Animals

Sixteen spontaneously dead puppies aged up to 28 days and belonging to breeds categorized as small-sized according to the standard breed adult body weight <7 kg¹³ were examined (Supplementary Table 1) and stored at -20°C.

Puppies were full term and died as a consequence of hypoxia during birth and/or bacterial diseases in the first days of life. Their body weight was normal in relation to the breed and the age at the time of death. Two puppies were premature (49 days of gestation). A spontaneously dead skeletally mature small-sized dog (15-year-old) was enrolled as positive control of ossification. The presence of skeletal abnormalities was excluded by macroscopic examination and confirmed by radiographic investigation: the expected appearance of the ossification centers was confirmed and no sign of degenerative joint disease in the shoulder and in the hip joint was detected in the mature dog.

All cadavers were obtained from breeder prior their informed consent signature; the research was approved by the Ethic Committee of the University of Milan (OPBA, 58/2016).