4.3.4. Testing Procedure

Testing procedures were performed in a room designed specifically to suit a model of noise-induced anxiety with recorded thunderstorm sounds, and to collect measures of anxiety and fear-related behaviors, such as activity or inactivity duration and frequency, and time spent in a hide box, as previously described [11]. The thunderstorm test consisted of an open field-testing room and an audio-recording of thunderstorm sounds [11]. Each testing session lasted nine minutes, where the first three minutes provided baseline data, whereas during minutes 4–6 the dogs were subjected to a taped presentation of thunder. No audible stimulus was provided during the final three minutes, which served as the post-thunder interval [11]. The entire study was designed in a manner such that each animal entered the test room on five occasions: once during the adaptation phase (no audio stimuli during 4–6 min); once during the thunder-test baseline phase (audio stimuli during 4–6 min); once during the open-field baseline phase (no audio stimuli during 4–6 min); once during the thunder-test treatment phase (audio stimuli during 4–6 min); and once during the open-field treatment phase (no audio stimuli during 4–6 min) (Table 1). The study procedures were carried out in cohorts for each phase following parallel-matched design so that each animal in each experimental group was tested with the same number of days between tests. Following thunderstorm and open-field baseline tests, dogs were dosed for five consecutive days with either placebo, diazepam, or a specific dose of the test product. After five days of dosing, all dogs were subjected to another thunderstorm test (thunder-treatment phase), followed by the open-field treatment phase, after which the anxiolytic properties of the tested products were determined.