Oxytocin Pathways and the Health Effects of Human-Animal Interaction
Phase: NA
Conditions: Human-Animal Bond, Human-Animal Interaction
Sponsor: University of Arizona

 
 
 
 
 
 
 
 
Oxytocin Pathways and the Health Effects of 
Human-animal interaction 
 
 
 
Protocol Number: 1808883345 
National Clinical Trial (NCT) Identified Number:  NCT03852264 
Principal Investigator: Evan MacLean, Ph.D. 
Sponsor: University of Arizona 
Grant Title: Oxytocin Pathways and the Health Effects of Human-animal interaction 
Grant Number: 1R21HD095217 
Funded by: National Institute of Child Health & Human Development 
Version Date: 15 March 2024 
 

 
 
 
 
 
https://clinicaltrials.gov/ct2/show/study/NCT03929367?term=oxytocin&rslt=With&cntry=U
S&draw=2&rank=1 
BACKGROUND 
Studies across more than three decades have revealed diverse health benefits from 
human-animal interaction, including effects on social behavior, mood, stress and anxiety, 
and physical health1-3. However, relatively little is known about the underlying biological 
mechanisms which mediate these outcomes, precluding a deeper understanding of how 
and why human-animal interaction works4. Recent studies indicate that human-animal 
interaction can lead to acute release of oxytocin in human adults, a neuropeptide that 
plays critical roles in the development and expression of social behavior, cognition, stress 
physiology, and physical health. Dysregulation of the oxytocin system has been 
implicated in a range of diseases and/or disabilities, including autism, post-traumatic 
stress disorder, and anxiety disorders5. Exogenous oxytocin administration has shown 
some promise as an experimental therapeutic6-8, but knowledge about its long-term safety 
or efficacy is limited9, and current administration routes rely on flooding the periphery with 
supraphysiological concentrations of synthetic oxytocin (only minute amounts of which 
cross the blood-brain barrier10). In contrast, human-animal interaction may provide a safe 
and effective method for stimulating endogenous oxytocin activity in the central nervous 
system11,12. However, no studies have investigated how human-animal interaction affects 
oxytocin release in children.  This study was designed to assess the effects of human-
animal interaction on oxytocin release in children.  Because oxytocin can be released in 
response to both pleasurable stimuli and stress, salivary cortisol was also measured as 
a biomarker of acute stress, to contextualize findings regarding oxytocin.  
PROTOCOL 
Participants 
 
Eligibility criteria: Children were eligible for inclusion if they were between 8-10 years of 
age, lived with a companion dog who had been in the household for at least 6 months, 
and did not meet any of the exclusionary criteria.  Exclusionary criteria for children 
included current use of psychoactive medications or diagnosis with neurodevelopmental 
or endocrine diseases / disorders.  
 
Recruitment: Participants were recruited from the local community through email listservs, 
print advertisements, social media, and flyers distributed to schools, libraries, museums, 
and veterinary clinics. Parents received monetary compensation and children were 
allowed to select a small toy to bring home after each study visit.  
 
Procedure 
 

 
 
 
 
 
Materials 
 
The study was conducted at the Arizona Canine Cognition Center (ACCC) in Tucson, 
Arizona in an indoor room with padded floor mats. A chair was set up in the corner of the 
room. Video was recorded from two overhead cameras and two tripod-mounted cameras 
and audio was captured using two room microphones. Brown noise was played through 
two wall-mounted speakers to mask distracting noises from outside the experiment room.  
 
General Procedure 
 
Participants visited the ACCC three times and completed one of three conditions at each 
visit: Pet Dog (PD), Unfamiliar Dog (UD), and Control (CT). Conditions were scheduled 
in two fixed orders (UD-CT-PD or PD-CT-UD), and the order was counterbalanced across 
participants. 
 
Upon arrival, participants were greeted by two experimenters. Baseline (T1) saliva 
samples were then collected from the child (see Sample Collection). The parent was 
provided with an iPad and headphones and instructed to sit in a chair facing a corner of 
the room and to ignore the child and dog (when applicable) until the end of the final 
sample collection. The experimenters then left the child in the room to engage in the target 
activity and returned 15 minutes later to collect timepoint 2 (T2) saliva samples from the 
child. Experimenters then left the room and children were allowed an additional 10 
minutes to engage in the activity. Timepoint 3 (T3) saliva samples were collected fifty 
minutes after the start of the behavioral activity.  
 
Pet Dog (PD) condition: In the PD condition, children were given the opportunity to interact 
naturally with their pet dog. Children were informed that they would be left in the room 
with their dog, and to “keep the dog company” during this time. They were further 
instructed that they could play with their dog however they liked and were briefly reminded 
about appropriate and inappropriate interactions with dogs (e.g. not okay to pull a dog’s 
tail or step on a dog; okay to pet a dog gently or to play with a ball together).  
 
Unfamiliar Dog (UD) condition: The UD condition was identical to the PD condition with 
the exception that children interacted with a dog that they had not met prior to the 
study.  This dog was a Labrador retriever who was released from a service dog program 
for a benign medical condition. Children were shown a photo of the dog prior to meeting 
her and informed that she had a mellow personality and enjoyed belly rubs.  
 
Control condition (CT): In the CT condition, a table and chair were set up in the middle of 
the experiment room, and children were provided with a box of toys that included LEGOs, 
kinetic sand, a Lite-Brite, various puzzles, and colored pencils and drawing paper. 
Children were instructed that they could play with whichever toys they liked. 
Sample Collection, Processing, and Analysis 
Saliva Collection. Saliva was collected using Saliva Collection Aids from Salimetrics®, or 
in rare cases where this device presented challenges to children, using a weigh boat. 

 
 
 
 
 
Before collection began, the experimenter instructed the child on how they should drool 
into the tube and indicated that the 1 mL mark on the tube is the amount of saliva they 
are aiming to produce; they were instructed to passively drool into the straw by letting 
saliva fall into the straw and not to actively spit into it. Children were also told that if they 
were having trouble producing saliva, they could think of things such as “biting into a 
lemon” or “thinking of their favorite food.” The experimenter then recorded the initial 
weight of the micro tube, inserted the collection aid into the tube, and gave it to the child 
to begin sample collection. When the volume of the saliva had reached or exceeded the 
1 mL mark, the straw was removed, and the sample was weighed. If the sample was not 
at least 1.0 g heavier than the initial weight of the tube, the child was instructed to attempt 
to produce more saliva. A maximum of ten minutes were allotted for child saliva collection. 
If the child could not produce enough saliva to reach 1 mL or weigh at least 1.0 g, the 
weight was recorded and the experiment continued. If a child struggled with the Saliva 
Collection Aid, a weigh boat was used instead (providing a larger opening in which to 
deposit saliva); saliva in the weigh boat was then transferred to a microtube using a 
transfer pipet. 
 
Saliva processing. Samples were thawed and centrifuged in a microcentrifuge at 10,000 
RPM for 10 minutes. Supernatants from each sample were collected into a new 
microcentrifuge tube and the pellet was discarded. The supernatant volume was then 
aliquoted in the following volumes for corresponding analysis: 500 uL for oxytocin 
measurement and 150 uL for cortisol measurement. All aliquots were stored again in the 
-80 ⁰C freezer until analysis. 
 
Quantitation of salivary oxytocin. Prior to analysis, salivary samples (500 uL) were 
lyophilized using a CentriVap (Labconco model #7810016) with the following settings: 
CentriVap: unheated, cold trap: 80–85 °C, vacuum: 0.3–0.4 mbar. When lyophilization 
was complete, and immediately before assay, samples were reconstituted in 250 uL 
assay buffer (resulting in a two-fold concentration). Samples were assayed in duplicate 
using a competitive enzyme-linked immunosorbent assay manufactured by Arbor 
Assays™ (product number K048).  
 
Quantitation of urinary oxytocin. Prior to analysis we used a mixed-mode solid-phase 
extraction following the protocol described and validated by Gnanadesikan et al.13. 
Samples were assayed in duplicate using a competitive enzyme-linked immunosorbent 
assay manufactured by Arbor Assays™ (product number K048). Urinary oxytocin 
concentrations were corrected for the specific gravity of the sample using the method 
described by Gnandesikan et al.14.  
 
Quantitation of salivary cortisol. Salivary cortisol was measured using a competitive 
enzyme-linked immunosorbent assay manufactured by Arbor Assays™ (product 
number K003).  Samples were analyzed in duplicate.  
 
 
STATISTICAL ANALYSIS PLAN 
 

 
 
 
 
 
The primary outcome measure was the oxytocin output (relative to baseline) in 
conditions involving human-animal interaction (pet dog, unfamiliar dog) relative to the 
nonsocial control condition. The area under the curve with respect to the initial value 
(AUCi) was used as the measure of oxytocin output15. We compared AUCi values across 
conditions using a Bayesian linear mixed model. In addition to the focal predictor variable 
(experimental condition), the model included covariates for participant sex, age (years), 
assay plate, and study visit number. Participant ID was included as a random intercept in 
the model.  
 
The secondary outcome measure was the cortisol output (relative to baseline) in the 
conditions involving human-animal interaction (pet dog, unfamiliar dog) relative to the 
nonsocial control conditions. Analysis of this endpoint was conducted identically to that 
for oxytocin, as described above, with the exception that the model did not include a 
covariate for assay plate.  
 
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