Eggshell ultrastructure and mineral composition during the osteogenesis of Kinosternon scorpioides (Testudines: Kinosternidae)

Abstract

One of the challenges in the management of Kinosternon scorpioides is related to egg quality, since mineral deficiencies affect egg production. The aim of this study was to evaluate the ultrastructure and mineral composition of eggshells, and their relation to K. scorpioides embryonic skeletogenesis. Eggs were collected, incubated and distributed into three groups: 1- pre-ossification (PO); 2- bone ossification (BO); and 3- complete ossification (CO). Nine samples were included in the PO and BO groups, and six in the CO group, for a total of 24 eggs. The morphological and mineral analyses of the eggs were performed by scanning electron microscopy coupled with energy-dispersive spectroscopy. The embryos from the BO and CO groups were cleared with potassium hydroxide and stained with alcian blue and alizarin red to observe osteogenesis. The results showed a significant difference (p < 0.05) in the amount of shell calcium between the PO (63.14%) and CO (41.95%) groups, as well as in the shell thickness between the same groups (PO: 305.38 ± 60.27 μm; CO: 197.67 ± 40.28 μm), and BO (289.56 ± 55.87 μm) and CO groups. It is suggested that the gradual mobilization of calcium by the embryo is reflected in the decrease in shell thickness. In addition, the first structures to ossify were the dermatocranium and mandible. The CO group, in which the individuals all had established bone structures, had the lowest calcium concentration of eggshells. Approximately 21% of the shell calcium was absorbed, which suggests participation in the embryo skeletogenesis. Understanding calcium dynamics as they relate to shell quality and embryonic development of K. scorpioides highlights the importance of including this mineral in the diet of breeding females and, consequently, in improving offspring production.