Measurement of Salivary Cortisol in Two New World Primate Species.

Glucocorticoids (GCs) are mammalian steroid hormones involved in a variety of physiological processes, including metabolism, the immune response, and cardiovascular functions. Due to their link to the physiological stress response, GC measurement is a valuable tool for conservation and welfare assessment in animal populations. GC levels can be measured from different matrices, such as urine and feces. Moreover, especially in captive settings, measuring GCs from saliva samples proved particularly useful as those samples can be collected non-invasively and easily from trained animals. Salivary GC levels can be measured using a variety of analytical methods, such as enzyme immunoassays. However, it is crucial to validate the analytical method for each specific application and species when using a new matrix. Using high-pressure liquid chromatography and a cortisol enzyme immunoassay, we show that the main glucocorticoids secreted in the saliva of squirrel monkeys and brown capuchin monkeys are cortisol and cortisone. Our biological validation found the expected salivary cortisol level to decline throughout the day. Our findings support the reliability of salivary cortisol measurements and their potential to be used as a valid tool in research and welfare assessment for these non-human primates.

A Comparative Study of Chest CT With Lung Ultrasound After SARS-CoV-2 Infection in the Assessment of Pulmonary Lesions in Rhesus Monkeys (Macaca Mulatta).

Lung ultrasound (LUS) is a fast and non-invasive modality for the diagnosis of several diseases. In humans, LUS is nowadays of additional value for bedside screening of hospitalized SARS-CoV-2 infected patients. However, the diagnostic value of LUS in SARS-CoV-2 infected rhesus monkeys, with mild-to-moderate disease, is unknown. The aim of this observational study was to explore correlations of the LUS appearance of abnormalities with COVID-19-related lesions detected on computed tomography (CT). There were 28 adult female rhesus monkeys infected with SARS-CoV-2 included in this study. Chest CT and LUS were obtained pre-infection and 2-, 7-, and 14-days post infection. Twenty-five animals were sub-genomic PCR positive in their nose/throat swab for at least 1 day. CT images were scored based on the degree of involvement for lung lobe. LUS was scored based on the aeration and abnormalities for each part of the lungs, blinded to CT findings. Most common lesions observed on CT were ground glass opacities (GGOs) and crazy paving patterns. With LUS, confluent or multiple B-lines with or without pleural abnormalities were observed which is corresponding with GGOs on CT. The agreement between the two modalities was similar over the examination days. Pleural line abnormalities were clearly observed with LUS, but could be easily missed on CT. Nevertheless, due to the air interface LUS was not able to examine the complete volume of the lung. The sensitivity of LUS was high though the diagnostic efficacy for mild-to-moderate disease, as seen in macaques, was relatively low. This leaves CT the imaging modality of choice for diagnosis, monitoring, and longitudinal assessment of a SARS-CoV-2 infection in macaques.

Measuring salivary mesotocin in birds - Seasonal differences in ravens' peripheral mesotocin levels.

Oxytocin is involved in a broad array of social behaviours. While saliva has been used regularly to investigate the role of oxytocin in social behaviour of mammal species, so far, to our knowledge, no-one has tried to measure its homolog, mesotocin, in birds' saliva. Therefore, in this study we measured salivary mesotocin in common ravens (Corvus corax), and subsequently explored its link to three aspects of raven sociality. We trained ravens (n = 13) to voluntarily provide saliva samples and analysed salivary mesotocin with a commercial oxytocin enzyme-immunoassay kit, also suitable for mesotocin. After testing parallelism and recovery, we investigated the effect of bonding status, sex and season on mesotocin levels. We found that mesotocin was significantly more likely to be detected in samples taken during the breeding season (spring) than during the mating season (winter). In those samples in which mesotocin was detected, concentrations were also significantly higher during the breeding than during the mating season. In contrast, bonding status and sex were not found to relate to mesotocin detectability and concentrations. The seasonal differences in mesotocin correspond to behavioral patterns known to be associated with mesotocin/oxytocin, with ravens showing much more aggression during the mating season while being more tolerant of conspecifics in the breeding season. We show for the first time that saliva samples can be useful for the non-invasive determination of hormone levels in birds. However, the rate of successfully analysed samples was very low, and collection and analysis methods will benefit from further improvements.

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.

A comparative approach to affect and cooperation.

A central premise of the science of comparative affect is that we can best learn about the causes and consequences of affect by comparing affective phenomena across a variety of species, including humans. We take as a given that affect is widely shared across animals, but a key challenge is to accurately represent each species' affective experience. A common approach in the comparative study of behavior and cognition is to develop standardized experimental paradigms that can be used across species, with the assumption that if the same task is being used, we can directly compare behavioral responses. This experimental approach rests on two underlying assumptions: first, that different species' perception of and affective response to these paradigms are the same; and second, that behavioral and physiological (including endocrine and neural) responses to these paradigms are homologous; if either of these assumptions is not true, then the comparison becomes much less straightforward. Our goal in the present paper is to summarize the dominant paradigms that have been used for such comparative research, with a particular focus on paradigms common in the cooperation literature, and to critically discuss dominant assumptions about what affective states these tasks can or should measure. We then consider the advantages and drawbacks of this experimental method, and consider alternatives that may improve our understanding. We hope that this will help scholars recognize and avoid pitfalls inherent in studying affect, and stimulate them to create novel, ecologically relevant paradigms for examining affect across the animal kingdom.

Behavioural and Hormonal Stress Responses to Social Separation in Ravens, Corvus corax.

Social life is profitable, but it facilitates conflicts over resources and creates interdependence between individuals. Separating highly social animals triggers intense reactions aimed at re-establishing lost connections. Less is known, however, about behavioural and physiological responses to separation in socially facultative species, where individuals temporarily form groups and may subsequently leave them. Non-breeding common ravens (Corvus corax) gather in large numbers at feeding and roosting sites, but otherwise spend time seemingly solitary or in small subgroups. We here studied how ravens cope with being socially isolated, and investigated the life characteristics that might explain potential individual differences. For this, we individually separated captive subadult ravens (n = 25) and housed them in physical and visual isolation from their group members across 4 d. During the separation period, we collected behavioural data and measured the amount of immunoreactive corticosterone metabolites from bird droppings to assess the ravens' physiological stress response. We found behavioural indicators of stress at the start of the separation, when ravens showed higher levels of tension than of comfort - a pattern that reversed at the end of the separation. Furthermore, we found that the upbringing of ravens affected their behaviour during separation. Hand-raised birds produced more vocalisations in the beginning of the separation, and were less active at the end, while the reverse pattern occurred with parent-raised ravens. Contrary to our predictions, we did not find differences in hormonal responses between the beginning and end of the separation period or any link between hormonal responses and behaviours. Ravens' behavioural responses to social separation stress seem to be dependent on their arousal states, although possible links with hormonal reactions remain unclear. Our results show that behavioural reactions are not always linked with hormonal responses to stress, and further emphasise the importance of investigating effects of early-life experiences.

Using photographs to study animal social cognition and behaviour: Do capuchins' responses to photos reflect reality?

Behavioural responses to photos are often used to infer what animals understand about their social environment, but are rarely validated against the same stimuli in real life. If subjects' responses to photos do not reflect responses to the same live stimuli, it is difficult to conclude what happens in reality based on photo responses alone. We compared capuchins' responses to photos versus live stimuli in an identical scenario within research cubicles. Subjects had the opportunity to approach food placed in front of an alpha group member and, in a separate condition, photos depicting the same individual. Subjects' latencies to approach food when placed in front of the real alpha negatively correlated with time subjects spent in close proximity to the alpha in their main enclosure. We therefore predicted subjects' latencies to approach food in the presence of photos would positively correlate with their latencies to approach food in the presence of the real alpha inside the cubicles, but negatively correlate with time they spent in proximity to the alpha in their enclosure. Neither prediction was supported. While not necessarily surprising, we explain why these results should be an important reminder that care is needed when interpreting results from photo studies.

Loner or socializer? Ravens' adrenocortical response to individual separation depends on social integration.

Non-breeding common ravens (Corvus corax) live in complex social groups with a high degree of fission-fusion dynamics. They form valuable relationships and alliances with some conspecifics, while taking coordinated action against others. In ravens, affiliates reconcile their conflicts, console each other after conflicts with a third party, and provide each other with social support - all behaviors that presumably reduce corticosterone levels and alleviate stress. However, how well an individual is socially integrated in a (sub)group might vary substantially. This raises the question whether the social integration of a raven affects its stress responses to fission-fusion dynamics. The present study aims to investigate this effect experimentally by separating single ravens (n=16) individually from their group for four days and subsequently reintroducing them. To determine stress response patterns in the separated individuals we measured the amounts of immunoreactive corticosterone metabolites (CM) in droppings. We compared two enzyme immunoassays, which we validated by conducting an ACTH challenge, and finally decided to apply an 11-oxoetiocholanolone enzyme immunoassay. Additionally, we determined levels of social integration using focal observations. Our findings suggest that a strong social integration is related to low CM levels when the individuals are within the group and high levels during separations, implying that separation leads to stress in these birds. In contrast, poorly socially integrated ravens seem to exhibit the opposite pattern, indicating that to them group living is more stressful than being temporarily separated. We, therefore, conclude that the birds' adrenocortical activity is modulated by their social integration.