Measurement of salivary cortisol in two New World primate species

Funding: R.S. was supported by the Austrian Science Fund (FWF, Young Independent Researcher Group (YIRG) grant; Grant Number ZK 66) and ERC Grant 230604 SOMACCA (to W. Tecumseh Fitch).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.Publisher PDFPeer reviewe

Shallow Torpor Expression in Free-Ranging Common Hamsters With and Without Food Supplements

Energy expenditure during winter can be reduced by expressing torpor, which is characterized by decreased metabolic rate and body temperature. In addition to deep, multiday torpor bouts alternating with short arousal episodes during the hibernation period, common hamsters can also enter shallow torpor bouts (STBs), lasting for < 24 h at a minimum body temperature between 30 and 20°C. Food supplements provided shortly before winter have been shown to shorten hibernation in males, but did not affect hibernation duration in females. In the presented study, we analyzed the expression of STBs and compared supplemented to unsupplemented common hamsters. Body temperature during winter was recorded using subcutaneously implanted data loggers (iButtons). The results revealed that supplemented males showed more STBs and thus spent more time in shallow torpor than unsupplemented individuals. The duration of STBs, however, was shorter in supplemented males and both mean and minimum body temperature were significantly higher compared to unsupplemented males. In females, shallow torpor expression did not differ between individuals with and without food supplements. STBs were mainly expressed before the onset of the first deep, multiday torpor bout, but the number of STBs was not related to that of deep torpor bouts. These results indicate that in males, shallow torpor combined with feeding on food stores could be the more appropriate overwintering strategy when sufficient external energy reserves are available. Females generally cache more food than males and are therefore assumed to be less affected by the additional food stores. These results underline the flexibility of the species in the use of heterothermy and could enable adequate and rapid responses to changes in food availability

Stress behaviour and physiology of developing Arctic barnacle goslings (<i>Branta leucopsis</i>) is affected by legacy trace contaminants

Natural populations are persistently exposed to environmental pollution, which may adversely impact animal physiology and behaviour and even compromise survival. Responding appropriately to any stressor ultimately might tip the scales for survival, as mistimed behaviour and inadequate physiological responses may be detrimental. Yet effects of legacy contamination on immediate physiological and behavioural stress coping abilities during acute stress are virtually unknown. Here, we assessed these effects in barnacle goslings (Branta leucopsis) at a historical coal mine site in the Arctic. For three weeks we led human-imprinted goslings, collected from nests in unpolluted areas, to feed in an abandoned coal mining area, where they were exposed to trace metals. As control we led their siblings to feed on clean grounds. After submitting both groups to three well-established stress tests (group isolation, individual isolation, on-back restraint), control goslings behaved calmer and excreted lower levels of corticosterone metabolites. Thus, legacy contamination may decisively change stress physiology and behaviour in long-lived vertebrates exposed at a young age.</p

Mitochondrial polymorphism m.3017C>T of SHLP6 relates to heterothermy

Heterothermic thermoregulation requires intricate regulation of metabolic rate and activation of pro-survival factors. Eliciting these responses and coordinating the necessary energy shifts likely involves retrograde signalling by mitochondrial-derived peptides (MDPs). Members of the group were suggested before to play a role in heterothermic physiology, a key component of hibernation and daily torpor. Here we studied the mitochondrial single-nucleotide polymorphism (SNP) m.3017C&gt;T that resides in the evolutionarily conserved gene MT-SHLP6. The substitution occurring in several mammalian orders causes truncation of SHLP6 peptide size from twenty to nine amino acids. Public mass spectrometric (MS) data of human SHLP6 indicated a canonical size of 20 amino acids, but not the use of alternative translation initiation codons that would expand the peptide. The shorter isoform of SHLP6 was found in heterothermic rodents at higher frequency compared to homeothermic rodents (p &lt; 0.001). In heterothermic mammals it was associated with lower minimal body temperature (Tb, p &lt; 0.001). In the thirteen-lined ground squirrel, brown adipose tissue—a key organ required for hibernation, showed dynamic changes of the steady-state transcript level of mt-Shlp6. The level was significantly higher before hibernation and during interbout arousal and lower during torpor and after hibernation. Our finding argues to further explore the mode of action of SHLP6 size isoforms with respect to mammalian thermoregulation and possibly mitochondrial retrograde signalling

Non-invasive cortisol measurements as indicators of physiological stress responses in guinea pigs

Non-invasive measurements of glucocorticoid (GC) concentrations, including cortisol and corticosterone, serve as reliable indicators of adrenocortical activities and physiological stress loads in a variety of species. As an alternative to invasive analyses based on plasma, GC concentrations in saliva still represent single-point-of-time measurements, suitable for studying short-term or acute stress responses, whereas fecal GC metabolites (FGMs) reflect overall stress loads and stress responses after a species-specific time frame in the long-term. In our study species, the domestic guinea pig, GC measurements are commonly used to indicate stress responses to different environmental conditions, but the biological relevance of non-invasive measurements is widely unknown. We therefore established an experimental protocol based on the animals’ natural stress responses to different environmental conditions and compared GC levels in plasma, saliva, and fecal samples during non-stressful social isolations and stressful two-hour social confrontations with unfamiliar individuals. Plasma and saliva cortisol concentrations were significantly increased directly after the social confrontations, and plasma and saliva cortisol levels were strongly correlated. This demonstrates a high biological relevance of GC measurements in saliva. FGM levels measured 20 h afterwards, representing the reported mean gut passage time based on physiological validations, revealed that the overall stress load was not affected by the confrontations, but also no relations to plasma cortisol levels were detected. We therefore measured FGMs in two-hour intervals for 24 h after another social confrontation and detected significantly increased levels after four to twelve hours, reaching peak concentrations already after six hours. Our findings confirm that non-invasive GC measurements in guinea pigs are highly biologically relevant in indicating physiological stress responses compared to circulating levels in plasma in the short- and long-term. Our approach also underlines the importance of detailed investigations on how to use and interpret non-invasive measurements, including the determination of appropriate time points for sample collections

Sex-Specific Effects of Diets High in Unsaturated Fatty Acids on Spatial Learning and Memory in Guinea Pigs.

Unsaturated fatty acids (UFAs), including omega-3, omega-6 polyunsaturated and omega-9 monounsaturated fatty acids, are essential components and modulators of neuromembranes and may affect various aspects of physiology and cognition. UFAs are suggested to positively affect spatial learning and memory and also to diminish the negative consequences of physiological stress on cognitive abilities. Due to pronounced sex differences in neurophysiological functions, we hypothesize that these UFA-related effects might differ between male and female individuals. We therefore determined the effects of dietary UFAs on cognitive performances in a radial-Y-maze in male and female guinea pigs in relation to saliva cortisol concentrations, a marker for physiological stress. Animals were assigned to four treatment groups and maintained on diets enriched in either chia seeds (omega-3), walnuts (omega-6), or peanuts (omega-9), or a control diet. Female learning abilities throughout a three-day learning phase were positively affected by omega-3 and omega-9, as determined by a decreasing latency to pass the test and the number of conducted errors, while males generally showed distinct learning abilities, irrespective of the diet. A sex difference in learning performances was found in the control group, with males outperforming females, which was not detected in the UFA-supplemented groups. This was paralleled by significantly increased saliva cortisol concentrations in males throughout the cognition test compared to females. Three days after this learning phase, UFA-supplemented males and all females showed unchanged performances, while control males showed an increased latency and therefore an impaired performance. These results were corroborated by pronounced differences in the plasma UFA-status, corresponding to the different dietary treatments. Our findings indicate sex-specific effects of dietary UFAs, apparently enhancing spatial learning abilities only in females and protecting males from long-term memory impairment, while male learning abilities seem to be more strongly affected by an acute physiological stress response to the maze task

Heart rate, heart rate variability and salivary cortisol as indicators of arousal and synchrony in clients with intellectual disability, horses and therapist during equine-assisted interventions

The aim of the study was to analyse interaction processes in equine-assisted therapy (EAT) sessions with ten female clients in the period of emerging adulthood with intelectual disability (ID). Heart rate (HR), heart rate variability and salivary cortisol levels have been analysed in humans and horses before, during and after a standardised therapy session as well as in a control condition. There was a trend of lower cortisol levels and higher variability and parasympathetic tone induced by horses. During challenge however, there was a significant lower HR in the horse condition. Significant correlations in heart rate between therapist, client and horse were found with stronger interaction with a familiar horse. Our findings suggest that EAT may effectively modulate stress in humans with ID. Our results further elucidate synchronisation patterns in HR highlighting the pivotal role of relationship quality and intensity as modulators of synchrony