Forced swimming test:

Porsolt and colleagues (18) are first authors that originally developed the Rat Forced Swimming test (FST), this is the most used test for animal models to assess antidepressant-like behavior (19), active or passive behavior of rats that are forced to swim and can’t escape from the swimming tank. Forced swimming test for rats later was modified for mice (20). The mouse version of the forced swim test is a relatively short and low-cost behavioral test that requires no training of the mice and can be conducted with minimal equipment. This is in contrast to the rat version of the test, which generally involves exposure to the water tank one day prior to the test day (21). There are two versions that are used, namely the traditional and modified FST which differ in their experimental setup, for both versions, a pretest of 10-15 min is included, as this accentuates the different behaviors in the 5-min swim test following drug treatment (19).

Rats were exposed to forced swim test the swimming program included two phases: adaptation and training. In the first week (adaptation), the rats swam 60-90 min per day for 6 days (1 week). The training period began from the beginning of the second-week swimming duration was progressively increased from 50 min to approximately 90 min per day. Rats were exposed to a swimming test between 08.30h till 12.00h AM. This intensity was maintained to the end of the training program which lasted 6 days per week for a total of three weeks (21 days), during weekdays Monday-Saturday, while Sunday was without activity for experimental rats (Picture 1).

Forced swimming stress was induced in rats by forcing them to swim in a cylindrical swim tank which was filled with tepid water (±34°C). All swimming protocol was recorded by a video camera and photo camera. The length of the cylindrical swim tank was approximately 80-90 cm which was filled with water till 50-60 cm of depth, this depth exceeded the length of the rat including tail (the length of the rats was approximately 40cm), the width of the tank was 60cm. During the experiment, it was measured body mass of the rats. Body mass was measured with a standard weighting scale of 1 kg, the measurement was taken every day at the same time in the morning approximately at 08.30 AM on day 2- 21.