Cooperation with closely bonded individuals reduces cortisol levels in long-tailed macaques

Many animal species cooperate with conspecifics in various social contexts. While ultimate causes of cooperation are being studied extensively, its proximate causes, particularly endocrine mechanisms, have received comparatively little attention. Here, we present a study investigating the link between the hormone cortisol, cooperation and social bonds in long-tailed macaques (Macaca fascicularis). We tested 14 macaques in a dyadic cooperation task (loose-string paradigm), each with two partners of different social bond strength and measured their salivary cortisol before and after the task. We found no strong link between the macaques' cortisol level before the task and subsequent cooperative success. By contrast, we did find that the act of cooperating in itself led to a subsequent decrease in cortisol levels, but only when cooperating with closely bonded individuals. Two control conditions showed that this effect was not due to the mere presence of such an individual or the pulling task itself. Consequently, our study shows an intricate way in which the hypothalamic–pituitary–adrenal axis is involved in cooperation. Future studies should reveal whether and how our findings are driven by the anxiolytic effect of oxytocin, which has been associated with social bonding.

We observed the male in the group for 30 min before starting the challenge to ensure that at least no obvious stressors occurred. Then we let the subject into the testing compartment and took 13 saliva samples in an interval of 2.5 min, hence for 30 min (approx. 1 min/sample, 1.5 min in-between). In the stress challenge, the experimenter left the room after collection of the first sample. An animal keeper (to the subject unfamiliar) immediately entered the room, stood in front of the subject and made noise with a large paper bag. We chose to present a paper bag because animal keepers have previously observed that this macaque group shows an alert/aversive, though not extremely intense responses to that. After one minute the animal keeper left and the experimenter returned and continued to collect saliva samples.
To determine the influence of the training itself and the presence of the experimenter, we conducted a control test employing the same procedure with the same individuals, but without presenting a stressor. The male was tested in June 2016, first in the control condition, and two days later in the stressor condition, both between 11 and 12 am.
The analysis of the saliva samples collected during the test situations was conducted as described in the Salivary cortisol -Hormone analysis section (IBL Cortisol Saliva ELISA, intra-assay CV: 1.56 %).

Results and discussion
The results show a time lag between the start of the stressor and the appearance of the cortisol peak of 15 min (Fig. SC). During that time, the male had a 2.6-fold increase (4.7 ng/ml) in cortisol (3.7-fold (5.6 ng/ml) from the lowest point). During the first half of the control condition the individual showed a highly varying and overall increasing cortisol level (sample 0 -12.5: mean = 4.13 ± 1.86 SD), which Cooperation with closely bonded individuals reduces cortisol levels in long-tailed macaques -ESM 2 2 might be due to interactions in the group or the experimenter starting the session. The level, however, stabilized 12.5 to 15 min after the session has started (sample 15 -30: mean = 5.72 ng/ml ± 0.42 SD), indicating, first, that the stable cortisol level in the second half represents the stable situation during the control session, and second, that also training and test sessions lead to an elevated level of arousal. In the stress condition the cortisol level finally (after 30 min) drops to the stable, but elevated level shown in the control. As a precaution regarding animal welfare, we did not keep the monkeys in the training compartment for more than 30 min. Therefore, we were not able to investigate full recovery time in the stress challenge and the second sample during test conditions already had to be taken 10 min after the end of the tests instead of 15 min later. We are aware that one sub-adult male is not necessarily representative for a whole species, but the time lag of 15 min in his salivary cortisol response corresponds with what was shown in blood samples after ACTH challenges in the same species [1,2]. Considering that the peak cortisol concentration in saliva follows the one in blood with a time lag of 2-3 min [3] one might have expected the peak in our male to occur after 17.5 min. However, blood samples after the ACTH challenges were collected only at two time points (probably due to animal welfare concerns) -15 and 30 min after ACTH administration [1,2], whereas the sampling interval in our stress challenge was much higher, leading to a more detailed temporal resolution. Moreover, the response to ACTH is most likely much more intense than to the rather mild stressor we used in our study.
The male's elevated but stable cortisol level in the stress challenge control indicates that subjects might also experience slightly increased arousal/stress when interacting with an experimenter. However, the mean cortisol level of the second half of this control session (reflecting the subject-experimenter interaction), which coincides with the overall average level in this study, is somewhat lower than what was found in other studies investigating salivary cortisol in macaques (e.g., long-tailed macaques, Macaca fascicularis [4]; rhesus macaques, M. mulatta [5]. This suggests that, probably due to regular training with positive reinforcement, most subjects tested in this study did not experience extreme stress during training and test sessions. During the stress test, in which the subject not only showed an endocrine response, but also clear behavioural signs of arousal (piloerection, lip-smacking [1]) that were absent in the control test, salivary cortisol concentrations increased by 4.7 ng/ml. Accordingly, even though changes in cortisol during the stress challenge were rather small, we consider them meaningful.