [1,5,6] . As commercial laying hens are continually selected for production cycles that exceed 100 weeks, maintaining shell quality throughout the extended laying period will become increasingly more challenging for both breeding programs and commercial egg production [6] . A major research priority will be to improve our understanding of the temporal coordination between ovulation, oviposition, and uterine biomineralization. Considerable progress has been made in the characterization of the hormonal regulation of the hypothalamicpituitary-gonadal axis, but important questions remain regarding ovary-oviduct communication, circadian synchronization, and endocrine regulation of shell gland activity during prolonged laying cycles [1,6] . Future investigations that integrate chronobiology with reproductive endocrinology may identify new physiological targets for improving shell formation efficiency. Another area of rapid advancement is in the field of multi-omics technologies, which include genomics, transcriptomics, proteomics, metabolomics, epigenomics, and single-cell sequencing. These have been used to complement each other, and in doing so, they have proven that eggshell quality is not regulated by one or a few candidate genes but rather by very complex networks that include calcium transport, bicarbonate secretion, extracellular matrix assembly, mitochondrial metabolism, response to oxidative stress, regulation of immunity, and homeostasis of the epithelium. Systems biology and network analysis are thus projected to play ever-increasing roles in the identification of biomarkers linked to not only superior shell quality but also laying persistency. The use of artificial intelligence and precision technologies in poultry farming is expected to change commercial eggshell management as well [6] . Automated environmental sensors, computer vision systems, real-time production monitoring, and machine-learning algorithms can constantly assess egg production as well as shell quality and even compare feed intake by the hens to environmental conditions and flock health, so that producers can implement precision nutritional and management interventions before measurable shell deterioration sets in [6] . and thus, significantly improve prediction accuracy for shell defects while enhancing production efficiency and animal welfare.

It is expected that future nutritional strategies would shift from fixed diet formulation to dynamic precision feeding systems wherein calcium, vitamin D, phosphorus, and other necessary nutrients would be provided at the physiological stage of shell formation. Automatic feeding technologies could be integrated with sensor-based monitoring and predictive algorithms to optimize nutrient utilization, reduce unnecessary mineral excretion, cut production costs, and improve environmental sustainability without compromising shell quality. The reference list is as mentioned in the original article [9] [8] [9] [8] . Genomic selection is also projected to make the transition from conventional selection based on phenotypic performance to genome-assisted breeding, genomic estimated breeding values (GEBVs), and pathway-based selection facilitated by artificial intelligence and systems biology [6] . Future breeding programs are expected to prioritize biological resilience, uterine functional capacity, skeletal integrity, and persistent shell quality throughout extended laying cycles rather than early egg production as it was in the past [6] . Perhaps, the major advancements in eggshell research will be achieved when endocrinologists chronicle uterine physiologists, and those undertaking multi-omics studies, precision nutritionists, digital phenotypes, and genomic selectors finally get their acts together as a big family of precision production. Such interdisciplinary approaches are anticipated to improve eggshell quality, as well as laying persistency, while reducing the economic losses linked to shell defects and promoting more sustainable commercial egg production in nextgeneration laying hens [1,5,6,8,9] .

Conclusion

Sequential oviposition in modern laying hens demonstrates the operation of an integrated biological system in endocrine regulation, follicular development, oviductal transport, ion regulation in the uterus, and biomineralization of the eggshell. In this integrated reproductive framework, the shell gland is deemed the primary functional center for shell formation due to its activities in calcium transport and bicarbonate secretion with pH regulation and extracellular matrix assembly; together with calcite crystal growth, they define the structural integrity and mechanical strength of the eggshell. Current evidence indicates that the quality of the shell is determined by gene networks and not by single genes. Good mineralization of the shell in quality shells implies integrated actions of calcium homeostasis, bicarbonate metabolism, matrix proteins, mitochondrial function, immune regulation, and integrity of the epithelium. Also, the initial phase of shell calcification can be considered as a critical period during which the mammillary layer is formed and the crystalline structure is organized to provide a foundation for the final quality of the shell. Since the genetic selection programs for commercial laying hens have been directed toward increasing the length of productive life, eggshell quality in late lay has become a major problem. This would require an integrated approach in advancing our knowledge of reproductive physiology, molecular biology, genomics, and nutrition. In practice, such strategies as precision calcium supplementation, split-feeding systems, vitamin D optimization, improved gut health, and environmental management can be implemented to improve shell quality while persistency and sound skeletal condition can be maintained. The next breakthroughs in eggshell quality will result from successful integration of all these measures into an overall production system on an interdisciplinary and holistic basis. Systems biology, multi-omics technologies, genomic selection, artificial intelligence, precision nutrition, and digital poultry management are the next breakthroughs in eggshell quality. This will lead to better laying persistence and production efficiency with enhanced animal welfare under conditions of sustainability in commercial egg production for the next generation of high-performing laying hens.

References

1. Scanes CG. Discontinuities in understanding follicular development, the ovulatory cycle and the oviposition cycles in the hen: Advances, opportunities, slow downs and complete stops. Frontiers in Physiology. 2022;13:1023528.https://doi.org/10.3389/fphys.2022.1023 528 2. Sah N, Mishra B. Regulation of egg formation in the oviduct of laying hen. World's Poultry Science Journal. 2018;74(3):509522.https://doi.org/10.1017/s0043933918000442
2. Jonchère V, Brionne A, Gautron J, Nys Y. Identification of uterine ion transporters for mineralisation precursors of the avian eggshell. BMC Physiology. 2012;12(1):10.https://doi.org/10.1186/1472-6793-12-10
3. Zhang J, Wang Y, Zhang C, Xiong M, Rajput SA, Liu Y, et al. The differences of gonadal hormones and uterine transcriptome during shell calcification of hens laying hard or weak-shelled eggs. BMC Genomics. 2019;20(1):707.https://doi.org/10.1186/s12864-
4. Brionne A, Nys Y, Hennequet-Antier C, Gautron J. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process. BMC Genomics. 2014;15(1):220.https://doi.org/10.1186/1471-2164-15-220
5. Yang L, Yang Y, Jing Y, Zhang M, Zhang M, Zhang S, et al. Research Progress on Genetic Factors of Poultry Egg Quality: A Review. Animals. 2025;15(24):3652.https://doi.org/10.3390/ani15243652
6. Akter N, Dao TH, Crowley TM, Sukirno, Nawab A, Moss AF. Afternoon Calcium and Vitamin D Supplementation in Water: A Targeted Approach to Improve Laying Hen Nutrition. Animals. 2025;15(5):720.https://doi.org/10.3390/ani15050720
7. Akter N, Dao TH, Crowley TM, Moss AF. Optimization of Split Feeding Strategy for Laying Hens Through a Response Surface Model. Animals. 2025;15(5):750.https://doi.org/10.3390/ani15050750
8. Onbaşılar EE, Yalçın S, Bakıcı C, Batur B, Kartal YK, Ahlat O, Kılıçlı İB, Yalçın S. Comprehensive Evaluation of Probiotic Effects on Laying Hen Physiology: From Performance to Bone and Gut Morphology. Animals. 2025;15(16):2408.https://doi.org/10.3390/ani15162408
9. Hincke MT, Gautron J, Tsang CPW, McKee MD, Nys Y. Molecular Cloning and Ultrastructural Localization of the Core Protein of an Eggshell Matrix Proteoglycan, Ovocleidin-116. Journal of Biological Chemistry. 1999;274(46):3291532923.https://doi.org/10.1074/jbc.274.46.32915
10. Stapane L, Le Roy N, Ezagal J, Rodriguez-Navarro AB, Labas V, Combes-Soia L, et al. Avian eggshell formation reveals a new paradigm for vertebrate mineralization via vesicular amorphous calcium carbonate. Journal of Biological Chemistry. 2020;295(47):1585315869.https://doi.org/10.1074/jbc.ra120.014542
11. Nys Y, Gautron J, Garcia-Ruiz JM, Hincke MT. Avian eggshell mineralization: biochemical and functional characterization of matrix proteins. Comptes Rendus Palevol. 2004;3(6-7):549562.https://doi.org/10.1016/j.crpv.2004.08.002
12. Rodríguez-Navarro AB, Marie P, Nys Y, Hincke MT, Gautron J. Amorphous calcium carbonate controls avian eggshell mineralization: A new paradigm for understanding rapid eggshell calcification. Journal of Structural Biology. 2015;190(3):291303.https://doi.org/10.1016/j.jsb.2015.04.014
13. Baron R, Rawadi G. Targeting the Wnt/β-Catenin Pathway to Regulate Bone Formation in the Adult Skeleton. Endocrinology. 2007;148(6):26352643.https://doi.org/10.1210/en.2007-0270
14. Dacke CG, Arkle S, Cook DJ, Wormstone IM, Jones S, Zaidi M, et al. Medullary Bone and Avian Calcium Regulation. Journal of Experimental Biology. 1993;184(1):63-88.https://doi.org/10.1242/jeb.184.1.63
15. Whitehead CC. Overview of bone biology in the egg-laying hen. Poultry Science. 2004;83(2):193-
16. Fleming RH, McCormack HA, Whitehead CC. Bone structure and strength at different ages in laying hens and effects of dietary particulate limestone, vitamin K and ascorbic acid. British Poultry Science. 1998;39(3):434440.https://doi.org/10.1080/00071669889024
17. Kebreab E, France J, Kwakkel RP, Leeson S, Kuhi HD, Dijkstra J. Development and evaluation of a dynamic model of calcium and phosphorus flows in layers. Poultry Science. 2009;88(3):680-
18. Bar A. Calcium transport in strongly calcifying laying birds: Mechanisms and regulation. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology. 2009;152(4):447-
19. Bain MM, Nys Y, Dunn IC. Increasing persistency in lay and stabilising egg quality in longer laying cycles. What are the challenges?. British Poultry Science. 2016;57(3):330338.https://doi.org/10.1080/00071668.2016.1161727
20. Roberts JR. Factors Affecting Egg Internal Quality and Egg Shell Quality in Laying Hens. The Journal of Poultry Science. 2004;41(3):161177.https://doi.org/10.2141/jpsa.41.161
21. Preisinger R. Innovative layer genetics to handle global challenges in egg production. British Poultry Science. 2017;59(1):16.https://doi.org/10.1080/00071668.2018.1401828
22. Bain MM. Recent advances in the assessment of eggshell quality and their future application. World's Poultry Science Journal. 2005;61(2):268277.https://doi.org/10.1079/wps200459
23. Rodriguez-Navarro A, Kalin O, Nys Y, Garcia-Ruiz JM. Influence of the microstructure on the shell strength of eggs laid by hens of different ages. British Poultry Science. 2002;43(3):395403.https://doi.org/10.1080/00071660120103675
24. de Matos R. Calcium Metabolism in Birds. Veterinary Clinics of North America: Exotic Animal Practice. 2008;11(1):5982.https://doi.org/10.1016/j.cvex.2007.09.005
25. Abd El Hack ME, El Saadony MT, Shafi ME, Qattan SYA, Batiha GE, Khafaga AF, et al. Probiotics in poultry feed: A comprehensive review. Journal of Animal Physiology and Animal Nutrition. 2020;104(6):18351850.https://doi.org/10.1111/jpn.13454
26. Kaspers B. An egg a day -the physiology of egg formation. Lohmann Information. 2016;50(2):12-17.https://lohmannbreeders.com/lohmanninfo/an-egg-a-day-thephysiologyof-egg-formation/
27. Al-Baidhani AKM, Humam AM, Al-Mashhadani HA, AlHammed SAM, Joodi AA, Abd Al-Razak NZ. Effect of Supplementation Different Levels of Fermented Barley by Kiwi Juice in Broiler Diets on Growth Performance, Physiological Traits, Gut Microbiota and Liver Enzymes Activity. IOP Conference Series: Earth and Environmental Science. 2025;1487(1):012170.https://doi.org/10.1088/17551315/1487/1/012170
28. Al-Mhanawi AAJ, Al-Hayani WKA. The effect of adding nano concentrations of hydrogen peroxide to the diluted semen of Iraqi local roosters on some laboratory characteristics after cooling and cryopreservation. Plant Archives. 2020;20(Suppl 2):26972703.http://www.plantarchives.org/SPL
29. Yang YY, Dai D, Zhang HJ, Wu SG, Qi GH, Wang J. The characterization of uterine calcium transport and metabolism during eggshell calcification of hens laying high or low breaking strength eggshell. Poultry Science. 2025;104(6):105111.https://doi.org/10.1016/j.psj.2025.10 5111
30. Fu Y, Zhou J, Schroyen M, Zhang H, Wu S, Qi G, et al. Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens. Journal of Animal Science and Biotechnology. 2024;15(1):37.https://doi.org/10.1186/s40104-023-009862
31. Johnson AL, Van Tienhoven A. Plasma Concentrations of Six Steroids and LH During the Ovulatory Cycle of the Hen, Gallus domesticus1. Biology of Reproduction. 1980;23(2):386393.https://doi.org/10.1095/biolreprod23.2.386
32. Gao S, Qiu K, Zheng J, Zhang H, Wang J, Qi X, et al. Dietary 25-Hydroxycholecalciferol Supplementation as a Vitamin D3 Substitute Improves Performance, Egg Quality, Blood Indexes, Jejunal Morphology, and Tibia Quality in Late-Phase Laying Hens. Animals. 2024;14(6):878.https://doi.org/10.3390/ani14060878
33. Rose-Martel M, Du J, Hincke MT. Proteomic analysis provides new insight into the chicken eggshell cuticle. Journal of Proteomics. 2012;75(9):26972706.https://doi.org/10.1016/j.jprot.2012.03.019
34. Oster M, Qasir H, Reyer H, Ponsuksili S, Trakooljul N, Sommerfeld V, et al. Transcriptional and endocrine orchestration of medullary bone formation and mineral turn-over in female chickens. Poultry Science. 2025;104(11):105798.https://doi.org/10.1016/j.psj.2025.1 05798
35. Fu Y, Delezie E, Qi G, Wang J. Optimizing eggshell quality in extended laying periods: Insights and strategies. Animal Nutrition. 2026;24:492506.https://doi.org/10.1016/j.aninu.2025.11.003
36. Hincke MT, Tsang CPW, Courtney M, Hill V, Narbaitz R. Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin 17). Calcified Tissue International. 1995;56(6):578583.https://doi.org/10.1007/BF00298593
37. Benavides-Reyes C, Folegatti E, Dominguez-Gasca N, Litta G, Sanchez-Rodriguez E, Rodriguez-Navarro AB, et al. Research Note: Changes in eggshell quality and microstructure related to hen age during a production cycle. Poultry Science. 2021;100(9):101287.https://doi.org/10.1016/j.psj.2021.10 1287
38. Li W, Ma X, Li X, Zhang X, Sun Y, Ning C, et al. Integrating Single-Cell RNA-Seq and ATAC-Seq Analysis Reveals Uterine Cell Heterogeneity and Regulatory Networks Linked to Pimpled Eggs in Chickens. International Journal of Molecular Sciences. 2024;25(24):13431.https://doi.org/10.3390/ijms25241343
39. Hincke MT. The eggshell: structure, composition and mineralization. Frontiers in Bioscience. 2012;17(1):1266.https://doi.org/10.2741/3985
40. Gautron J, Stapane L, Le Roy N, Nys Y, RodriguezNavarro AB, Hincke MT. Avian eggshell biomineralization: an update on its structure, mineralogy and protein tool kit. BMC Molecular and Cell Biology. 2021;22(1):11.https://doi.org/10.1186/s12860-021-
41. Marie P, Labas V, Brionne A, Harichaux G, HennequetAntier C, Nys Y, et al. Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization. Data in Brief. 2014;1:6569.https://doi.org/10.1016/j.dib.2014.09.006
42. Mann K, Siedler F. The amino sequence of ovocleidin 17, a major protein of the avian eggshell calcified layer. IUBMB Life. 1999;47(6):9971007.https://doi.org/10.1080/15216549900202123
43. Mann K, Maček B, Olsen JV. Proteomic analysis of the acid soluble organic matrix ofthe chicken calcified eggshell layer. PROTEOMICS. 2006;6(13):38013810.https://doi.org/10.1002/pmic.200600120
44. Mann K. The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata). Proteome Science. 2015;13(1):29.https://doi.org/10.1186/s12953015-0086-1
45. Hanlon C, Takeshima K, Bédécarrats GY. Changes in the Control of the Hypothalamic-Pituitary Gonadal Axis Across Three Differentially Selected Strains of Laying Hens (Gallus gallus domesticus). Frontiers in Physiology. 2021;12:651491.https://doi.org/10.3389/fphys.2021.65149 1 47. do Nascimento CH, de Lima AV, de Souza PEL, de Souza Filho DF, Santos Silva Rd, Rodrigues AB, Neto RdCL, de Lima MR, Guerra RR, Ribeiro AG, Carvalho LRRA, Costa FGP. Split feeding for laying hens: a step beyond precision nutrition. Poultry Science. 2025;104(7):105158.https://doi.org/10.1016/j.psj.2025.10 5158
46. Yin Z, Lian L, Zhu F, Zhang ZH, Hou ZC, Tang YZ, et al. The transcriptome landscapes of ovary and three oviduct segments during chicken (Gallus gallus) egg formation. Genomics. 2020;112(2):17141725.https://doi.org/10.1016/j.ygeno.2019.02.003
47. Oketch EO, Yu M, Hong JS, Chaturanga NC, Seo E, Lee H, et al. Laying hen responses to multi-strain Bacillusbased probiotic supplementation from 25 to 37 weeks of age. Animal Bioscience. 2024;37(8):14181427.https://doi.org/10.5713/ab.23.0495