## 3 Results and Discussion  
Morphological and Elemental Analysis of Eggshell Components by Scanning Electron Microscopy and Energy Dispersive Spectroscopy  
It has been documented that the composition of mineralized eggshells forms roughly 96% of calcium carbonate (calcite). In comparison, the remaining 4% represents such minor components as magnesium, phosphorus, and various trace elements located mainly in the organic matrix [26]. From the outside direction to the inside, the eggshell comprises histological layers such as outer and inner shell membranes, mammillary layer, palisade, and cuticle layers (Figure 1A-C). The eggshell membrane represents significant importance since it provides the shell foundation and prevents microbial invasion [27]. The eggshell membrane is located between the albumen fraction and eggshell, and attached to the latter with its organic-rich rounded surface structures and mammillary knobs [28]. The eggshell membrane is represented as a fibrous network consisting of collagen type I, dermatan sulfate, chondro sulfate, and glucosamine [29]. The discovery of the eggshell membrane as a source of multiple health-promoting compounds makes it practical and safe for treating pain and stiffness associated with knee osteoarthritis [30]. The presence of a high vacuum, though, in the SEM system used to prevent electrical discharge in the gun assembly and to allow the electrons to travel within the instrument unimpeded caused a slight detachment of the eggshell membrane from the eggshell. However, we successfully measured the eggshell and membrane thickness. As seen, the thickness of the eggshell varied in the range from 356.2 to 366.4 \(\upmu\)m, with the eggshell derived from eggs of cage hens H/LS/CCE having a thinner layer, and the eggshell derived from eggs of cage-free hens H/D/BWE having a thicker layer (Table 2). It is worth noting, though, no statistically significant differences (\(p\geq 0.05\)) were observed between the eggshell thicknesses of four eggs. The observed values are consistent with [PERSON] et al. [27], reporting the range of chicken eggshell thickness from 240 to 370 \(\upmu\)m. The authors also indicated that the eggshell thickness of domestic hen eggs was not uniform over the whole egg surface, i.e., thickness at the poles was thicker than at the equator. Therefore, its thickness must be measured within the large end, equator, and small end of the egg. However, [PERSON] et al. [31] reported a nearly similar thickness of the chicken eggshell layer. In turn, statistically significant differences (\(p\leq 0.05\)) were observed between the eggshellmembrane thickness, which was found in the range from 20.0 to 59.9 \(\upmu\)m. Similarly, as in the case of eggshell thickness, cage eggs produced from H/LS/CCE hens demonstrated a tangibly thinner layer of eggshell membrane, while cage-free eggs derived from H/D/BWE hens had a thicker membrane. Thinner eggshell membranes observed in caged hens are most likely associated with the deficiency of minerals, especially Cu. This element is reported to act as a promoter and component of crucially essential enzymes involved in the formation of the eggshell membrane, such as Cu-containing amine oxidase [20]. The relevance of the palisade layer thickness and the organization of calcite crystals have been highlighted by [PERSON] et al. [19], as these factors strongly contribute to the strength of the eggshell. However, as seen (Figure 1A-C), the calcite columns in all four eggs had a wedge-shaped structure, and no significant difference was observed between the samples. Furthermore, on a more detailed microscopic examination of eggshell specimens, no prominent large and uniform type B mammillary knobs were seen, confirming the lack of endometrial tissue damages of those documented to take direct involvement in  
Figure 1: Radial or cross-sectional scanning electron microscopy micrographs of rigid laying hen eggshells showing tightly abutting shell units. The fibrous shell membrane has pulled away from the calcareous layer.