A new insight for investigating the prenatal and postnatal ossification centers of pelvic and femur bones in white New Zealand rabbits (Oryctolagus cuniculus) using 3D CT, double stain technique, and morphometry.

Background: The ossification centers in rabbit limbs are related to fetal age and bone maturation.
Objective: To address the limited studies on ossification in the hind limbs of New Zealand rabbits, we investigated the prenatal and postnatal development of the pelvic and femur bones.
Methods: Double staining with Alcian Blue and Alizarin Red, computed tomography (CT), and 3D reconstruction were employed to visualize and analyze ossification centers in detail.
Results: Using double staining, we observed these patterns: At prenatal days 18 and 21, ossification centers appeared in the ilium. By prenatal days 23 and 25, ossification began in the ischium. On postnatal day 1, ilium ossification centers spread across most of the ilium wings, except for the iliac crest, and new centers appeared in the pubis and cotyloid bones. Most bones had ossified by the third week and one month postnatal, except for the iliac crest and ischial tuberosity. At 1.5 months, both were fully ossified. On day 18 post coitum, an ossification center was visible in the middle of the femur shaft. By day 28 post coitum, ossification extended through the shaft, and postnatally, new ossification spots appeared at the extremities by day one and week one. By the third week, complete ossification of the femur head, lesser trochanter, third trochanter, medial condyle, and lateral condyle was observed. At 1.5 months, the entire proximal extremity was ossified.
Conclusion: 3D CT provided clear imaging of ossification progression in the pelvic and femur bones. This study enhances our understanding of vertebrate skeletal development.
Competing Interests: Declaration of Competing Interest None of the authors has financial or personal relationships that could inappropriately influence or bias the paper’s content.
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