Reproductive status affects stress-induced hormonal and immune changes in male and female Rhinella diptycha toads

Species with seasonal reproduction exhibit marked variations in physiological processes, including reproductive physiology and immune functions. In unpredictable situations, the emergency life-history stage is characterized by a stress response, primarily mediated by glucocorticoids, which regulate the balance between survival and reproduction. To better understand the impact of stressful events on endocrine and immune physiology in amphibians, we subjected male (calling or foraging) and female (foraging) toads (Rhinella diptycha) captured in the field during a reproductive event to a restraint protocol (0 h, 1 h, and 24 h, repeated samples). Before the restraint, a blood sample was collected to obtain reference values for plasma hormone levels (corticosterone, the main glucocorticoid in amphibians; testosterone; and estradiol) and immune measures (plasma bacterial killing ability and the neutrophil-to-lymphocyte ratio). Plasma corticosterone levels increased 1 and 24 h post-restraint in females and males (regardless of activity). We found decreased estradiol and testosterone plasma levels 24 h post-restraint, regardless of sex or activity. The plasma bacterial killing ability increased 1 h post-restraint in calling males and decreased 24 h post-restraint irrespective of sex or activity. The neutrophil to lymphocyte ratio increased only in foraging males 24 h post-restraint compared with 1 h post-restraint. These findings indicate that while corticosterone responses to acute stress are rapid and persist over time, the suppression of sex steroids and certain immune functions becomes more evident under prolonged stress exposure. Moreover, the transient enhancement of bacterial killing ability in calling males and the delayed increase in neutrophil-to-lymphocyte ratio in foraging males suggest that stress-induced immunomodulation may be influenced by reproductive behavior in R. diptycha.