The estrous cycle of female guinea pigs is characterized by a spontaneous ovulation and a subsequent active luteal phase that is physiologically well described. In contrast to other rodents, however, the effects of the cycle on stress responses and behavior are understudied in this species. This study examines the extent to which cortisol concentrations and behavior differ between the estrus and diestrus phases of two consecutive cycles in twelve female guinea pigs. In addition, the parameters were analyzed regarding repeatability to determine how far both physiological parameters and behavior are individually consistent throughout the cycle. Saliva samples to analyze cortisol concentrations were collected before and after a 15-minute dark-light test. The dark-light test and a 15-min sociality test were performed to analyze exploration, anxiety, and social interest. Cortisol concentrations were increased in estrus compared to diestrus. In the dark-light test, the cycle phase had no significant influence on locomotion or anxiety-like behavior. Locomotion and time spent with a conspecific in the sociality test were partly increased in estrus compared to diestrus. All behaviors were repeatable throughout the two investigated cycles. The results show that the estrous cycle of female guinea pigs can influence physiology and behavior to some extent, but that individual differences have a stronger influence on behavior than does the cycle. These findings contribute to the general understanding of physiological and behavioral changes during the estrous cycle, which are understudied not only in guinea pigs but in many mammalian species.

The estrous cycle of female mammals does not only cause regular changes in steroid hormone concentrations but can also influence behavior and hence (social) life. A valuable model organism to study such effects is the guinea pig: its long 16-day estrous cycle with an active luteal phase represents a perfect opportunity to investigate the estrous cycle in detail, but information on estrous cycle effects is scarce. This study investigated the proestrus/estrus and diestrus phases of two consecutive cycles in twelve socially housed female guinea pigs. Following cycle monitoring, sociopositive and aggressive behavior, general activity patterns, anxiety-like behavior, and saliva cortisol concentrations were investigated on three days around estrus (day -1, day 0 = estrus, day 1) and in diestrus (day 7, day 8, day 9) each. Aggressive behavior significantly increased on day 0 (estrus) and the guinea pigs spent less time in the shelters and more time feeding on day 9 compared to day 7 in diestrus. Cortisol concentrations were increased around the time of estrus versus diestrus and body mass was decreased on day 0. None of the behavioral parameters affected cortisol concentrations considerably, except for a weak negative effect of the time spent feeding. This study demonstrates that the estrous cycle exerts an effect not only on physiological parameters but also on the behavior of group-living female guinea pigs. This represents an important starting point for future research and highlights stronger consideration of the estrous cycle when investigating (social) behavior and physiology in female guinea pigs.

Repeatability estimation is the proportion of variance explained by between-individual variation, as opposed to within-individual variation, and allows quantifying the reliability of a measurement. With regard to behaviour, this enables finding consistency in behavioural traits, as well as looking into the importance of behavioural flexibility. It has been shown that behavioural repeatability decreases with longer inter-test intervals, and while many species are well investigated in this context, the guinea pig, representing an important laboratory model, remains understudied. In the present study, male guinea pigs were tested in three behavioural tests (light-dark test, sociality test, open-field test) four times, including a short (2-week), a long (2-month), and another short inter-test interval. The tests aimed at general activity/exploration, anxiety-like and risk-taking behaviour, and social interest. The resulting repeatability estimations were compared between time intervals to determine the degree of consistency over different time periods. Repeatability estimates were very high for risk-taking and anxiety-like behaviour as well as for general activity. Repeatability increased over time, showing lower repeatability for the first short-term and the long-term interval compared to the second short-term interval. The sociality test showed lower repeatability estimates throughout, which is discussed in regard to the experimental setup. Overall, the performed tests other than the sociality test reliably showed clear individual behavioural consistency in the short- and long-term. This is important knowledge not only for future studies on behaviour or personality in guinea pigs but also regarding the applicability of the behavioural tests for adequately assessing specific behaviours and individual differences.

Sex differences in hypothalamic-pituitary-adrenal (HPA)-axis functions and related stress responsiveness can strongly depend on influences of gonadal hormones and their fluctuations during the female estrous or menstrual cycle. Females usually show higher HPA-axis activity and glucocorticoid concentrations in the preovulatory phases of the cycle than in the other phases and compared to males. The guinea pig represents a potential model species in this context, but little and inconclusive information on sex differences and estrous cycle effects on HPA-axis activity is available in this species. This study investigated basal saliva cortisol concentrations and stress responses to a 1-h social separation stressor in an open-field-like novel environment in female (n = 16) and male (n = 16) guinea pigs aged 0.5-2.5 years. All guinea pigs were sampled and tested repeatedly: females during diestrus and estrus of two estrous cycles, and males twice at comparable intervals. Basal cortisol concentrations were significantly increased in females during estrus compared to diestrus and males. Cortisol concentrations increased strongest in response to the stress test in females during diestrus and the smallest increase was found during estrus. This resulted in significantly higher cortisol concentrations after the test in females during diestrus compared to males. The results were highly robust with regard to repeated testing and individual age. The estrous cycle of female guinea pigs clearly contributes to sex differences in cortisol concentrations and stress responses, which strongly calls for consideration of both in future investigations.

Nutrition represents an important aspect regarding neuronal and cognitive functions. Especially the essential omega-3 polyunsaturated fatty acids (PUFAs) can positively affect cognitive abilities as indicated by various findings in humans and rodent models. Some important PUFA sources have been well investigated in this context while others received less attention, which also applies to walnut oil. Moreover, the majority of studies in rodents used similar types of mazes to assess learning and memory abilities and rarely investigated long-term memory abilities. For this pilot study, rats were supplemented with walnut oil for eight weeks (PUFA group) or received a control diet (control group) before they were trained to a three-choice multiple-T-maze task to test long-term memory abilities four weeks afterwards. In the end of the training phase as well as in the long-term memory test, no differences between diet groups were detected in the time to finish the task or the number of conducted errors. However, comparing the training phase and the memory test revealed an increase in the time and errors in the control group while the PUFA group remained constant in its performance. The findings presented here indicate that supplementation of walnut oil high in PUFAs can positively affect long-term memory formation in rats in a multiple-T-maze. Future studies may similarly consider alternative and more challenging maze tasks to assess the magnitude of cognitive influences.

BACKGROUND:This exploratory study aimed to analyse physiological interaction processes in equine-assisted-therapy (EAT) between client, therapy horse and therapist. METHODS:We measured heart rate (HR), heart rate variability (HRV) and cortisol levels before, during and after a standardized therapy session and a control condition in one therapist, four therapy horses and ten female clients in emerging adulthood (Mn = 21.8 years, SD = 3.39). The clients were diagnosed with mild (N = 5) to moderate (N = 5) intellectual disability (ID). RESULTS:There was no significant change in the client's HR, HRV and cortisol levels during an EAT session. No difference was observed between therapy sessions with or without a therapy horse, except during the challenge phase of the EAT protocol, where clients had a significantly lower HR when interacting with the therapy horse. HR between therapist and client correlated significantly, as well as between therapist and horse. This effect was greater when therapists interacted with a familiar horse. Clients' and horses' HRs also correlated, but only when the horse was the clients' familiar and preferred horse. CONCLUSIONS:These results indicate that relationship intensity is an important factor for the synchronization process. Moreover, the inclusion of horses in a therapeutic setting can lead to a decreased HR in young adults with intellectual disability while mastering a challenge. Future research should investigate this potential benefit of EAT, considering the reciprocal influences and the relationship between client, therapist and horse.

Hibernation is a remarkable physiological adaptation in many mammals, characterized by prolonged torpor and profound metabolic suppression. Despite its importance, the molecular mechanisms regulating mitochondrial-derived gene expression during hibernation remain poorly understood. In this study, we analysed mitochondrial gene expression across multiple tissues of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) using publicly available RNA sequencing (RNA-seq) data. We assessed all known mitochondrial DNA-derived transcripts—including mitochondrial mRNAs, mitochondrial-derived peptides and proteins (MDPs), rRNAs, and long non-coding RNAs (lncRNAs)—in the liver, adrenal gland, three brain regions, and brown adipose tissue (BAT) across different hibernation states. Our findings reveal distinct tissue-specific expression patterns of mitochondrial transcripts. Differential expression was observed in three of the six tissues analysed (liver, adrenal gland, and BAT) while no significant changes were detected in the three brain regions. In tissues exhibiting differential expression, a consistent pattern emerged: lncRNAs such as Mdl1, Mdl1as, and lncCyb were generally upregulated, whereas mRNAs, including a novel transcript encoding the putative mitochondrial protein Rudel, were predominantly downregulated. These results provide new insights into mitochondrial gene regulation during hibernation and highlight tissue-specific adaptations at the level of mitochondrial gene expression.

Birds that migrate from temperate areas to the Arctic to breed lose their strongest Zeitgeber of circadian organization when they cross the Arctic circle in spring – the 24h light-dark cycle. Under continuous daylight, diverse behavioral and physiological patterns have been detected in both free-ranging and laboratory animals. To better understand the evolution of plasticity in circadian clocks, it is essential to study behavioral and physiological rhythmicity in the context of a species' ecology. Employing a multifaceted approach, which included wildlife cameras, accelerometers, and non-invasive sampling of hormone metabolites, we investigated activity patterns and corticosterone rhythmicity in a migratory herbivore, the barnacle goose (Branta leucopsis), during its Arctic breeding season on Svalbard. We found that females showed a combination of both ultradian and diel rhythmicity in nest recesses and sleep during incubation. In both parents, these rhythms in activity continued also during the gosling rearing phase. During molt, many geese aligned activity with the prevailing tidal rhythm. Barnacle geese showed weak diel rhythmicity in excreted corticosterone metabolites (CORTm). This suggests that while Arctic geese may adopt an alternative Zeitgeber during the Arctic summer to maintain a diel rhythm, ultradian rhythmicity remains essential, allowing the geese to flexibly adjust their rhythms to environmental conditions.

Research on equine-assisted therapy (EAT) has primarily been centered on human health. Relatively few studies have addressed the impact of EAT on horses. This study sought to monitor four experienced therapy horses' cardiovascular and glucocorticoid activity over the course of standardized EAT sessions designed to support women with intellectual disability. In the control condition, horses completed the EAT protocol solely with the therapist, thereby resembling a training session. Descriptive data analysis revealed higher levels of heart rate during an experimental EAT session and increased salivary cortisol when horses were navigated by the client through an obstacle course during the "challenge" phase of the protocol, pointing at a greater physical demand due to the recipient on horseback. Given the parasympathetic activity and overall heart rate variability across experimental EAT sessions and the cortisol recovery after the sessions, the findings do not give rise to any acute animal welfare concerns. For a more holistic interpretation of the present research results, further investigation into the horse perception of EAT, based on a bigger sample size and additional markers of welfare, is needed.

An individual’s energetic demands and hence metabolic rate can strongly change during adolescence, a phase characterized by profound morphological, physiological, and endocrine changes. Glucocorticoid hormones (e.g. cortisol) are released in response to hypothalamic–pituitary–adrenal–axis activity, modulate several metabolic processes, and can also be linked to increased metabolic rate. In domestic guinea pigs (Cavia aperea f. porcellus) housed in same-sex groups, cortisol concentrations increase during adolescence in males but remain stable in females, which was suggested to be related to different energetic demands by age. We therefore measured metabolic rate through oxygen (O2) consumption over 2.5 h in male and female guinea pigs housed in same-sex groups during adolescence at ages of 60, 120, and 180 days, which was paralleled by analyses of saliva cortisol concentrations before and after the measurement. The statistical analyses involved whole body metabolic rate (ml O2/h), body mass-corrected metabolic rate (ml O2/h/kg), and body mass-independent metabolic rate (ml O2/h statistically corrected for body mass). We found increasing cortisol concentrations with age in males only, but none of the three metabolic rate analyses revealed a sex difference by age. On the individual level, repeatability across ages was found in metabolic rate as well as in body mass and cortisol concentrations after the measurement, but not in “basal” cortisol concentrations. Our results suggest no sex-specific changes in metabolic rate and hence equal energetic demands in male and female guinea pigs during adolescence. Moreover, metabolic rate clearly represents a highly stable physiological trait already early in a guinea pig’s life irrespective of rather fluctuating cortisol concentrations.

Birds that migrate from temperate areas to the Arctic for breeding lose their strongest Zeitgeber of circadian organization when they cross the Arctic circle in spring — the 24h geophysical light-dark cycle. Under continuous daylight various behavioural and physiological patterns have been detected in both free-ranging and laboratory animals. To better understand the evolution of plasticity in circadian clocks, it is essential to study behavioural and physiological rhythmicity in the context of a species' ecology. Employing a multifaceted approach, which included wildlife cameras, accelerometers, and non-invasive sampling of hormone metabolites, we investigated activity patterns and corticosterone rhythmicity in a migratory herbivore, the Svalbard barnacle goose ( Branta leucopsis ), during its Arctic breeding season. We detected that females showed a combination of both ultradian and diel rhythmicity in incubation recesses and sleep, respectively. In both parents, rhythms in activity continued over the period, when goslings were present. During moult, many geese aligned activity with the tidal rhythm. Barnacle geese showed weak diel rhythmicity in excreted corticosterone metabolites. This suggests that while Arctic geese may adopt an alternative Zeitgeber during the Arctic summer to keep a diel rhythm, ultradian rhythmicity is crucial and geese are able to tune their rhythms flexibly to environmental conditions. ### Competing Interest Statement The authors have declared no competing interest.

Enriching mitochondrial DNA (mtDNA) for sequencing entire mitochondrial genomes (mitogenomes) can be achieved by single long-range PCR. This avoids interference from the omnipresent nuclear mtDNA sequences (NUMTs). The approach is currently restricted to the use of samples collected from humans and ray-finned fishes. Here, we extended the use of single long-range PCR by introducing back-to-back oligonucleotides that target a sequence of extraordinary homology across vertebrates. The assay was applied to five hibernating rodents, namely alpine marmot, Arctic and European ground squirrels, and common and garden dormice, four of which have not been fully sequenced before. Analysis of the novel mitogenomes focussed on the prediction of mitochondrial-derived peptides (MDPs) providing another level of information encoded by mtDNA. The comparison of MOTS-c, SHLP4 and SHLP6 sequences across vertebrate species identified segments of high homology that argue for future experimentation. In addition, we evaluated four candidate polymorphisms replacing an amino acid in mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) system that were reported in relation to cold-adaptation. No obvious pattern was found for the diverse sets of mammalian species that either apply daily or multiday torpor or otherwise cope with cold. In summary, our single long-range PCR assay applying a pair of back-to-back primers that target a consensus sequence motif of Vertebrata has potential to amplify (intact) mitochondrial rings present in templates from a taxonomically diverse range of vertebrates. It could be promising for studying novel mitogenomes, mitotypes of a population and mitochondrial heteroplasmy in a sensitive, straightforward and flexible manner.

Reproduction in female mammals is characterized by major changes in steroid hormone concentrations, which can be linked to fluctuations in energy expenditure (EE). Estradiol and cortisol can increase EE and metabolic rates (MRs), but knowledge on MR changes during the estrous cycle and gestation is scarce for many species. This also applies to the domestic guinea pig, a species exhibiting an exceptional estrous cycle among rodents. In this study, MRs were measured through oxygen (O2) consumption in female guinea pigs during different reproductive stages. Mean O2 consumption over 2.5 h, resting metabolic rate (RMR, lowest and most stable O2 consumption over 3 min), body mass, fecal estrogen and progesterone, and saliva cortisol concentrations were measured in twelve female guinea pigs in a repeated measurements design during diestrus, estrus, and the second trimester of gestation. In estrus, body mass was significantly lower and estrogen and cortisol concentrations were significantly higher compared to diestrus and gestation. Mean O2 consumption and RMR both were significantly increased in estrus compared to diestrus. Additionally, a positive effect of body mass on MRs detected during diestrus and gestation was not found during estrus. Mean O2 consumption was also higher during gestation compared to diestrus, and a significant increase in cortisol concentrations during the 2.5-h MR measurement was recorded. The results indicate that estrus in guinea pigs is energetically demanding, which probably reflects catabolic effects of estrogens and cortisol that uncoupled MRs from body mass. Knowledge on the energetic requirements associated with different reproductive stages is important for future physiological and behavioral studies on female guinea pigs.

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>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 < 0.001). In heterothermic mammals it was associated with lower minimal body temperature (Tb, p < 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.

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.

Hibernators save energy during winter by expressing torpor bouts characterized by strongly reduced body temperature and metabolic rate. Polyunsaturated fatty acids (PUFAs), specifically n-6 PUFAs, are known to positively affect hibernation performance and thereby energy savings predominantly in fat-storing hibernators. Accordingly, hibernators usually retain PUFAs and mobilize monounsaturated fatty acids (MUFAs) or saturated fatty acids (SFAs) during hibernation. In food-storing common hamsters (Cricetus cricetus), however, we previously found that PUFA proportions in white adipose tissue (WAT) decreased during winter, indicating that individuals did mobilize PUFAs. To further investigate these patterns, we analyzed PUFA changes in WAT during hibernation as well as hibernation performance in free-ranging and captive common hamsters with lower prehibernation PUFA proportions compared to those in the previous study. Under controlled conditions, total PUFAs, n-6 PUFAs, and SFAs increased while n-3 PUFAs and MUFAs decreased during hibernation. Higher prehibernation n-6 PUFA proportions resulted in fewer torpor bouts and less time spent in torpor. In free-ranging hamsters, n-6 PUFAs increased while n-3 PUFAs and SFAs decreased during winter. Prehibernation n-6 PUFA proportions, however, did not affect hibernation performance. In summary, these results indicate that the mobilization or retention of n-6 PUFAs during hibernation could depend on their availability in WAT or in the diet before the onset of the hibernation period.

Common hamsters (Cricetus cricetus) are hibernators that rely both on body fat reserves and food stores for the winter period. They face an ongoing population decline in most parts of their distribution and recently were classified as critically endangered. Knowledge on individual body fat proportions in this species is of particular interest for conservation, because it could contribute to better understand the high plasticity in overwintering strategies, overwinter mortality rates, individual variations in reproductive output, and give information on the animals' health state. To calculate body fat proportions, we validated a method that can be applied in the field without the use of anesthesia. To develop this method, we first analyzed the body fat in carcasses of common hamsters using Soxhlet extractions and measured four morphometric parameters (body mass, head length, tibia length, foot length). The morphometric measurements were then integrated in a linear regression model to predict body fat proportions based on the measured values. The morphometric variables yielded an explained variance (adjusted R 2) of 96.42% and body fat proportions were predicted with a mean absolute error of 1.27 ± 0.11% from measured values. We applied the model to predict body fat for available field data, which consistently produced reliable values. By measuring the four morphometric parameters and following the provided instructions, body fat proportions can be reliably and noninvasively estimated in captive or free-ranging common hamsters. Furthermore, the method could be applicable to other rodents after species-specific validation.

Dietary intake of polyunsaturated fatty acids (PUFAs) is crucial for neuronal functions, can positively affect cognition, and reduce glucocorticoid (e.g. corticosterone) concentrations in response to stress. We investigated the effects of walnut oil high in PUFAs on spatial cognition and fecal corticosterone metabolite (FCM) concentrations under non-stressed conditions in rats. Unexpectedly, PUFA-supplemented rats had higher FCM concentrations and elevated concentrations generally impaired learning in the subsequent T-maze task. Statistically adjusting for individual FCM concentrations, however, revealed that learning performance was improved in PUFA-supplemented rats. The results suggest that glucocorticoids can modulate the effects of PUFAs on spatial learning under normal (non-stressed) conditions and call for consideration of basal physiological conditions in spatial learning tasks.

The hypothalamic-pituitary-adrenal (HPA)-axis and related glucocorticoid concentrations regulate physiology and behavior, which can be modulated by nutritional conditions, particularly by the dietary fatty acid composition. Omega-3 polyunsaturated fatty acids (PUFAs) have been shown to promote hypothalamic-pituitary adrenal (HPA)-axis functions, whereas saturated fatty acids (SFAs) in general produce adverse effects and even increase baseline glucocorticoid concentrations. Glucocorticoids (e.g. cortisol) were further documented to modulate the establishment of dominance relationships, while the involvement of dietary fatty acids remains understudied. This study focused on different effects of PUFAs and SFAs on cortisol concentrations and social dominance in male guinea pigs. Three groups of animals were maintained on diets high in PUFAs (10 % w/w walnut oil), SFAs (10 % w/w coconut fat), or on an untreated control diet starting already prenatally. During adolescence, at an age of 60, 90, and 120 days, each individual's saliva cortisol concentrations and hierarchy index (calculated by initiated and received agonistic behavior) were measured during basal group housing conditions and stressful social confrontations with unfamiliar individuals of the other groups. SFA males showed highest baseline cortisol concentrations, lowest cortisol responses to social confrontations, and became subdominant. PUFA and control males showed significant cortisol responses. However, while control males became dominant during social confrontations, the hierarchy index in PUFA males decreased with age. Individual hierarchy indices during consecutive social confrontations revealed a high consistency. The findings presented here indicate that dietary fatty acids differently affect HPA-axis functions and social dominance but the underlying mechanisms remain to be determined.