Developmental changes in bone mechanics from Florida manatees (Trichechus manatus latirostris), obligate swimming mammals

Mammals living in aquatic environments load their axial skeletons differently than their terrestrial counterparts. The structure and mechanical behavior of trabecular bone can be especially indicative of varying habitual forces. Here, we investigate vertebral trabecular bone mechanical properties (yield strength, stiffness, and toughness) throughout development in Florida manatees (Trichechus manatus latirostris), obligate undulatory swimmers. Thoracic, lumbar, and caudal vertebrae were dissected from manatees (N=20) during necropsies. We extracted 6 mm3 samples from vertebral bodies and tested them in compression in three orientations (rostrocaudal, dorsoventral, and mediolateral) at 2 mm min-1. We determined variation in mechanical properties between sexes, and among developmental stages, vertebral regions, and testing orientations. We also investigated the relationships between vertebral process lengths and properties of dorsoventrally and mediolaterally-tested bone. Rostrocaudally-tested bone was the strongest, stiffest, and toughest, suggesting that this is the principle direction of stress. Our results showed that bone from female subadults was stronger and stiffer than their male counterparts; based on these data we hypothesize hormonal shifts at sexual maturity may partially drive these differences . In calves, bone from the posterior region was stronger and tougher than from the anterior region. We hypothesize that since animals grows rapidly throughout early development, bone in the posterior region would be the most ossified to support the rostrocaudal force propagation associated with undulatory swimming .

Methods
We destructively tested bone cubes (from manatee vertebrae) in compression to yield at a displacement rate of 2 mm min-1 using an Instron E1000 material tester. We converted force-displacement curves to engineering stress-strain (σ-ε) curves, which standardizes the compressive load (N) by dividing the force by the cross-sectional area (width • height: mm2) and displacement by dividing the change in length (mm) by the original length (mm). Yield was defined as the point where the sample transitioned from elastic to plastic deformation, as seen on the σ-ε curve (Fig. S1). We calculated stiffness (E: Young’s Modulus), or the resistance to deformation, as the slope of the steepest point of the linear portion of the σ-ε curve. Yield strength (σy) indicates σ when the material transitions from elastic to plastic deformation (stress at permanent deformation). Toughness (Ur) is the ability to absorb energy and was derived as the area under the σ-ε up until a yield behavior. We calculated mechanical properties using Bluehill Universal Software v.3.67 (Instron, Norwood, MA, USA).