The structure and composition of the eggshell and embryonic membranes of Alligator mississippiensis

Summary The structure and chemical composition of the eggshell and eggshell membrane of Alligator mississippiensis were investigated utilizing macroscopy, scanning electron microscopy, energy dispersive analysis by X-rays, atomic absorption flame spectroscopy and spectrographic techniques on age known eggs fixed at daily intervals throughout the 65-day incubation period. Macroscopic observations were also made on the structure and disposition of the extraembryonic membranes. The freshly laid alligator egg is pure white and translucent. Within 24 h of egg-laying the embryo attaches to the top of the egg, so inducing an opaque, chalky white, central band. As incubation progresses, this band expands in length, eventually reaching the ends of the egg by about the fifth week of development. The origin and expansion of eggshell banding are multifactorial, being caused by: (1) a drying out of the eggshell due to polarization of the watery albumen towards the ends of the egg and an increase in porosity of the shell; (2) the numerous pores in the central region of the shell which render the calcite opaque; (3) the development of erosion craters which render the calcite opaque and alter the optical properties of the shell; (4) the presence of fewer mammillae at the ends of the egg; (5) a progressive decrease in the ratio of calcite crystals to organic matrix in the organic layers; and (6) the expansion of the vascular chorio-allantoic membrane around the inside of the shell which mobilizes minerals from the latter (for use in embryonic calcification), so causing the eggshell membrane to become chalky white. The alligator egg consists of the following layers from outside in: (1) an outer densely calcified layer (100–200 μm thick) consisting of small vertically stacked calcite crystals orientated with their crystallographic c axes at right angles to the shell surface; (2) a honeycomb layer (300–400 μm thick) consisting of horizontally stacked calcite crystals with their crystallographic c axes parallel to the shell surface; (3) an organic layer (approximately 10 μm thick) containing a higher percentage of organic matrix to calcite crystals, and through which the shell cleaves and falls away from the egg shortly before hatching; (4) a mammillary layer (20–30 μm thick) which is more pronounced in the central opaque region of the shell and which attaches the latter to the eggshell membrane; and (5) an eggshell membrane (150–250 μm thick) consisting of an interwoven mesh of fibres, separated from the albumen by an amorphous limiting membrane, in which there are numerous pores. The crystals of the outer densely calcified layer undergo extrinsic acidic dissolution (by organic acids produced by nest micro-organisms and by hydrated carbon dioxide) to produce erosion craters and cratered pore orifices. The extent, shape and depth of these craters is governed by the strict crystallographic alignment of calcite crystals in the outer densely calcified and honeycomb layers, by the anisotropic properties of these crystals and by diffusion control. These erosion craters expose at their broad bases the underlying honeycomb layer which contains large numbers of vesicular holes, interconnecting with other cavities throughout the shell. Thus, development of erosion craters renders the entire egg more porous as incubation proceeds. In addition, these craters, together with the mobilization of calcite crystals out of the organic layer for use in embryonic mineralization, progressively weaken the shell causing it to crack and cleave off the eggshell membrane and mammillary layer, so facilitating hatching. No air space is present in the alligator egg, neither are there any chalzae. The embryo has the usual amniote arrangement of extra-embryonic membranes which are firmly fused along two longitudinally opposite ribs, so anchoring the embryo in a constant position within the shell. The structure of the alligator egg is beautifully adapted to both its function and the nesting biology of the animal. The present investigation documents for the first time in any animal: (1) extrinsic degradation of an eggshell as incubation progresses; (2) a gross change in the external appearance of an egg as development proceeds; (3) two types of shell structure (mammillae plus pores, no mammillae no pores) within different regions of the same egg; and (4) a marked change in the porosity of an egg as incubation proceeds.