Cryo-SEM documentation of intact frozen-fractured avian eggs reveals species-dependent albumen freezing morphologies.

Cryo-scanning electron microscopy (cryo-SEM) was used to examine intact frozen-fractured eggs from five avian species-quail, hen, duck, turkey, and goose-spanning two taxonomic orders (Galliformes and Anseriformes) and a wide range of egg sizes. Whole eggs were frozen in liquid nitrogen within 24 h of laying, fractured at cryogenic temperature, and observed in the frozen-hydrated state after vacuum sublimation and gold coating. Multiple structural regions were examined, including yolk spheres, the albumen body, the shell-shell membrane-albumen interface, and the yolk-vitelline membrane-albumen interface where suitable fracture surfaces were available. Yolk spheres displayed a conserved polyhedral close-packing geometry across species, with larger eggs generally showing larger yolk sphere dimensions. The frozen albumen displayed species-dependent ice crystal morphologies: an isotropic spongy network in quail, fine parallel lamellae in hen, coarser lamellae in turkey, a multi-domain columnar-dendritic pattern in duck, and coarse lamellae in goose. These morphological differences did not simply scale with egg size and suggest that species-dependent differences in albumen composition and freezing behavior may influence ice crystal growth during cryogenic fixation. Because freezing rate and egg geometry were not independently controlled, the observed structures should be interpreted as cryo-fixed freezing morphologies rather than direct representations of unfrozen albumen microstructure. To our knowledge, systematic cryo-SEM comparison of albumen freezing morphology within intact avian eggs has not previously been reported. This study provides a rare comparative cryo-SEM atlas of intact frozen-fractured avian eggs and a morphological basis for future controlled investigations of albumen composition, freezing behavior, and ice crystal growth.