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In this study, we analyzed selected morphological traits of eggs, as well as structure, strength, and protein composition of the vitelline membrane (VM) of ostrich, emu, and greater rhea eggs. Ninety eggs (30 for species) were analyzed for the following parameters: egg weight, yolk weight, yolk ratio, and yolk index. In addition, pH value, water activity, consistency index, and flow behavior index were determined. The strength of VM was measured using the TA.HDPlus Texture Analyzer. Micrograph images were taken via a scanning electron microscope. Polyacrylamide gel electrophoresis was conducted under denaturing conditions. Ostrich eggs were characterized by the highest egg and yolk weight compared with those of emu and greater rhea eggs, whereas emu eggs had the highest yolk ratio compared with those of ostrich and greater rhea eggs (P > 0.05). Yolk content differed among the species in terms of water activity; it was found to be higher in emu eggs than in ostrich and greater rhea eggs (P > 0.05). Based on flow curves, yolks of the ratites were classified as pseudoplastic non-Newtonian fluids. The consistency index was significantly higher in yolks of ostrich and emu than that of greater rhea eggs, whereas the VM of yolks of greater rhea eggs was the most resistant (had the highest breaking force = 26.4 g). All species differed significantly regarding the structure of VM, the outer layer (OL) in particular, which was found to constitute fibers of various thicknesses that were differently arranged. Fibers of the OL of the VM of emu, whose fibers were the least differentiated but formed the most compact network, were the most diverse in characterization. An electropherogram of the VM of ostrich revealed 11 primary protein bands: 6 for the OL and 5 for the inner layer (IL), that of emu revealed 9 bands: 5 for the OL and 4 for the IL, and that of greater rhea revealed 10 bands: 6 for the OL and 4 for the IL.