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The equine hoof wall has a complex, hierarchical structure that can inspire designs of impactresistant materials. In this study, we utilized micro-computed tomography (µ-CT) and serial blockface scanning electron microscopy (SBF-SEM) to image the microstructure and nanostructure of the hoof wall. We quantified the morphology of tubular medullary cavities by measuring equivalent diameter, surface area, volume, and sphericity. High-resolution µ-CT revealed that tubules are partially or fully filled with tissue near the exterior surface and become progressively empty towards the inner part of the hoof wall. Thin bridges were detected within the medullary cavity, starting in the middle section of the hoof wall and increasing in density and thickness towards the inner part. Porosity was measured using three-dimensional (3D) µ-CT, twodimensional (2D) µ-CT, and a helium pycnometer, with the highest porosity obtained using the helium pycnometer (8.07%), followed by 3D (3.47%) and 2D (2.98%) µ-CT. SBF-SEM captured the 3D structure of the hoof wall at the nanoscale, showing that the tubule wall is not solid, but has nano-sized pores, which explains the higher porosity obtained using the helium pycnometer. The results of this investigation provide morphological information on the hoof wall for the future development of hoof-inspired materials and offer a novel perspective on how various measurement methods can influence the quantification of porosity. 

The equine hoof wall has a unique hierarchical structure that allows it to survive high‐impact scenarios. Previous authors have explored the compressive, viscoelastic, and fracture control properties of the hoof wall and suggested that this complex structure plays a vital role in the hoof's behavior. However, the link between the structure and the behavior of the hoof wall has been made primarily with the use of post‐fracture analysis. Here, periodic microcomputed tomography scans are used to observe the temporal behavior of the hoof's meso and microstructures during compression, fracture, and relaxation. These results shed light on the structural anisotropy of the hoof wall and how its hollow tubules behave when compressed in different directions, at different hydration levels, and in various locations within the hoof wall. The behavior of tubule bridges during compression is also reported for the first time. This study elucidates several fracture phenomena, including the way cracks are deflected at tubule interfaces and tubule bridging, tubule arresting, and fiber bridging. Finally, relaxation tests are used to show how the tubule cavities can regain their shape after compression.