Effects of dietary vitamin D supplementation on bone microarchitecture, mineralization, and mechanical properties in Wistar rat animal model

Vitamin D is essential for proper skeletal development, yet the specific dose-dependent effects of its dietary supplementation during the transition from late adolescence through young adulthood remain unclear. Because the standard diet already supplies 1,000 IU/kg vitamin D₃ and is widely regarded as adequate for growing rats, poorer skeletal outcomes at 0 IU/kg and favorable outcomes at 1,000 IU/kg are expected. The studies objective was therefore to quantify the magnitude and map which dimensions of bone quality respond to vitamin D across the adolescent-to-young-adult transition, and to test whether a supraphysiological intake (5,000 IU/kg) confers additional benefit and we hypothesized that moderate vitamin D would improve bone outcomes versus deficiency, whereas a high dose would offer no added advantage. Male and female Wistar rats (≈ 7 weeks old; n = 6/sex/group) were fed for 12 weeks diets containing 0, 1000, or 5000 IU/kg vitamin D. The right femur was assessed by DXA (BMD), three-point bending (strength, energy absorption, stiffness), growth-plate morphometry, elemental composition, and X-ray diffraction of hydroxyapatite; RANKL/OC/VEGF immunoexpression and serum 25(OH)D were measured. Diets produced graded systemic exposure: 25(OH)D increased with dose in both sexes. Across outcomes, sex showed a strong main effect (males larger/stronger), while dose effects were selective. Compared with 0 IU/kg, 1000 IU/kg increased BMD and several mechanical endpoints (e.g., maximum force/energy) primarily in males, and improved material behavior (post-yield strain) without altering elastic modulus. Growth plate effects were limited: the hypertrophic (Zone IV) thickness was lower with deficiency and greater with supplementation, with sex- and dose-specific patterns; other zones showed minimal dose effects. The 5000 IU/kg diet raised 25(OH)D but did not consistently outperform 1000 IU/kg and showed no broad detriments across measured variables. During late growth, moderate vitamin D (1000 IU/kg) improves femoral mass and selected mechanical/biomechanical properties relative to deficiency, with sex-specific magnitudes of effect. A higher dose (5000 IU/kg) increases circulating vitamin D metabolite, but provides no clear additional skeletal benefit. These data support moderate supplementation as optimal in this model.