Attachment-like behavioral expressions to humans in puppies are related to oxytocin and cortisol: A comparative study of Akitas and Labrador Retrievers.

This study investigated the relationship between urinary hormone concentrations and attachment-related behaviors in two dog breeds, the Akitas and Labrador Retrievers, to elucidate the hormonal and behavioral mechanisms underlying domestication and interspecies attachment to humans. By measuring cortisol and oxytocin concentrations, and conducting the Strange Situation Test (SST), we aimed to investigate breed differences in endocrine secretions associated with domestication and how these differences influence dog behavior toward humans. Our results showed significant breed differences in urinary cortisol concentrations, with Akitas exhibiting higher levels than Labrador Retrievers. This suggests a breed-specific stress response related to genetic proximity to wolves. However, oxytocin concentrations did not differ significantly, which suggests a complex interplay between factors influencing the domestication process and the formation of attachment behaviors. Behavioral observations during the SST revealed breed-specific patterns, with Labrador Retrievers showing more playful and attachment-like behaviors and Akitas showing more exploratory and passive behaviors. The study found correlations between hormones and behaviors within breeds, particularly in Labrador Retrievers, where oxytocin concentrations were associated with attachment-like behaviors, and cortisol concentrations reflected individual differences in physical activity rather than stress responses to social situations. These findings contribute to the understanding of the evolutionary and adaptive processes underlying the ability of domestic dogs to form close relationships with humans while highlighting the role of hormonal mechanisms in mediating attachment behaviors and the influence of breed-specific genetic backgrounds on these processes.

Urinary oxytocin and cortisol concentrations vary by group type in male western lowland gorillas (Gorilla gorilla gorilla) in North American zoos.

Evaluating how primates in human care function within their social environment is important for understanding and optimizing their management and welfare. The neuroendocrine hormone oxytocin is associated with affiliation and bonding, suggesting it can be used to evaluate the affiliative nature of social groupings. When paired with cortisol concentrations, social stressors can simultaneously be assessed, providing a more complete picture of primate social environments than if measuring either hormone independently. Here, we measured both oxytocin and cortisol in urine within a large subset of male western lowland gorillas (Gorilla gorilla gorilla; n = 71) living in North American zoos. Both endocrine measures were compared between social group types, with an emphasis on comparing bachelor and mixed-sex groupings to understand how these broad management practices affect male gorillas in zoos. Oxytocin concentrations were greater in bachelor group males than mixed-sex group males and singly housed males, providing physiological evidence that males in bachelor groups form comparatively stronger affiliative relationships than males in other group types. Cortisol concentrations did not differ between bachelor and mixed-sex group males and males in both group types had lower cortisol concentrations than singly housed males. These results indicate that males are similarly capable of coping with group-specific social stressors, and single management may expose males to additional stressors for which further study is needed. These data contribute to a larger body of research highlighting the value of bachelor groups from both a population management and individual welfare perspective.

What are oxytocin assays measuring? Epitope mapping, metabolites, and comparisons of wildtype & knockout mouse urine.

Oxytocin has become a popular analyte in behavioral endocrinology in recent years, due in part to its roles in social behavior, stress physiology, and cognition. Urine samples have the advantage of being non-invasive and minimally disruptive to collect, allowing for oxytocin measurements even in some wild populations. However, methods for urinary oxytocin immunoassay have not been sufficiently optimized and rigorously assessed for their potential limitations. Using samples from oxytocin knockout (KO) and wildtype (WT) mice, we find evidence of considerable interference in unextracted urine samples, with similar distributions of measured oxytocin in both genotypes. Importantly, although this interference can be reduced by a reversed-phase solid-phase extraction (SPE), this common approach is not sufficient for eliminating false-positive signal on three immunoassay kits. To better understand the source of the observed interference, we conducted epitope mapping of the Arbor Assays antibody and assessed its cross-reactivity with known, biologically active fragments of oxytocin. We found considerable cross-reactivity (0.5-52% by-molarity) for three fragments of oxytocin that share the core epitope, with more cross-reactivity for longer fragments. Given the presence of some cross-reactivity for even the tripeptide MIF-1, it is likely that many small protein metabolites might be sufficiently similar to the epitope that at high concentrations they interfere with immunoassays. We present a new mixed-mode cation-exchange SPE method that minimizes interference-with knockout samples measuring below the assay's limit of detection-while effectively retaining oxytocin from the urine of wildtype mice. This method demonstrates good parallelism and spike recovery across multiple species (mice, dogs, sifakas, humans). Our results suggest that immunoassays of urine samples may be particularly susceptible to interference, even when using common extraction protocols, but that this interference can be successfully managed using a novel mixed-mode cation exchange extraction. These findings imply that previous conclusions based on urinary oxytocin measurements-especially those involving unextracted samples-may need to be reassessed.

Analytical and physiological validation of an enzyme immunoassay to measure oxytocin in dog, wolf, and human urine samples.

Oxytocin (OT) promotes pro-sociality, bonding, and cooperation in a variety of species. Measuring oxytocin metabolite (OTM) concentrations in urine or saliva provides intriguing opportunities to study human and animal behaviour with minimal disturbance. However, a thorough validation of analytical methods and an assessment of the physiological significance of these measures are essential. We conducted an analytical validation of a commercial Enzyme Immunoassay (EIA; Arbor OT assay kit) to measure OTM concentrations in dog, wolf, and human urine samples. To test the assay's ability to detect changes in OTM concentrations, we administered oxytocin intranasally to 14 dogs. Assay performance with regard to parallelism was acceptable. Assay accuracy and extraction efficiency for dog and wolf samples were comparable to a previously validated assay (Enzo OT assay kit) but variation was smaller for human samples. Binding sensitivity and antibody specificity were better in the Arbor assay. Average OTM concentrations were more than twice as high as in comparable samples measured with the Enzo assay, highlighting a lack of comparability of absolute values between different assays. Changes in OTM concentrations after intranasal treatment were detected reliably. The Arbor assay met requirements of a "fit-for-purpose" validation with improvement of several parameters compared to the Enzo assay.

Endocrine changes related to dog domestication: Comparing urinary cortisol and oxytocin in hand-raised, pack-living dogs and wolves.

Dogs are exceptionally well adapted to life close to humans, and alterations in their endocrine system during the domestication process may be an underlying mechanism. In particular, it has been suggested that low circulating cortisol concentrations in conjunction with simultaneously high oxytocin concentrations may have resulted in dogs' increased docility ('selection for tameness' hypothesis) and heightened propensity to interact and form relationships with humans ('hypersociability' hypothesis) compared to wolves. To investigate this, we analyzed cortisol and oxytocin metabolite concentrations from urine samples of hand-raised, pack-living domestic dogs and their non-domestic relatives, grey wolves. Based on the hypotheses outlined above, we predicted lower cortisol but higher oxytocin concentrations in dogs than wolves. In contrast to our prediction, we found higher cortisol concentrations in dogs than wolves. However, oxytocin concentrations were higher in dogs compared to wolves although the effect was relatively small. Indeed, male dogs had the highest oxytocin concentrations while female dogs' oxytocin concentrations were comparable to wolves'. Feeding status, reproductive phase, and conspecific social interactions also significantly affected cortisol and oxytocin concentrations. Furthermore, we compared two methods of correcting for variable water content of urine samples. We discuss our results in light of physiological and behavioral changes during domestication and highlight the importance of accounting for confounding variables in future studies.

Parental divorce in childhood is related to lower urinary oxytocin concentrations in adulthood.

Oxytocin has been shown to be important for social behavior and emotional attachments in early life and may also mediate effects of early experiences on social motivation in adulthood. In animal models, early maternal separation results in alterations in the oxytocin system, with effects on sexual, maternal, and stress reactivity behaviors in adulthood. Studies of children experiencing parental divorce find effects on mood disorders, substance abuse, and other behaviors in adulthood. Here, we examine the effect of divorce on adult urine oxytocin levels. To stimulate oxytocin release, participants, aged 18 to 62, were asked to complete a set of questionnaires on attachment style, parental history of divorce (age at parental divorce ranged from 0 to 20), and other measures. A sample of urine was then collected for the oxytocin assay. Urine oxytocin concentrations were substantially lower (p = .016) in subjects who experienced parental divorce (M = 3.70, Standard Error of the Mean = 0.73), compared to those who did not (M = 8.00, Standard Error of the Mean = 1.21), and correlated with responses on several attachment instruments. These results suggest that oxytocin levels are adversely affected by parental divorce in humans and may be related to attachment measures in adulthood. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

The Cascade System of Osmotic Homeostasis Regulation.

The human and animal osmoregulation system is aimed at stabilizing serum osmolality in order to maintain cell volume. It has been shown that the introduction of 5 mL water per 100 g of body weight into the stomach of rats decreases serum osmolality and the concentration of Na and Ca, but not K and Mg. The cascade system of osmotic homeostasis increases secretion of glucagon-like peptide-1 (GLP-1) and oxytocin, and decreases secretion of vasopressin, which reduces the osmotic permeability of collecting duct. After water loading and the injection of 0.015 nM exenatide (GLP-1 mimetic), the time of excretion of 50% of water was halved from 112 ± 4 to 57 ± 5 min (p < 0.01), and after the injection of 0.015 nM oxytocin, it decreased to 83 ± 6 min (p < 0.01). The physiological mechanism of renal effect of the aforementioned hormones which accelerate the recovery of osmotic homeostasis has been found.

Consistency and efficacy of two methods of intranasal oxytocin application in dogs.

Over the last few years, oxytocin (OT) administration to investigate the role of the oxytocinergic system in the social behavior of dogs has become of more and more interest. To date, the most common OT administration method for dogs is the intranasal spray commonly used for humans. Due to the different nasal conformation of dogs and the unpleasantness of the procedure, most dogs need to be restrained to allow administration. This has 2 main drawbacks-it may hinder reliable administration, which might lead to tremendous variance in the uptake of OT across individuals and it is likely to be stressful for the dogs. Alternatively, a vaporizer mask can be used to administer aerolized OT and dogs can be trained to voluntarily enter the mask, which might enable a more reliable administration without having to restrain the dogs. The aim of this study was to compare the efficacy of these 2 methods to identify a reliable non-invasive method for exogenous OT administration, thereby assisting future research on the role of OT in canines. We administered OT to pet dogs using either an intranasal spray bottle or a vaporizer mask and assessed urinary OT concentrations as a measure of OT uptake. We found that only when administered using a vaporizer mask, OT significantly increased in all subjects, while using a spray bottle led to considerable variance in OT uptake and an inconsistent increase in urinary OT concentrations across individuals. These results suggest that using a vaporizer mask should be preferred over using an intranasal spray bottle for OT administration in dogs. If not available, experimenters should at least monitor OT uptake after administration using spray bottles, to evaluate the success of the method.

A neurobiological association of revenge propensity during intergroup conflict.

Revenge during intergroup conflict is a human universal, but its neurobiological underpinnings remain unclear. We address this by integrating functional MRI and measurements of endogenous oxytocin in participants who view an ingroup and an outgroup member's suffering that is caused mutually (Revenge group) or by a computer (Control group). We show that intergroup conflict encountered by the Revenge group is associated with an increased level of oxytocin in saliva compared to that in the Control group. Furthermore, the medial prefrontal activity in response to ingroup pain in the Revenge group but not in the Control group mediates the association between endogenous oxytocin and the propensity to give painful electric shocks to outgroup members, regardless of whether they were directly involved in the conflict. Our findings highlight an important neurobiological correlate of revenge propensity, which may be implicated in conflict contagion across individuals in the context of intergroup conflict.

Oxytocin increases after affiliative interactions in male Barbary macaques.

Mammals living in stable social groups often mitigate the costs of group living through the formation of social bonds and cooperative relationships. The neuropeptide hormone oxytocin (OT) is proposed to promote both bonding and cooperation although only a limited number of studies have investigated this under natural conditions. Our aim was to assess the role of OT in bonding and cooperation in male Barbary macaques (Macaca sylvanus). First, we tested for an effect of affiliation - grooming and triadic male-infant-male interactions - with bond and non-bond partners on urinary OT levels. Second, we tested whether grooming interactions (and thus increased OT levels) increase a male's general propensity to cooperate in polyadic conflicts. We collected >4000 h of behavioral data on 14 adult males and measured OT levels from 139 urine samples collected after affiliation and non-social control periods. Urinary OT levels were higher after grooming with any partner. By contrast, OT levels after male-infant-male interactions with any partner or with bond partners were not different from controls but were higher after interactions with non-bond partners. Previous grooming did not increase the likelihood of males to support others in conflicts. Collectively, our results support research indicating that OT is involved in the regulation of adult affiliative relationships. However, our male-infant-male interaction results contradict previous studies suggesting that it is affiliation with bond rather than non-bond partners that trigger the release of OT. Alternatively, OT levels were elevated prior to male-infant-male interactions thus facilitating interaction between non-bond partners. The lack of an association of grooming and subsequent support speaks against an OT linked increase in the general propensity to cooperate.

The cooperative sex: Sexual interactions among female bonobos are linked to increases in oxytocin, proximity and coalitions.

In some species habitual same-sex sexual behavior co-occurs with high levels of intra-sexual alliance formation, suggesting that these behaviors may be linked. We tested for such a link by comparing behavioral and physiological outcomes of sex with unrelated same- and opposite-sex partners in female bonobos (Pan paniscus). We analyzed behavioral outcomes following 971 sexual events involving n = 19 female and n = 8 male adult and sub-adult members of a wild, habituated bonobo community. We additionally collected n = 143 urine samples before and after sexual interactions to non-invasively measure oxytocin (OT), which modulates female sexual behavior and facilitates cooperation in other species. The majority of sexual events (65%) consisted of female same-sex genito-genital rubbing (or GG-rubbing). Female dyads engaged in significantly more sexual interactions than did inter-sexual dyads, and females were more likely to remain within close proximity to their partners following GG-rubbing. Females also exhibited greater increases in urinary OT following GG-rubbing compared with copulations, indicating a physiological basis for increased motivation to cooperate among females. The frequency of coalitionary support among non-kin was positively predicted by the frequency of sexual interactions for female as well opposite-sex dyads, although coalitionary support tended to be more frequent among females. The emergence of habitual same-sex sexual behavior may have been an important step in the evolution of cooperation outside of kinship and pair-bonds in one of our closest phylogenetic relatives.

Demographic, sampling- and assay-related confounders of endogenous oxytocin concentrations: A systematic review and meta-analysis.

Studies on endogenous oxytocin concentrations are often criticized for the debatable comparability between specimens and the variation in reported values. We performed meta-regressions on k = 229 studies (n = 12 741 participants), testing whether specimen, extraction, sex, age, time of day, or fasting instructions influenced oxytocin measurements. Predicted oxytocin concentrations differed depending on specimen and extraction: Measurements were extremely high in unextracted blood, compared to extracted blood and other specimens. Measurements were higher in samples with more female participants and higher age. Instructions not to smoke before sampling were correlated with higher oxytocin in unextracted samples. There was no impact of instructions to refrain from eating, drinking, consume caffeine, alcohol or exercising. Oxytocin concentrations increased from morning to afternoon. Our results showed that oxytocin is differentially reflected in blood, saliva, urine and cerebrospinal fluid. Extraction impacts oxytocin measurements, particularly in blood. Considering relevant confounders might increase comparability between studies.

Analytical validation of an Enzyme Immunoassay for the measurement of urinary oxytocin in dogs and wolves.

Assessing changes in oxytocin (OT) levels in response to a variety of social stimuli has become of major interest in the field of behavioral endocrinology. OT is involved in the regulation of various aspects of social behavior such as tolerance, and the formation and maintenance of social bonds but also the regulation of stress. All of these aspects have been identified as potential targets of selection during the domestication process. Therefore, comparing the role of the oxytocinergic system in various aspects of dog and wolf social behavior, might help to understand whether this system was involved in the domestication process. Studies assessing OT levels in dogs and wolves have used invasively collected plasma and serum samples and non-invasively collected urine samples. However, when using an assay system on a new species a careful and complete validation of the method is of crucial importance, and to date no proper validation, to assess urinary OT levels in dogs and wolves, has been reported. We therefore conducted an analytical validation of an Enzyme Immunoassay (EIA) for the measurement of OT in urine of dogs and wolves, using a commercially available EIA. Stability tests revealed that OT levels degrade over time when stored at 4 °C, but are little affected by repeated thawing. In addition, our results indicate that the variance in OT levels is slightly lower when phosphoric acid is added following collection to prevent OT degradation. Long term storage tests revealed that urinary OT levels are least variable when stored as extracts in ethanol at -20 °C, rather than as unextracted urine samples. Validation results were acceptable with regard to parallelism, but values for accuracy and extraction efficiency were not meeting the standard criteria usually applied to steroid EIAs, especially when assessed for the lower range of the assay. The results of this study highlight the importance of an analytical assay validation, since even if validation parameters are not optimal, if published, they allow readers to estimate the relevance of studies using the validated method.

Cortisol and oxytocin show independent activity during chimpanzee intergroup conflict.

The oxytocinergic system is involved in a range of functions, from attachment and social bonding to aggression and stress responses. Whether oxytocin is released in response to a stressor, shows contradictory results across species and potential contexts-dependent differences. To avoid unintended contextual changes due to experimental procedures, we tested this question non-invasively in wild chimpanzees in an ecologically valid context. We collected endogenous hormonal measures during exposure to a known natural stressor, intergroup conflict. Specifically, we tested for potential synchronous activation patterns between urinary oxytocin and cortisol in male and female chimpanzees during stressor exposure. Oxytocinergic system reactivity during chimpanzee intergroup conflict has already been established in this study population. Thus, we first investigated urinary cortisol levels during border patrol and intergroup encounter days, in comparison to another potential stressor, hunting, and control days. We found higher urinary cortisol levels during intergroup encounter days compared with control and hunting days. We then compared secretion patterns of oxytocin and cortisol in relation to increased levels of out-group contact and hostility ('out-group risk') during intergroup conflict. We found that increased 'out-group risk' was associated with higher cortisol levels, especially when involving direct visual or physical contact with rival groups. Although urinary oxytocin levels were high across intergroup conflict contexts, increasing levels of out-group risk showed no significant variation. Taken together, results indicate independent secretion of oxytocin and cortisol during chimpanzee intergroup conflict, emphasizing that stressor exposure in this context is not the main trigger of oxytocin secretion.

Oxytocin, vasopressin and trust: Associations with aggressive behavior in healthy young males.

Oxytocin enhances trust during social interactions and reduces the tendency for social betrayal. Animal studies have revealed that oxytocin is also an important factor in the establishment and regulation of aggression, for which social interaction is a critical precondition. In humans, however, the effects of oxytocin appear more nuanced and influenced by social context and personality traits. Moreover, the pro-social effects of oxytocin are not mirrored by vasopressin, despite their high chemical similarity. Rather, vasopressin has been associated with an increase of aggressive responses. Therefore, we sought to investigate the association of oxytocin and vasopressin with trust and aggressive behavior. Overnight urinary oxytocin and vasopressin levels were obtained from 62 healthy males (age range: 18-26 years) to compare with trait measures of trust and aggressive behavior. We found a significant interaction of oxytocin and trust on aggression in which low trait measures of trust, in combination with low levels of oxytocin, were associated with a history of aggressive behavior. Notably, we found no significant associations for vasopressin. Although both oxytocin and vasopressin have been shown to be important in the emergence of violent behavior, our study suggests that oxytocin may be particularly modified by affiliative behaviors. These findings provide insights into the neuropsychological influences of oxytocin, and highlights the potential for clinical translation regarding the treatment of patients who exhibit recurrent aggressive behavior.

Oxytocin analysis from human serum, urine, and saliva by orbitrap liquid chromatography-mass spectrometry.

Oxytocin (OT) is a neurohormone that has gained interest recently due to its emerging role in cognition and social/emotional behaviors, including possibly depression and autism. OT is commonly measured using enzyme- or radio-based immunoassays (RIA, ELISA), which lack specificity or are complicated to perform and involve hazardous radioactive material. We have developed a high resolution accurate-mass (HRAM) liquid chromatography-mass spectrometry (LC-MS) method that separates interferences and selectively and accurately quantitates native OT from human serum, urine, and saliva after solid phase extraction. The doubly protonated OT ion m/z 562.25503 was selected for quantitation due its high signal intensity. With our method lower limit of detection (LLOD) of 5-25 pg/mL, we measured native OT in serum from pregnant women (16-24 pg/mL) and rats (350 pg/mL), and in serum, urine, and saliva from a healthy male after intranasal (IN) OT application of 100 IU and 20 IU and from a healthy post-menopausal female after IN OT application of 100 IU. Peak levels were detected in serum, urine, and saliva 15-30 minutes after each dose then decreased to below detection limits 1-2 hours thereafter. We were unable to detect native OT in serum from non-pregnant/non-lactating/non-medicated women due to levels known to occur below 5 pg/mL. The fast elimination of OT we found is in excellent agreement with the pharmacokinetics of OT in other studies. The effects on the central nervous system occurring after IN OT administration remains to be determined.

Exploring associations between perinatal depression, anxiety, and urinary oxytocin levels in Latinas.

Perinatal depression has been associated with lower oxytocin (OT) levels. However, few studies have explored this topic in relation to Latinas who are at high risk of perinatal depression. The objective of this study was to explore these associations in Latinas. A total of 108 Latinas in the third trimester of pregnancy participated in the study. Depression and urinary OT levels were assessed in pregnancy and 6 weeks postpartum. Nonparametric tests were implemented to test the proposed associations. Results revealed that 28% of the participants had probable depression in pregnancy, and 23% at 6 weeks postpartum. OT levels significantly decreased from prenatal to postpartum in the whole sample; however, participants with probable prenatal depression did not exhibit a significant change in OT levels. Participants who were depressed or anxious at 6 weeks postpartum exhibited persistently higher mean OT levels over time. A distinct pattern of higher levels of OT in depressed Latinas suggests that OT levels may be an important neuroendocrine factor contributing to depressive and anxious symptoms.

Social bonds facilitate cooperative resource sharing in wild chimpanzees.

Why share when access to benefits is uncertain is crucial to our understanding of the evolution of humans' extensive cooperation. Here, we investigated some of the different human sharing hypotheses and potential neuroendocrine mechanisms, in one of our closest living relatives, chimpanzees. The strongest predictor of sharing across food types was the presence of enduring and mutually preferred grooming partners, more than harassment, direct signalling, or trade. Moreover, urinary oxytocin levels were higher after the sharing of both individually and jointly acquired resources compared with controls. We conclude that the emotional connection inherent in social bonds was a key factor determining sharing patterns, with the oxytocinergic system potentially facilitating long-term cooperative exchanges. Testing for the role of social bonds in increasing predictability of sharing behaviour, a feature frequently overlooked, may help us to identify the evolutionary drivers of resource sharing and mechanisms that sustain delayed reciprocity between non-kin.

Urinary oxytocin levels in relation to post-conflict affiliations in wild male chimpanzees (Pan troglodytes verus).

Many animals living in social groups have evolved behaviors to resolve conflicts between group members, behaviors thought crucial for maintaining stable group life. Several hypotheses, based mainly on observational data, aim to explain how post-conflict (PC) affiliations, such as reconciliation and consolation, resolve conflicts by restoring relationships and/or alleviating anxiety. To examine a potential endocrinological mechanism of PC affiliations, we used an experimental-like procedure to investigate whether the oxytocinergic system is activated during naturally observed reconciliations, receiving bystander PC affiliations and aggressions not followed by PC affiliations in wild male chimpanzees. We compared urinary oxytocin (uOT) levels after reconciliations, receiving bystander PC affiliations or aggressions without affiliations with two control conditions: affiliations without previous aggression and after time periods without social interactions. We furthermore tested the 'valuable relationship' hypothesis of reconciliation, as well as the influence of relationship quality between individuals engaged in each of the three behavioral conditions involving aggression on uOT levels. We found that the probability to reconcile a conflict increased with increasing relationship quality between opponents, thus our results support the 'valuable relationship' hypothesis. However, relationship quality did not influence uOT levels, while behavioral condition had a significant effect on uOT levels. uOT levels after reconciliations, receiving bystander PC affiliations and affiliations not related to conflicts were higher than after aggressions alone and time periods without social interactions. Overall, our results indicate that the oxytocinergic system is activated during affiliative interactions, whether occurring as reconciliation, bystander PC affiliation or affiliation alone. We conclude that the oxytocinergic system, in addition to building and maintaining social relationships, also takes part in repairing them.

Validating the use of a commercial enzyme immunoassay to measure oxytocin in unextracted urine and saliva of the western lowland gorilla (Gorilla gorilla gorilla).

The neuroendocrine hormone oxytocin, which is an important physiological driver of social behavior and bonding, is increasingly being measured in conjunction with behavior to better understand primate sociality. To date no data are available on oxytocin concentrations within the genus Gorilla; however, as a result of their close genetic relatedness to humans, and tolerance-based social system, Gorilla represents an important group of study. The purpose of this study was to validate the measurement of urinary and salivary oxytocin in western lowland gorillas (Gorilla gorilla gorilla) to help facilitate future study of the interaction between oxytocin and behavior within the subspecies. The primary validation procedure was an intranasal challenge. Elevated oxytocin concentrations were observed in saliva samples taken 15-120 min post challenge. Urine levels remained within baseline range approximately 30 and 90 min following the challenge; however, elevated levels were observed 24 h post challenge. No diurnal variation was found in salivary samples taken at regular intervals throughout the day; however, morning urine samples had higher concentrations than afternoon samples. In addition, samples were collected opportunistically following three social events: play, breeding, and the death of a conspecific. Following the play bouts, salivary oxytocin was almost three times greater than baseline. Salivary oxytocin was also significantly higher 15 min post breeding compared to match-control samples. Following the death of a conspecific, the group mate's urinary oxytocin concentrations decreased by half compared to a baseline period when the group was intact. This study provides a biological validation of the measurement of urinary and salivary oxytocin in western lowland gorillas. These results suggest that urinary oxytocin measurements are suitable for establishing baseline levels, as they represent the build up of the previous day's concentrations, and salivary oxytocin measurements are suitable for assessing changes following specific events.