: The test composed of a 3-day training
and the tests were done on the following day and
then 7 days later. This training period was done in
order to reduce the fear and anxiety of the rats when
exposed to a new environment. This was done by
placing each pair of rat from the same cage into the
apparatus and allowing them to freely explore the
open field arena for 30 minutes for 3 consecutive
days. The next day, each pair of rats was placed in the
apparatus at 06.00 pm and their behaviors were
observed for 24 hrs using closed circuit video recorder for further analysis. During this time, rats had free
access to water. Seven days later, each pair of rats was
exposed to the same repellent or control (blank). The
behavioral analysis was done every six hours for 30
min, at 06.00-06.30 pm, 12.00-12.30 am, 06.00-06.30 am
and 12.00-12.30 pm designated as P1, P2, P3 and P4,
respectively. The parameters were numbers of visits
and time spent in the inner, middle and outer zones.
If the tested substances could repel the rats, the
numbers of visits and time spent would be fewer in
the inner zone and higher in the outer zone.
Moreover, the total number of line crossed during 30
min was also recorded and considered as locomotor
activity of the rats. In order to test the efficacy of
potential repellent, the fear of novelty as being
exposed to a new environment was reduced by the
training period and by placing the rats in pair as rats
are social animals. Because it had been shown that
memory could last for at least 5 days following
learning (Rossato et al., 2006), a 7 day post-exposure
was then used to test whether the rats could
remember the substances. For each substance, 3 pairs
of rat were used and the behavioral data were
analyzed and scored by 2 experimenters blinded to
the experiment.