Periodic Reporting for period 1 - BonEggHens (Investigation of coupled bone and eggshell (de)mineralization during laying cycle of hens)
Reporting period: 2023-09-01 to 2025-08-31
Bone is a rigid organ that provides structural support and acts as a reservoir of essential minerals. In laying hens, this function is particularly demanding: each day they mobilize about 10% of their body calcium to build an eggshell. To meet this demand, hens form a special type of bone, called medullary bone (located in the medullary cavity), which serves as a calcium storage. Repeated mobilization of calcium for eggshell formation disrupts the balance between bone resorption and formation, potentially slowing turnover in the structural compartments and altering the organization of the mineral–collagen composite. This may compromise the quality of the cortical bone and modify the distribution and intensity of the bone anisotropic mechanical response.
This skeletal disorder poses serious challenges for animal welfare, the sustainability of egg production, and the economic viability of the poultry industry (it especially affects commercial flocks). Beyond poultry farming, understanding how bone forms, dissolves, and adapts to age physiological changes also provides insights relevant to human bone health, particularly for diseases such as osteoporosis.
BonEggHens project aims to address these issues through a systematic study of hen bone and eggshells across different life and production (laying cycle) stages by high-resolution techniques. The main objectives are:
• To characterize bone and eggshell composition and structure from the macro- to the nanoscale.
• To determine the role of collagenous and non-collagenous proteins in controlling bone dissolution.
• To assess how labile versus crystalline mineral phases influence bone fragility.
• To establish correlations between i) micro and nanoscopic characteristics of hen’s bones and bone quality ii) bone structural and compositional changes and eggshell quality.
By combining advanced microscopy, mineralogical analyses, organic matrix investigation, and statistical approaches, the project will uncover the mechanisms leading to hen bone degradation and the links between bone quality and eggshell formation. These findings will contribute to improving poultry health and sustainability, while also providing knowledge relevant to human bone biology and disease.
This skeletal disorder poses serious challenges for animal welfare, the sustainability of egg production, and the economic viability of the poultry industry (it especially affects commercial flocks). Beyond poultry farming, understanding how bone forms, dissolves, and adapts to age physiological changes also provides insights relevant to human bone health, particularly for diseases such as osteoporosis.
BonEggHens project aims to address these issues through a systematic study of hen bone and eggshells across different life and production (laying cycle) stages by high-resolution techniques. The main objectives are:
• To characterize bone and eggshell composition and structure from the macro- to the nanoscale.
• To determine the role of collagenous and non-collagenous proteins in controlling bone dissolution.
• To assess how labile versus crystalline mineral phases influence bone fragility.
• To establish correlations between i) micro and nanoscopic characteristics of hen’s bones and bone quality ii) bone structural and compositional changes and eggshell quality.
By combining advanced microscopy, mineralogical analyses, organic matrix investigation, and statistical approaches, the project will uncover the mechanisms leading to hen bone degradation and the links between bone quality and eggshell formation. These findings will contribute to improving poultry health and sustainability, while also providing knowledge relevant to human bone biology and disease.
The implementation of BonEggHens envisaged the analysis of a large array of hen bone samples to investigate all the aspects that could have an impact on the skeletal condition of aging hens. At the same time, eggshell samples were analysed to establish a link between bone degradation and egg production. Several state-of-the art techniques were employed, each of which provided key information on the bone tissue and eggshell variability with the age:
• Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR)
General information: molecular bonds, composition
Specific information: content of lipids, phosphate, polysaccharides, carbonates, collagen (amide I).
• Thermogravimetry (TGA)
General information: composition and thermal stability.
Specific data: content of water, organic matrix, mineral phase, carbonates.
• X-ray diffraction (XRD)
General information: crystal structure, phases
Specific data: crystalline phases, estimation of unit cell parameters, approximate crystallite size, information about crystal orientation (2D mode).
• µ-Computed Tomography (µCT)
General information: 3D internal morphology
Specific data: eggshell porosity, eggshell thickness, bone surface density, bone volume fraction, average cortical area fraction, cortical porosity, average cortical thickness, trabeculae number, average trabecular thickness.
• Scanning Electron Microscopy (SEM)
General information: high-resolution surface topography
Specific data: surface morphology, microstructure, qualitative and semi-quantitative elemental composition, Ca/P ratio (stoichiometry), trace element detection, chemical-microstructural correlation.
• Transmission Electron Microscopy (TEM)
General information: lattice-scale crystallography
Specific data: ultrastructure, size, shape and orientation of nanocrystals, spatial relationship between the organic phase (collagen) and the mineral phase, periodicity of collagen fibrils in bone, electron diffraction pattern, point analysis and elemental mapping.
• Atom Probe Tomography (APT)
General Information: elemental composition and 3D distribution
Specific data: 3D reconstruction of atomic composition, distribution of light and heavy elements, interface composition, precise stoichiometry of the mineral, nanosegregation of elements, defect analysis.
Furthermore, an investigation was carried out on the bone remodelling process (bone resorption and formation). Specifically, laboratory experiments were conducted to study the dissolution process of hen cortical and medullary bone tissues by using close systems and mild acid solution (1% hydrochloric acid) to mimic the natural process of bone resorption but under controlled conditions. The bone tissues were examined under a scanning electron microscope (SEM) to monitor their structural changes, while the aqueous solution was analysed with the technique Inductively Coupled Plasma (ICP- Optical Emission Spectroscopy) to measure the amount of calcium, phosphorus, magnesium, sodium, and other ions released as the bone dissolved.
The bone samples were also grouped according to two main features:
1. The maturity of the collagen fibres, which is a measure of how tightly the structural organic matrix of the bone is cross-linked (number and types of chemical bonds that connect individual collagen molecules).
2. The amount of “labile” mineral phase, meaning the fraction of bone mineral that is amorphous or poorly crystalline.
This classification helped understand how the bone’s organic framework influences mineral behaviour and what is the role of the less mineralized phase on the resorption process. Finally, selected cortical bone samples of different ages were analysed using Synchrotron FTIR spectroscopy at the ALBA Synchrotron in Barcelona (Spain). This powerful technique allowed to observe, in great detail, how the chemical composition of the bone changed as the dissolution process advanced.
All the data gathered were processed by statistical analysis software to state i) the correlations between bone structure and composition and mechanical properties ii) the correlations between bone properties and eggshell quality.
• Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR)
General information: molecular bonds, composition
Specific information: content of lipids, phosphate, polysaccharides, carbonates, collagen (amide I).
• Thermogravimetry (TGA)
General information: composition and thermal stability.
Specific data: content of water, organic matrix, mineral phase, carbonates.
• X-ray diffraction (XRD)
General information: crystal structure, phases
Specific data: crystalline phases, estimation of unit cell parameters, approximate crystallite size, information about crystal orientation (2D mode).
• µ-Computed Tomography (µCT)
General information: 3D internal morphology
Specific data: eggshell porosity, eggshell thickness, bone surface density, bone volume fraction, average cortical area fraction, cortical porosity, average cortical thickness, trabeculae number, average trabecular thickness.
• Scanning Electron Microscopy (SEM)
General information: high-resolution surface topography
Specific data: surface morphology, microstructure, qualitative and semi-quantitative elemental composition, Ca/P ratio (stoichiometry), trace element detection, chemical-microstructural correlation.
• Transmission Electron Microscopy (TEM)
General information: lattice-scale crystallography
Specific data: ultrastructure, size, shape and orientation of nanocrystals, spatial relationship between the organic phase (collagen) and the mineral phase, periodicity of collagen fibrils in bone, electron diffraction pattern, point analysis and elemental mapping.
• Atom Probe Tomography (APT)
General Information: elemental composition and 3D distribution
Specific data: 3D reconstruction of atomic composition, distribution of light and heavy elements, interface composition, precise stoichiometry of the mineral, nanosegregation of elements, defect analysis.
Furthermore, an investigation was carried out on the bone remodelling process (bone resorption and formation). Specifically, laboratory experiments were conducted to study the dissolution process of hen cortical and medullary bone tissues by using close systems and mild acid solution (1% hydrochloric acid) to mimic the natural process of bone resorption but under controlled conditions. The bone tissues were examined under a scanning electron microscope (SEM) to monitor their structural changes, while the aqueous solution was analysed with the technique Inductively Coupled Plasma (ICP- Optical Emission Spectroscopy) to measure the amount of calcium, phosphorus, magnesium, sodium, and other ions released as the bone dissolved.
The bone samples were also grouped according to two main features:
1. The maturity of the collagen fibres, which is a measure of how tightly the structural organic matrix of the bone is cross-linked (number and types of chemical bonds that connect individual collagen molecules).
2. The amount of “labile” mineral phase, meaning the fraction of bone mineral that is amorphous or poorly crystalline.
This classification helped understand how the bone’s organic framework influences mineral behaviour and what is the role of the less mineralized phase on the resorption process. Finally, selected cortical bone samples of different ages were analysed using Synchrotron FTIR spectroscopy at the ALBA Synchrotron in Barcelona (Spain). This powerful technique allowed to observe, in great detail, how the chemical composition of the bone changed as the dissolution process advanced.
All the data gathered were processed by statistical analysis software to state i) the correlations between bone structure and composition and mechanical properties ii) the correlations between bone properties and eggshell quality.
Through remodeling processes, the amount and composition of both structural and more labile (medullary) bone tissues are regulated during the hen’s life cycle. In younger hens, the active remodeling of bone tissues (especially the medullary bone), which provides the calcium needed for eggshell calcification, results in a lower degree of bone mineralization. However, the increased mineralization and carbonate substitution associated with ageing result in bones that are stiffer but also more brittle (less capable of dissipating energy). Furthermore, data from BonEggHens investigation into susceptibility to bone dissolution indicates that with advancing age the release of Ca and P decreased from the onset of the laying cycle. This suggests i) a reduction in the dynamism of the bone remodeling process with age, which may in turn affect calcium availability and consequently eggshell quality, ii) the strong dependence of the Ca uptake on the labile, more hydrated mineral phase which also decreased with the bone age.
Overall, current findings indicate important variation in bone crystallinity and the mineral-organic matrix interface in ageing hens. Identifying these tissue quality biomarkers and correlating them with productive parameters (e.g. eggshell strength, bone density) can support the development of evidence-based strategies to improve bone health in laying hens.
Overall, current findings indicate important variation in bone crystallinity and the mineral-organic matrix interface in ageing hens. Identifying these tissue quality biomarkers and correlating them with productive parameters (e.g. eggshell strength, bone density) can support the development of evidence-based strategies to improve bone health in laying hens.