New advances in estimating the age of dog puppies for medico-legal purposes

Age determination of dog puppies represents a significant issue of animal welfare and forensic medicine. Despite the legislation in force, the illegal trade of puppies that are too young is expanding. To date, none of the available age estimation methods can be credited with a degree of accuracy sufficient for medical-legal purposes due to the many variability factors affecting the measured biological phenomena, such as teeth and skeletal development, and the lack of standardisation. This work aimed at quantifying the degree of correlation between the chronological age of puppies and the biological age that can be estimated by visual teeth examination and the radiographic examination of limb ossification centres (OCs), and combining the information in a predictive model to obtain greater accuracy in estimating the age. The study included 93 puppies of 10 different breeds, which were examined on a bi-weekly basis from 4 to 20 weeks of age and radiographed from 6 to 16 weeks, when not sold earlier. Teeth eruption and development was affected by a wide degree of variability, in line with the information found in the Literature. On the basis of the number of OCs present, the breeds included in the sample could be divided in “early” and “late” breeds, and no correlation between this variability and size or morphological type was observed. However, the radiographic examination of limb ossification centres allowed to determine, with a fair degree of accuracy, whether a puppy was younger or older than 12 weeks old. Contrarily, at eight weeks, it was not possible to determine the age with a good degree of precision, as various degrees of variability were observed. A model to predict if a puppy was 6 or 8 weeks old was constructed using random forests, which proved to be a powerful tool to be further developed