Measuring short-term changes in stress-associated salivary analytes in free-ranging Japanese macaques (Macaca...

Monitoring short-term changes of endocrine responses in non-human primates living in wild populations is a challenge. Saliva contains enzymes, steroids, and various analytes that can be broadly useful for helping us understand physiological responses to social and environmental sources of stress as well as other compromises to an individual's health homeostasis. Salivary alpha-amylase and salivary cortisol are known to respond rapidly to stress, which can allow us to use these analytes to monitor stress on a time scale of minutes. We developed a non-invasive methodology for the collection of saliva and verified its applicability by conducting short-term interval sampling of focal individuals under varying social conditions in a group of semi-provisioned free-ranging Japanese macaques (Macaca fuscata) living endemically on the island of Koshima, Miyazaki prefecture, Japan. Monkeys were habituated to sampling by using ingestive attractants applied to cotton ropes. Their receptivity to chewing on the saliva sampling material provided and chewing time was recorded ad libitum during the initial habituation period of the troop to the sampling procedure. Focal animal behavior observations were conducted in conjunction with saliva sampling. Salivary analytes associated with stress, alpha-amylase and cortisol, were measured via enzyme immunoassay. Short-term changes in salivary alpha-amylase and salivary cortisol were examined in relation to short-term changes in social behavior, in particular aggression or grooming. We tested flow-rate effects in both analytes and found strong correlations between original sample results and their respective flow-rate transformed equivalents. Additionally, temperature effects on samples were tested and both analytes showed nearly the same values when stored at - 20, 4, and 30 °C conditions for 6 h; important information expected to facilitate future sampling in field conditions where freezer storage is uncertain. Our study shows that saliva can be repeatedly sampled non-invasively to investigate short-term changes in stress-associated markers in free-ranging Japanese macaques.