Sex-specific stress and biobehavioral responses to human experimenters in rats

Abstract

Important factors influencing the outcome of animal experiments in preclinical research are often overlooked. In the current study, the reaction of female and male rats toward the biological sex of a human experimenter was investigated in terms of anxiety-like behaviors and physiological stress responses, as measured by infrared (IR) thermography, circulating corticosterone (CORT) and oxytocin levels. Female rats displayed consistently exacerbated anxiety-related behaviors along with elevated body surface temperature during repeated exposure to male experimenters. Experimental stress further intensified thermal responses to a male experimenter, especially in female rats. The behavioral responses to a male experimenter in females were associated with higher circulating CORT and lower oxytocin levels. Similar responses were induced by a T-shirt worn by a human male. The findings suggest that psychophysiological responses of female rats to a male experimenter are influenced by both visual and olfactory cues. The results emphasize the need to not only consider sex differences in experimental animals, but also standardize and report the experimenter's biological sex to avoid ambiguity in the generation and interpretation of results.

Keywords: corticosterone (CORT); cutaneous temperature; experimenter sex; hypothalamic-pituitary-adrenal (HPA) axis; infrared thermography; oxytocin; sex differences; stress response.

FIGURE 1

Experimental design. Female and male adult rats were exposed to female and male experimenters in four distinct experiments (Exp1–4). In Experiments 1–2, rats were exposed to opposite-sex experimenters or their T-shirts, whereas rats in the Experiments 3–4 were required to be handled and/or tested by same-sex experimenters. In all experiments animals were manipulated for handling (A), pre-stress open field testing (B), stress (C), stress and post-stress open field testing (D), and blood sampling (E). Rats’ thermal responses to the experimental procedures were recorded by an infrared (IR) thermographic camera in the presence or absence of the experimenters.

FIGURE 2

Open field testing and IR imaging. (A) Exploratory movements of female and male rats were analyzed for thigmotaxis or the repetitive pattern of exploration near to the wall in the open field. (B–D) Thigmotaxis in Experiment 1 was significantly impacted when female rats were required to explore the open field in the presence of an opposite-sex experimenter. The multipath shown in the panel (B) motion track graphics compares thigmotaxis taken from four representative rats during Exp1. Small squares represent individual rats in each group and experimental session. (E) The IR thermographic imaging showing two regions of interest [head (left and right) and back] in assessments of surface temperature during the open field exploration. (F) Because there were no differences in the thermal responses between the left and right sides of the head, the average of cutaneous temperatures for the left and right sides were used. Females showed consistently higher cutaneous thermal temperatures than males for the head and back (left panel). The inset IR graphic output (right panel) provide samples of thermal differences in females and males in both ROIs. However, when animals were tested by same-sex experimenters in Experiments 3 and 4, male rats showed higher thermal responses than females in the head. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; one-way and repeated-measure ANOVA, n = 6/group. Error bars show ± SEM.

FIGURE 3

Stress and post-stress open field exploration. (A) No significant difference was observed between females and males in the surface temperature when rats were stressed in the presence of opposite-sex experimenters or their T-shirts. Inset thermal graphics compare changes in cutaneous temperature in two representative rats in min 4. (B–D) Although thermal responses during stress in Experiment 1 showed no sex differences, post-stress thigmotaxis noticeably increased in females. The multipath taken from four representative rats in each group is shown in the inset motion track graphics. (E) A comparison of the pre- and post-stress thigmotaxis indicated females spent more times near to the wall than males at both time points in the presence of a male experimenter (left panel). (F) Interestingly, females displayed higher thermal changes than males in the head and back when exposed to a male experimenter and the male’s T-shirt (Exp1 and 2) after stress. The inset thermal graphics represent thermal changes in min 3 in a female and male rat accompanied by the pertinent oscilloscopes. Purple triangles in the panels represent D. IR recording (stress-open field). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; one-way and repeated-measure ANOVA, n = 6/group. Error bars show ± SEM.

FIGURE 4

(A,B) Thermal changes prior to and after stress. Both groups were susceptible to the stress procedure in the presence of opposite-sex experimenters. However, female thermal responses in both ROIs were noticeably different from males before and after stress indicating higher vulnerability of females to the impact of stress and experimenter sex. (C) The rate of changes (ROC) provides further support for exacerbated thermal responses in female rats to experimenter sex after stress relative to male rats. Note the trendlines of thermal changes in the head and back in both sexes that noticeably depict sex-specific differences in the regional thermal responses to stress and experimenter sex.

FIGURE 5

Corticosterone and oxytocin responses to stress and experimenter sex. (A) Average CORT levels in females across all experiments were higher than males. Exposure to an opposite-sex experimenter significantly elevated plasma CORT levels in female rats only. There were no significant differences between females and males in Experiments 3 and 4 when exposed to same-sex experimenters or their T-shirts. Red squares represent individual animals in each group. Gray boxes represent statistically significant differences between sexes (*p ≤ 0.05, Mann–Whitney U; n = 5–6/group). (B) Female rats responded to the presence of a male experimenter with reduced plasma OT levels. In contrast, male rats experienced marginal changes in plasma OT levels across experiments (n = 4–6/group).