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.
