First reports on the relationship between the eggshell and bone mineralization in Rhinoclemmys punctularia punctularia embryos.

Composed of various minerals, the eggshell provides ideal conditions for embryonic development, and understanding its composition and structure is essential for clarifying its relationship with bone formation. This study characterized the structure and mineral profile of the Rhinoclemmys punctularia punctularia eggshell in relation to embryonic osteogenesis. Fertilized eggs were collected and incubated under controlled humidity (80%) and temperature (28.5°C ± 0.5°C). From the 15th day of incubation onward, embryos (n = 39) were analyzed at different developmental stages using a clearing and double-staining technique. Eggshell ultrastructure and mineral composition (n = 23) were evaluated by scanning electron microscopy coupled with energy-dispersive spectroscopy. Three developmental periods were established: pre-ossification (PO), with no signs of mineralization; ossification in progress (EO), marked by the first ossification centers; and complete ossification (OC), in which all bones were mineralized. Osteogenesis began on the 30th day of incubation in the dermatocranium. Ultrastructural analysis revealed three shell layers: cuticle, mineral layer, and organic layer. Calcium weight percentage on the eggshell surface decreased significantly by 5% (p = 0.037) during embryonic development. Eggshell thickness also declined significantly (p = 0.00242), from 389.86 ± 40.14 μm in PO to 323.11 ± 37.00 μm in EO and 320.65 ± 40.23 μm in OC. These findings indicate that eggshell mineral dynamics play a fundamental role in embryonic osteogenesis, contributing to a better understanding of skeletal development and providing insights for evolutionary studies.