On PND 9, the pups were individually placed into a Plexiglas arena (30 (length) × 30 (width) × 30 (height) cm), located inside a sound-attenuating and temperature-controlled chamber, with a camera positioned above the arena. The ultrasonic vocalizations (USVs) emitted by the pup were detected for 3 min by an ultrasound microphone (Avisoft Bioacoustics, Glienicke, Germany) sensitive to frequencies between 10 and 200 kHz. Pup axillary temperature was measured before and after the test by a digital thermometer. The USVs were analyzed, both quantitatively and qualitatively, using Avisoft Recorder software (Version 5.1), and classified in six different groups according to number of syllables, frequency modulation and duration:44 (link), 45 (link), 46 (link) flat calls (calls with constant frequency with a maximum variation of ±5 kHz); up/downward (single syllable calls emitted at ±5 kHz with a single frequency modulation); flat high frequency calls (calls with similar constant frequency, but emitted at >75 kHz); syllable calls (calls composed by two or more syllables); complex calls (calls displaying complex frequency modulation); short calls (calls with durations shorter than 5 ms).
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