The use of micro‐CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination

Objectives In our laboratory we evaluated the use of micro‐computed tomography (micro‐CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). Methods To show concordance between traditional Alizarin Red S stain and micro‐CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro‐CT, then stained and re‐examined using traditional methods. Results A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low‐density skeletal bones may be visible by Alizarin Red stain but not by micro‐CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro‐CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro‐CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro‐CT scanned and traditional Alizarin Red S stained skeletons. Conclusion Micro‐CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.