, Volume 60, Issue 6, pp 547–558 | Cite as

Salivary alpha-amylase enzyme is a non-invasive biomarker of acute stress in Japanese macaques (Macaca fuscata)

  • Nelson BrocheJr.Email author
  • Rafaela S. C. Takeshita
  • Keiko Mouri
  • Fred B. Bercovitch
  • Michael A. Huffman
Original Article


Salivary alpha-amylase (sAA) enzyme functions as a digestive enzyme in many species that consume starch in their diet. Human studies have also revealed that sAA enzyme activity levels are positively correlated with the release of the stress hormone norepinephrine, allowing sAA to act as a biomarker for sympathetic nervous system activity. Recent non-human primate studies have incorporated sAA as a physiological stress marker. However, no published reports have investigated the time course of sAA from a stressful event to return to baseline levels in non-human primates. Furthermore, no validation of sAA as a stress biomarker has been reported for Japanese macaques (Macaca fuscata). This study had two primary aims: (1) to develop a systematic method for non-invasive saliva collection and, (2) to investigate sAA as a biomarker of acute stress in M. fuscata in order to better understand its acute stress-related characteristics. We developed a non-invasive method for cooperative saliva collection using positive reinforcement training (PRT) and tracked individual progress over 595 trials in ten individually housed Japanese macaques. We detected sAA enzyme in M. fuscata via kinetic reaction assay, then performed 22 acute stress tests. Four tests met conditions for interpreting sAA in response to an acute stressor and these results show that on average sAA activity rapidly increased post-stressor (mean ± SD = 4.2 ± 0.9 min) and returned to baseline shortly thereafter (10.4 ± 0.6 min). Our report reveals for the first time the temporal dynamics of sAA when applying acute stress to Japanese macaques and could be a useful tool for assessing animal welfare.


Positive-reinforcement sAA enzyme Stress Japanese macaque Biomarker 



The research reported here was greatly assisted by Mr. Josue A. Pastrana, Dr. Claire F. I. Watson, Dr. Ikuma Adachi, and Dr. Takao Oishi. Valuable advice and support in experiments were provided by Dr. Takako Miyabe-Nishiwaki, Dr. Munehiro Okamoto, and Ms. Naoko Suda-Hashimoto. This project would not have been possible without assistance and collaboration from the Center of Human Evolution Modeling Research at KUPRI. Financial support was provided by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Leading Graduate Program in Primatology and Wildlife Science (PWS) of Kyoto University; and the Graduate School of Science, Kyoto University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Experimental designs were approved under permit No. 2016-146, by the Animal Welfare and Animal Care Committee at the Primate Research Institute, Kyoto University and institutional guidelines for the care and use of nonhuman primates were followed (


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Copyright information

© Japan Monkey Centre and Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Primate Research InstituteKyoto UniversityInuyamaJapan
  2. 2.Wildlife Research CenterKyoto UniversityKyotoJapan
  3. 3.Save The GiraffesSan AntonioUSA
  4. 4.Universidade Federal Rural da AmazôniaBelémBrazil
  5. 5.Department of AnthropologyKent State UniversityKentUSA

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