2.4. Open Field Test

The Open field test was used to evaluate the level of anxiety through locomotor activities [36]. The apparatus consisted of a wooden box measuring 72 × 72 × 36 cm. The floor of the arena was divided into sixteen equal squares: four in the center and twelve in the periphery (four corner and eight wall) [37] (Figure 2). The total distance (the total distance traveled) and average speed (average movement speed) were measured to evaluate general locomotor activity. Additionally, the center time (duration of time spent in a central square), wall time (duration of time spent in a wall square), corner time (duration of time spent in a corner square) and number of excrements were measured to evaluate anxiety. The test duration was 5 min [37,38]. All measurements (times and distances) were recorded by an AHD camera 2-MP, Model No: cp-B20M3, Lens: 3.6 mm-3MP (Shenzhen MVTEAM Technology Co, Ltd, Shenzhen city, China). The analysis was performed in a blind manner. The apparatus was cleaned with a solution of 10% ethanol between trials to eliminate animal clues.

Open field test performance and box.