Animals
Experimental procedures were carried out following protocols approved by
the Ethical Committee for Use of Animals of the Federal University of
Lavras (Approval # 025/16), which complies with Brazilian and
international guidelines for animal use and welfare. Twenty-five male
Wistar rats (60 days old, weighing 230–270 g) were used in the present
study. The animals were obtained from the animal breeding facility of
São Paulo State University (UNESP) (Botucatu, SP, Brazil) and kept in
the Animal Care Unit of the Department of Pharmacology, School of
Medicine of Ribeirão Preto, University of São Paulo. The animals were
kept under a 12-h/12-h light/dark cycle (lights on between 7:00 AM and
7:00 PM) and had free access to water and standard rat food. We chose
all group size of all experiments shown in the present study based on
previously reported data which are comparable with our experiments
(Barretto-de-Souza, Benini, Reis-Silva, & Crestani, 2021;
Gomes-de-Souza, Costa-Ferreira, Oliveira, Benini, & Crestani, 2020).
The use of wistar rats for these experiments is justified by the fact
that this species of animal has some characteristics that allow us to
experiment with it, among them: Viability, similarities with human
beings (Leong, Ng, & Jaarin, 2015). Moreover, the choice of using male
wistar rats for these experiments was to avoid the possible interference
of hormonal variation throughout the estrous cycle of female and
autonomic responses during restraint stress (Mahmoodzadeh, Fliegner, &
Dworatzek, 2013; Miller et al., 2017; Miller et al., 2011).