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1.  Non-invasive assessment of adrenocortical activity as a measure of stress in giraffe (Giraffa camelopardalis) 
BMC Veterinary Research  2016;12:235.
Background
Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected.
Results
Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3β,11β-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3–7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day.
Conclusions
We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5β-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5β-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-016-0864-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12917-016-0864-8
PMCID: PMC5070010  PMID: 27756312
ACTH challenge; Validation; Ungulate; Ruminant; Zoo; Endocrine; Adrenal physiology; Glucocorticoid; Health
2.  Male Presence can Increase Body Mass and Induce a Stress-Response in Female Mice Independent of Costs of Offspring Production 
Scientific Reports  2016;6:23538.
Sexual reproduction in animals requires close interactions with the opposite sex. These interactions may generate costs of reproduction, because mates can induce detrimental physiological or physical effects on one another, due to their interest in maximising their own fitness. To understand how a male’s presence influences aspects of female physiology implicated in reproductive costs in mice, independent of offspring production, we paired females with vasectomised, castrated or intact males, or other females. Being paired with a male, irrespective of his gonadal status, increased female weight. This effect was transient in females paired with castrated males but more persistent in those with vasectomised males. Those paired with males also showed an increase in corticosterone, suggesting an increased stress response. However, this was dependent on the gonadal status of the male housing partner, since those housed with vasectomised males had lower corticosterone than those with castrated males. Altered energy metabolism was only detectable in pregnant females, and oxidative stress was not consistently affected by a female’s housing partner. These results suggest that a male’s presence alters female weight, and stresses associated with reproduction could be induced by simply the presence of a male, but reduced by mating and/or being solicited to mate.
doi:10.1038/srep23538
PMCID: PMC4804214  PMID: 27004919
3.  Loner or socializer? Ravens’ adrenocortical response to individual separation depends on social integration 
Hormones and behavior  2015;78:194-199.
Non-breeding common ravens (Corvus corax) live in complex social groups with a high degree of fission–fusion dynamics. They form valuable relationships and alliances with some conspecifics, while taking coordinated action against others. In ravens, affiliates reconcile their conflicts, console each other after conflicts with a third party, and provide each other with social support — all behaviors that presumably reduce corticosterone levels and alleviate stress. However, how well an individual is socially integrated in a (sub)group might vary substantially. This raises the question whether the social integration of a raven affects its stress responses to fission–fusion dynamics. The present study aims to investigate this effect experimentally by separating single ravens (n = 16) individually from their group for four days and subsequently reintroducing them. To determine stress response patterns in the separated individuals we measured the amounts of immunoreactive corticosterone metabolites (CM) in droppings. We compared two enzyme immunoassays, which we validated by conducting an ACTH challenge, and finally decided to apply an 11-oxoetiocholanolone enzyme immunoassay. Additionally, we determined levels of social integration using focal observations. Our findings suggest that a strong social integration is related to low CM levels when the individuals are within the group and high levels during separations, implying that separation leads to stress in these birds. In contrast, poorly socially integrated ravens seem to exhibit the opposite pattern, indicating that to them group living is more stressful than being temporarily separated. We, therefore, conclude that the birds’ adrenocortical activity is modulated by their social integration.
doi:10.1016/j.yhbeh.2015.11.009
PMCID: PMC4754940  PMID: 26631484
Social relationships; Fission–fusion dynamics; Social network; Separation; Stress; Glucocorticoids; Fecal corticosterone metabolites; Common raven; Corvus corax
4.  Mixed-strain housing for female C57BL/6, DBA/2, and BALB/c mice: validating a split-plot design that promotes refinement and reduction 
Background
Inefficient experimental designs are common in animal-based biomedical research, wasting resources and potentially leading to unreplicable results. Here we illustrate the intrinsic statistical power of split-plot designs, wherein three or more sub-units (e.g. individual subjects) differing in a variable of interest (e.g. genotype) share an experimental unit (e.g. a cage or litter) to which a treatment is applied (e.g. a drug, diet, or cage manipulation). We also empirically validate one example of such a design, mixing different mouse strains -- C57BL/6, DBA/2, and BALB/c -- within cages varying in degree of enrichment. As well as boosting statistical power, no other manipulations are needed for individual identification if co-housed strains are differentially pigmented, so also sparing mice from stressful marking procedures.
Methods
The validation involved housing 240 females from weaning to 5 months of age in single- or mixed- strain trios, in cages allocated to enriched or standard treatments. Mice were screened for a range of 26 commonly-measured behavioural, physiological and haematological variables.
Results
Living in mixed-strain trios did not compromise mouse welfare (assessed via corticosterone metabolite output, stereotypic behaviour, signs of aggression, and other variables). It also did not alter the direction or magnitude of any strain- or enrichment-typical difference across the 26 measured variables, or increase variance in the data: indeed variance was significantly decreased by mixed- strain housing. Furthermore, using Monte Carlo simulations to quantify the statistical power benefits of this approach over a conventional design demonstrated that for our effect sizes, the split- plot design would require significantly fewer mice (under half in most cases) to achieve a power of 80 %.
Conclusions
Mixed-strain housing allows several strains to be tested at once, and potentially refines traditional marking practices for research mice. Furthermore, it dramatically illustrates the enhanced statistical power of split-plot designs, allowing many fewer animals to be used. More powerful designs can also increase the chances of replicable findings, and increase the ability of small-scale studies to yield significant results. Using mixed-strain housing for female C57BL/6, DBA/2 and BALB/c mice is therefore an effective, efficient way to promote both refinement and the reduction of animal-use in research.
Electronic supplementary material
The online version of this article (doi:10.1186/s12874-016-0113-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12874-016-0113-7
PMCID: PMC4729181  PMID: 26817696
Mice; Refinement; Reduction; Animal Welfare; Experimental Design; Split-Plot
5.  Non-Invasive Measurement of Adrenocortical Activity in Blue-Fronted Parrots (Amazona aestiva, Linnaeus, 1758) 
PLoS ONE  2015;10(12):e0145909.
Parrots kept in zoos and private households often develop psychological and behavioural disorders. Despite knowing that such disorders have a multifactorial aetiology and that chronic stress is involved, little is known about their development mainly due to a poor understanding of the parrots’ physiology and the lack of validated methods to measure stress in these species. In birds, blood corticosterone concentrations provide information about adrenocortical activity. However, blood sampling techniques are difficult, highly invasive and inappropriate to investigate stressful situations and welfare conditions. Thus, a non-invasive method to measure steroid hormones is critically needed. Aiming to perform a physiological validation of a cortisone enzyme immunoassay (EIA) to measure glucocorticoid metabolites (GCM) in droppings of 24 Blue-fronted parrots (Amazona aestiva), two experiments were designed. During the experiments all droppings were collected at 3-h intervals. Initially, birds were sampled for 24 h (experiment 1) and one week later assigned to four different treatments (experiment 2): Control (undisturbed), Saline (0.2 mL of 0.9% NaCl IM), Dexamethasone (1 mg/kg IM) and Adrenocorticotropic hormone (ACTH; 25 IU IM). Treatments (always one week apart) were applied to all animals in a cross-over study design. A daily rhythm pattern in GCM excretion was detected but there were no sex differences (first experiment). Saline and dexamethasone treatments had no effect on GCM (not different from control concentrations). Following ACTH injection, GCM concentration increased about 13.1-fold (median) at the peak (after 3–9 h), and then dropped to pre-treatment concentrations. By a successful physiological validation, we demonstrated the suitability of the cortisone EIA to non-invasively monitor increased adrenocortical activity, and thus, stress in the Blue-fronted parrot. This method opens up new perspectives for investigating the connection between behavioural disorders and stress in this bird species, and could also help in their captive management.
doi:10.1371/journal.pone.0145909
PMCID: PMC4696673  PMID: 26717147
6.  Environmental and Intrinsic Correlates of Stress in Free-Ranging Wolves 
PLoS ONE  2015;10(9):e0137378.
Background
When confronted with a stressor, animals react with several physiological and behavioral responses. Although sustained or repeated stress can result in severe deleterious physiological effects, the causes of stress in free-ranging animals are yet poorly documented. In our study, we aimed at identifying the main factors affecting stress levels in free-ranging wolves (Canis lupus).
Methodology/Principal Findings
We used fecal cortisol metabolites (FCM) as an index of stress, after validating the method for its application in wolves. We analyzed a total of 450 fecal samples from eleven wolf packs belonging to three protected populations, in Italy (Abruzzo), France (Mercantour), and the United States (Yellowstone). We collected samples during two consecutive winters in each study area. We found no relationship between FCM concentrations and age, sex or social status of individuals. At the group level, our results suggest that breeding pair permanency and the loss of pack members through processes different from dispersal may importantly impact stress levels in wolves. We measured higher FCM levels in comparatively small packs living in sympatry with a population of free-ranging dogs. Lastly, our results indicate that FCM concentrations are associated with endoparasitic infections of individuals.
Conclusions/Significance
In social mammals sharing strong bonds among group members, the death of one or several members of the group most likely induces important stress in the remainder of the social unit. The potential impact of social and territorial stability on stress levels should be further investigated in free-ranging populations, especially in highly social and in territorial species. As persistent or repeated stressors may facilitate or induce pathologies and physiological alterations that can affect survival and fitness, we advocate considering the potential impact of anthropogenic causes of stress in management and conservation programs regarding wolves and other wildlife.
doi:10.1371/journal.pone.0137378
PMCID: PMC4580640  PMID: 26398784
7.  Lifetime Dependent Variation of Stress Hormone Metabolites in Feces of Two Laboratory Mouse Strains 
PLoS ONE  2015;10(8):e0136112.
Non-invasive measurement of stress hormone metabolites in feces has become routine practice for the evaluation of distress and pain in animal experiments. Since metabolism and excretion of glucocorticoids may be variable, awareness and adequate consideration of influencing factors are essential for accurate monitoring of adrenocortical activity. Reference values are usually provided by baselines compiled prior to the experiment and by age matched controls. The comparison of stress hormone levels between animals of different ages or between studies looking at hormone levels at the beginning and at the end of a long term study might be biased by age-related effects. In this study we analyzed fecal corticosterone metabolites (FCM) during the lifetime of untreated female mice of the strains C57BL/6NCrl and Crl:CD1. For this purpose feces for each individual mouse were collected every two months over a period of 24 hours, at intervals of four hours, until the age of 26 months. Results of the study revealed that age of the animals had a significant impact on the level and circadian rhythm of stress hormone metabolites. Furthermore, long-term observation of mice revealed a strain specific excretion profile of FCM influenced by strong seasonal variability.
doi:10.1371/journal.pone.0136112
PMCID: PMC4540567  PMID: 26284365
8.  Interaction of brain 5-HT synthesis deficiency, chronic stress and sex differentially impact emotional behavior in Tph2 knockout mice 
Psychopharmacology  2015;232(14):2429-2441.
Rationale
While brain serotonin (5-HT) function is implicated in gene-by-environment interaction (GxE) impacting the vulnerability-resilience continuum in neuropsychiatric disorders, it remains elusive how the interplay of altered 5-HT synthesis and environmental stressors is linked to failure in emotion regulation.
Objective
Here, we investigated the effect of constitutively impaired 5-HT synthesis on behavioral and neuroendocrine responses to unpredictable chronic mild stress (CMS) using a mouse model of brain 5-HT deficiency resulting from targeted inactivation of the tryptophan hydroxylase-2 (Tph2) gene.
Results
Locomotor activity and anxiety- and depression-like behavior as well as conditioned fear responses were differentially affected by Tph2 genotype, sex, and CMS. Tph2 null mutants (Tph2−/−) displayed increased general metabolism, marginally reduced anxiety- and depression-like behavior but strikingly increased conditioned fear responses. Behavioral modifications were associated with sex-specific hypothalamic-pituitary-adrenocortical (HPA) system alterations as indicated by plasma corticosterone and fecal corticosterone metabolite concentrations. Tph2−/− males displayed increased impulsivity and high aggressiveness. Tph2−/− females displayed greater emotional reactivity to aversive conditions as reflected by changes in behaviors at baseline including increased freezing and decreased locomotion in novel environments. However, both Tph2−/− male and female mice were resilient to CMS-induced hyperlocomotion, while CMS intensified conditioned fear responses in a GxE-dependent manner.
Conclusions
Our results indicate that 5-HT mediates behavioral responses to environmental adversity by facilitating the encoding of stress effects leading to increased vulnerability for negative emotionality.
Electronic supplementary material
The online version of this article (doi:10.1007/s00213-015-3879-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00213-015-3879-0
PMCID: PMC4480945  PMID: 25716307
Serotonin; Tryptophan hydroxylase-2 (Tph2); Chronic stress; Gene-by-environment interaction; Anxiety; Fear; Depression; Aggression
9.  Benefits of adversity?! How life history affects the behavioral profile of mice varying in serotonin transporter genotype 
Behavioral profiles are influenced by both positive and negative experiences as well as the genetic disposition. Traditionally, accumulating adversity over lifetime is considered to predict increased anxiety-like behavior (“allostatic load”). The alternative “mismatch hypothesis” suggests increased levels of anxiety if the early environment differs from the later-life environment. Thus, there is a need for a whole-life history approach to gain a deeper understanding of how behavioral profiles are shaped. The aim of this study was to elucidate the effects of life history on the behavioral profile of mice varying in serotonin transporter (5-HTT) genotype, an established mouse model of increased anxiety-like behavior. For this purpose, mice grew up under either adverse or beneficial conditions during early phases of life. In adulthood, they were further subdivided so as to face a situation that either matched or mismatched the condition experienced so far, resulting in four different life histories. Subsequently, mice were tested for their anxiety-like and exploratory behavior. The main results were: (1) Life history profoundly modulated the behavioral profile. Surprisingly, mice that experienced early beneficial and later escapable adverse conditions showed less anxiety-like and more exploratory behavior compared to mice of other life histories. (2) Genotype significantly influenced the behavioral profile, with homozygous 5-HTT knockout mice displaying highest levels of anxiety-like and lowest levels of exploratory behavior. Our findings concerning life history indicate that the absence of adversity does not necessarily cause lower levels of anxiety than accumulating adversity. Rather, some adversity may be beneficial, particularly when following positive events. Altogether, we conclude that for an understanding of behavioral profiles, it is not sufficient to look at experiences during single phases of life, but the whole life history has to be considered.
doi:10.3389/fnbeh.2015.00047
PMCID: PMC4347490  PMID: 25784864
life history; 5-HTT; anxiety-like behavior; predictive adaptive response hypothesis; match-mismatch
10.  Benefits of a Ball and Chain: Simple Environmental Enrichments Improve Welfare and Reproductive Success in Farmed American Mink (Neovison vison) 
PLoS ONE  2014;9(11):e110589.
Can simple enrichments enhance caged mink welfare? Pilot data from 756 sub-adults spanning three colour-types (strains) identified potentially practical enrichments, and suggested beneficial effects on temperament and fur-chewing. Our main experiment started with 2032 Black mink on three farms: from each of 508 families, one juvenile male-female pair was enriched (E) with two balls and a hanging plastic chain or length of hose, while a second pair was left as a non-enriched (NE) control. At 8 months, more than half the subjects were killed for pelts, and 302 new females were recruited (half enriched: ‘late E’). Several signs of improved welfare or productivity emerged. Access to enrichment increased play in juveniles. E mink were calmer (less aggressive in temperament tests; quieter when handled; less fearful, if male), and less likely to fur-chew, although other stereotypic behaviours were not reduced. On one farm, E females had lower cortisol (inferred from faecal metabolites). E males tended to copulate for longer. E females also weaned more offspring: about 10% more juveniles per E female, primarily caused by reduced rates of barrenness (‘late E’ females also giving birth to bigger litters on one farm), effects that our data cautiously suggest were partly mediated by reduced inactivity and changes in temperament. Pelt quality seemed unaffected, but E animals had cleaner cages. In a subsidiary side-study using 368 mink of a second colour-type (‘Demis’), similar temperament effects emerged, and while E did not reduce fur-chewing or improve reproductive success in this colour-type, E animals were judged to have better pelts. Overall, simple enrichments were thus beneficial. These findings should encourage welfare improvements on fur farms (which house 60-70 million mink p.a.) and in breeding centres where endangered mustelids (e.g. black-footed ferrets) often reproduce poorly. They should also stimulate future research into more effective practical enrichments.
doi:10.1371/journal.pone.0110589
PMCID: PMC4227648  PMID: 25386726
11.  Partial Reductions in Mechanical Loading Yield Proportional Changes in Bone Density, Bone Architecture, and Muscle Mass 
Although the musculoskeletal system is known to be sensitive to changes in its mechanical environment, the relationship between functional adaptation and below-normal mechanical stimuli is not well defined. We investigated bone and muscle adaptation to a range of reduced loading using the partial weight suspension (PWS) system, in which a two-point harness is used to offload a tunable amount of body weight while maintaining quadrupedal locomotion. Skeletally mature female C57Bl/6 mice were exposed to partial weight bearing at 20%, 40%, 70%, or 100% of body weight for 21 days. A hindlimb unloaded (HLU) group was included for comparison in addition to age-matched controls in normal housing. Gait kinematics was measured across the full range of weight bearing, and some minor alterations in gait from PWS were identified. With PWS, bone and muscle changes were generally proportional to the degree of unloading. Specifically, total body and hindlimb bone mineral density, calf muscle mass, trabecular bone volume of the distal femur, and cortical area of the femur midshaft were all linearly related to the degree of unloading. Even a load reduction to 70% of normal weight bearing was associated with significant bone deterioration and muscle atrophy. Weight bearing at 20% did not lead to better bone outcomes than HLU despite less muscle atrophy and presumably greater mechanical stimulus, requiring further investigation. These data confirm that the PWS model is highly effective in applying controllable, reduced, long-term loading that produces predictable, discrete adaptive changes in muscle and bone of the hindlimb.
doi:10.1002/jbmr.1814
PMCID: PMC4118556  PMID: 23165526
MECHANICAL LOADING; DISUSE; MECHANOSTAT; FUNCTIONAL ADAPTATION; WEIGHT BEARING
12.  Genetic Interactions with Sex Make a Relatively Small Contribution to the Heritability of Complex Traits in Mice 
PLoS ONE  2014;9(5):e96450.
The extent to which sex-specific genetic effects contribute to phenotypic variation is largely unknown. We applied a novel Bayesian method, sparse partitioning, to detect gene by sex (GxS) and gene by gene (GxG) quantitative loci (QTLs) in 1,900 outbred heterogeneous stock mice. In an analysis of 55 phenotypes, we detected 16 GxS and 6 GxG QTLs. The increase in the amount of phenotypic variance explained by models including GxS was small, ranging from 0.14% to 4.30%. We conclude that GxS rarely make a large overall contribution to the heritability of phenotypes, however there are cases where these will be individually important.
doi:10.1371/journal.pone.0096450
PMCID: PMC4014490  PMID: 24811081
13.  Environmental Enrichment Alters Splenic Immune Cell Composition and Enhances Secondary Influenza Vaccine Responses in Mice 
Molecular Medicine  2014;20(1):179-190.
Chronic stress has deleterious effects on immune function, which can lead to adverse health outcomes. However, studies investigating the impact of stress reduction interventions on immunity in clinical research have yielded divergent results, potentially stemming from differences in study design and genetic heterogeneity, among other clinical research challenges. To test the hypothesis that reducing glucocorticoid levels enhances certain immune functions, we administered influenza vaccine once (prime) or twice (boost) to mice housed in either standard control caging or environmental enrichment (EE) caging. We have shown that this approach reduces mouse corticosterone production. Compared with controls, EE mice had significantly lower levels of fecal corticosterone metabolites (FCMs) and increased splenic B and T lymphocyte numbers. Corticosterone levels were negatively associated with the numbers of CD19+ (r2 = 0.43, p = 0.0017), CD4+ (r2 = 0.28, p = 0.0154) and CD8+ cells (r2 = 0.20, p = 0.0503). Vaccinated mice showed nonsignificant differences in immunoglobulin G (IgG) titer between caging groups, although EE mice tended to exhibit larger increases in titer from prime to boost than controls; the interaction between the caging group (control versus EE) and vaccine group (prime versus boost) showed a strong statistical trend (cage-group*vaccine-group, F = 4.27, p = 0.0555), suggesting that there may be distinct effects of EE caging on primary versus secondary IgG vaccine responses. Vaccine-stimulated splenocytes from boosted EE mice had a significantly greater frequency of interleukin 5 (IL-5)-secreting cells than boosted controls (mean difference 7.7, IL-5 spot-forming units/106 splenocytes, 95% confidence interval 0.24–135.1, p = 0.0493) and showed a greater increase in the frequency of IL-5–secreting cells from prime to boost. Our results suggest that corticosterone reduction via EE caging was associated with enhanced secondary vaccine responses, but had little effect on primary responses in mice. These findings help identify differences in primary and secondary vaccine responses in relationship to stress mediators that may be relevant in clinical studies.
doi:10.2119/molmed.2013.00158
PMCID: PMC4002849  PMID: 24687160
14.  Are Motorways Potential Stressors of Roadside Wood Mice (Apodemus sylvaticus) Populations? 
PLoS ONE  2014;9(3):e91942.
Linear infrastructures represent one of the most important human impacts on natural habitats and exert several effects on mammal populations. Motorways are recognized as a major cause of habitat fragmentation and degradation and of biodiversity loss. However, it is unknown whether motorways lead to increased physiological stress reactions in wild animal populations. We analysed faecal corticosterone metabolites (FCM) in wild populations of wood mice (Apodemus sylvaticus) living in a well-preserved Mediterranean agro-pastoral woodland at different distances (verge, 500 m and 1000 m) from the AP-51 motorway in Spain. Wood mice were captured with Sherman live traps, and fresh faecal samples from 424 individuals were collected and analyzed in the laboratory. The quantification of FCM was performed by a 5α-pregnane-3β,11β, 21-triol-20-one enzyme immunoassay. Results showed that females had higher FCM levels than males, and these levels were higher in breeding females. In addition, FCM levels were positively correlated with body weight of individuals. Wood mice captured where cattle were present showed higher FCM levels than individuals living where cattle were not detected. FCM levels were higher in non-breeding individuals living close to the motorway compared with FCM levels in those individuals captured further from the motorway. This is the first study showing evidence of the motorways' impact on physiological stress reactions in wild wood mice populations. Understanding how free-living animals are influenced by human interventions could help to understand other subtle changes observed in wild animal populations. Since mice are used world-wide as research models these results could open new perspectives testing human influence on the natural environment and trade-offs of species in degraded ecosystems.
doi:10.1371/journal.pone.0091942
PMCID: PMC3956862  PMID: 24637740
15.  Development of an Optimal Diaphragmatic Hernia Rabbit Model for Pediatric Thoracoscopic Training 
Experimental Animals  2014;63(1):93-98.
Our objectives were to standarize the procedure needed to reproduce a similar surgical scene which a pediatric surgeon would face on repairing a Bochdalek hernia in newborns and to define the optimal time period for hernia development that achieve a realistic surgical scenario with minimimal animal suffering. Twenty New Zealand white rabbits weighing 3–3.5 kg were divided into four groups depending on the time frame since hernia creation to thoracoscopic repair: 48 h, 72 h, 96 h and 30 days. Bochdalek trigono was identified and procedures for hernia creation and thoracoscopic repair were standarized. Blood was collected for hematology (red blood cells, white blood cells, platelets, hemoglobin and hematocrit), biochemistry (blood urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and creatine kinase) and gas analysis (arterial blood pH, partial pressure of oxygen, partial pressure of carbón dioxide, oxygen saturation and bicarbonate) at baseline and before the surgial repairment. Glucocorticoid metabolites concentration in faeces was measured. Thoracoscopy video recordings were evaluated by six pediatric surgeons and rated from 0 to 10 according to similarities with congenital diaphragmatic hernia in newborn and with its thoracoscopic approach. Statistical methods included the analysis of variance, and comparisons between groups were followed by a post-hoc Tukey’s test. Fourty -eight h showed to be the optimal time frame to obtain a diaphragmatic hernia similar to newborn scenario from a surgical point of view with minimal stress for the animals.
doi:10.1538/expanim.63.93
PMCID: PMC4160932  PMID: 24521868
animal stress; congenital diaphragmatic hernia; rabbit model; thoracoscopy
16.  Environmentally Enriched Male Mink Gain More Copulations than Stereotypic, Barren-Reared Competitors 
PLoS ONE  2013;8(11):e80494.
Wild carnivores in zoos, conservation breeding centres, and farms commonly live in relatively small, unstimulating enclosures. Under these captive conditions, in a range of species including giant pandas, black-footed ferrets, and European mink, male reproductive abilities are often poor. Such problems have long been hypothesized to be caused by these animals' housing conditions. We show for the first time that rearing under welfare-improving (i.e., highly valued and stress-reducing) environmental enrichments enhances male carnivores' copulatory performance: in mate choice competitions, enriched male American mink (Neovison vison) mated more often than non-enriched males. We screened for several potential mediators of this effect. First was physiological stress and its impact on reproductive physiology; second, stress-mediated changes in morphology and variables related to immunocompetence that could influence male attractiveness; and third, behavioural changes likely to affect social competence, particularly autistic-like excessive routine and repetition (‘perseveration’) as is reflected in the stereotypies common in captive animals. Consistent with physiological stress, excreted steroid metabolites revealed that non-enriched males had higher cortisol levels and lower androgen levels than enriched conspecifics. Their os penises (bacula) also tended to be less developed. Consistent with reduced attractiveness, non-enriched males were lighter, with comparatively small spleens and a trend to greater fluctuating asymmetry. Consistent with impaired social competence, non-enriched males performed more stereotypic behaviour (e.g., pacing) in their home cages. Of all these effects, the only significant predictor of copulation number was stereotypy (a trend suggesting that low bodyweights may also be influential): highly stereotypic males gained the fewest copulations. The neurophysiological changes underlying stereotypy thus handicap males sexually. We hypothesise that such males are abnormally perseverative when interacting with females. Investigating similar problems in other taxa would be worthwhile, since many vertebrates, wild and domestic, live in conditions that cause stereotypic behaviour and/or impair neurological development.
doi:10.1371/journal.pone.0080494
PMCID: PMC3839975  PMID: 24282547
17.  Co-Housing Rodents with Different Coat Colours as a Simple, Non-Invasive Means of Individual Identification: Validating Mixed-Strain Housing for C57BL/6 and DBA/2 Mice 
PLoS ONE  2013;8(10):e77541.
Standard practice typically requires the marking of laboratory mice so that they can be individually identified. However, many of the common methods compromise the welfare of the individuals being marked (as well as requiring time, effort, and/or resources on the part of researchers and technicians). Mixing strains of different colour within a cage would allow them to be readily visually identifiable, negating the need for more invasive marking techniques. Here we assess the impact that mixed strain housing has on the phenotypes of female C57BL/6 (black) and DBA/2 (brown) mice, and on the variability in the data obtained from them. Mice were housed in either mixed strain or single strain pairs for 19 weeks, and their phenotypes then assessed using 23 different behavioural, morphological, haematological and physiological measures widely used in research and/or important for assessing mouse welfare. No negative effects of mixed strain housing could be found on the phenotypes of either strain, including variables relevant to welfare. Differences and similarities between the two strains were almost all as expected from previously published studies, and none were affected by whether mice were housed in mixed- or single-strain pairs. Only one significant main effect of housing type was detected: mixed strain pairs had smaller red blood cell distribution widths, a measure suggesting better health (findings that now need replicating in case they were Type 1 errors resulting from our multiplicity of tests). Furthermore, mixed strain housing did not increase the variation in data obtained from the mice: the standard errors for all variables were essentially identical between the two housing conditions. Mixed strain housing also made animals very easy to distinguish while in the home cage. Female DBA/2 and C57BL/6 mice can thus be housed in mixed strain pairs for identification purposes, with no apparent negative effects on their welfare or the data they generate. This suggests that there is much value in exploring other combinations of strains.
doi:10.1371/journal.pone.0077541
PMCID: PMC3810273  PMID: 24204864
18.  Differential behavioural and endocrine responses of common voles (Microtus arvalis) to nest predators and resource competitors 
BMC Ecology  2013;13:33.
Background
Adaptive behavioural strategies promoting co-occurrence of competing species are known to result from a sympatric evolutionary past. Strategies should be different for indirect resource competition (exploitation, e.g., foraging and avoidance behaviour) than for direct interspecific interference (e.g., aggression, vigilance, and nest guarding). We studied the effects of resource competition and nest predation in sympatric small mammal species using semi-fossorial voles and shrews, which prey on vole offspring during their sensitive nestling phase. Experiments were conducted in caged outdoor enclosures. Focus common vole mothers (Microtus arvalis) were either caged with a greater white-toothed shrew (Crocidura russula) as a potential nest predator, with an herbivorous field vole (Microtus agrestis) as a heterospecific resource competitor, or with a conspecific resource competitor.
Results
We studied behavioural adaptations of vole mothers during pregnancy, parturition, and early lactation, specifically modifications of the burrow architecture and activity at burrow entrances. Further, we measured pre- and postpartum faecal corticosterone metabolites (FCMs) of mothers to test for elevated stress hormone levels. Only in the presence of the nest predator were prepartum FCMs elevated, but we found no loss of vole nestlings and no differences in nestling body weight in the presence of the nest predator or the heterospecific resource competitor. Although the presence of both the shrew and the field vole induced prepartum modifications to the burrow architecture, only nest predators caused an increase in vigilance time at burrow entrances during the sensitive nestling phase.
Conclusion
Voles displayed an adequate behavioural response for both resource competitors and nest predators. They modified burrow architecture to improve nest guarding and increased their vigilance at burrow entrances to enhance offspring survival chances. Our study revealed differential behavioural adaptations to resource competitors and nest predators.
doi:10.1186/1472-6785-13-33
PMCID: PMC3847765  PMID: 24010574
Behavioural adaptations; Small mammals; Interspecific interactions; Nest predation; Stress response; Faecal corticosterone metabolites; Burrow system; Shrews; Voles
19.  A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice 
Oral gavage dosing can induce stress and potentially confound experimental measurements, particularly when blood pressure and heart rate are endpoints of interest. Thus, we developed a pill formulation that mice would voluntarily consume and tested the hypothesis that pill dosing would be significantly less stressful than oral gavage. C57Bl/6 male mice were singly housed and on four consecutive days were exposed to an individual walking into the room (week 1, control), a pill being placed into the cage (week 2), and a dose of water via oral gavage (week 3). Blood pressure and heart rate were recorded by radiotelemetry continuously for 5 hr after treatment, and feces collected 6–10 hr after treatment for analysis of corticosterone metabolites. Both pill and gavage dosing significantly increased mean arterial pressure (MAP) during the first hour, compared to control. However, the increase in MAP was significantly greater after gavage and remained elevated up to 5 hr, while MAP returned to normal within 2 hr after a pill. Neither pill nor gavage dosing significantly increased heart rate during the first hour, compared to control; however, pill dosing significantly reduced heart rate while gavage significantly increased heart rate 2–5 hr post dosing. MAP and heart rate did not differ 24 hr after dosing. Lastly, only gavage dosing significantly increased fecal corticosterone metabolites, indicating a systemic stress response via activation of the hypothalamic-pituitary-adrenal axis. These data demonstrated that this pill dosing method of mice is significantly less stressful than oral gavage.
doi:10.1016/j.taap.2012.01.025
PMCID: PMC3306547  PMID: 22326784
oral gavage; stress; blood pressure; alternative; corticosterone
20.  Plasma cortisol and faecal cortisol metabolites concentrations in stereotypic and non-stereotypic horses: do stereotypic horses cope better with poor environmental conditions? 
Background
Stereotypic behaviours, i.e. repetitive behaviours induced by frustration, repeated attempts to cope and/or brain dysfunction, are intriguing as they occur in a variety of domestic and captive species without any clear adaptive function. Among the different hypotheses, the coping hypothesis predicts that stereotypic behaviours provide a way for animals in unfavourable environmental conditions to adjust. As such, they are expected to have a lower physiological stress level (glucocorticoids) than non-stereotypic animals. Attempts to link stereotypic behaviours with glucocorticoids however have yielded contradictory results. Here we investigated correlates of oral and motor stereotypic behaviours and glucocorticoid levels in two large samples of domestic horses (NStudy1 = 55, NStudy2 = 58), kept in sub-optimal conditions (e.g. confinement, social isolation), and already known to experience poor welfare states. Each horse was observed in its box using focal sampling (study 1) and instantaneous scan sampling (study 2). Plasma samples (collected in study 1) but also non-invasive faecal samples (collected in both studies) were retrieved in order to assess cortisol levels.
Results
Results showed that 1) plasma cortisol and faecal cortisol metabolites concentrations did not differ between horses displaying stereotypic behaviours and non-stereotypic horses and 2) both oral and motor stereotypic behaviour levels did not predict plasma cortisol or faecal cortisol metabolites concentrations.
Conclusions
Cortisol measures, collected in two large samples of horses using both plasma sampling as well as faecal sampling (the latter method minimizing bias due to a non-invasive sampling procedure), therefore do not indicate that stereotypic horses cope better, at least in terms of adrenocortical activity.
doi:10.1186/1746-6148-9-3
PMCID: PMC3544618  PMID: 23289406
Stereotypic behaviours; Cortisol; Faeces; Plasma; Coping hypothesis; Horse
21.  The Calm Mouse: An Animal Model of Stress Reduction 
Molecular Medicine  2012;18(1):606-617.
Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a “calm mouse model” with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.
doi:10.2119/molmed.2012.00053
PMCID: PMC3388136  PMID: 22398685
22.  Non-invasive measurement of adrenocortical and gonadal activity in male and female guinea pigs (Cavia aperea f. porcellus) 
Taking blood samples is a common method in biomedical and biological research using guinea pigs. However, most blood sampling techniques are complicated and highly invasive and may therefore not be appropriate for certain research topics concerning stress and reproduction. Thus, a non-invasive method to measure steroid hormones is critically needed. The aim of this study was the biological validation of corresponding enzyme immunoassays for the measurement of fecal cortisol, progesterone, estrogen, and testosterone metabolites in guinea pigs. We examined the effect of subcutaneous injections of ACTH or saline on fecal cortisol metabolites to investigate the suitability of fecal samples to monitor adrenocortical activity. Furthermore, we investigated whether fecal sex steroid metabolites accurately reflected endocrine changes observed in plasma samples during female estrous cycles and male puberty, respectively. In addition, we compared fecal testosterone metabolites of intact males, castrated males, and females to investigate the reliability of fecal samples in discriminating gonadal status of males. Concentrations of fecal cortisol metabolites were significantly increased following ACTH challenge, indicating that adrenocortical activity can be monitored via fecal samples. Secondly, in females, plasma and fecal gonadal steroids were significantly correlated in most subjects. The assay for testosterone metabolites, on the other hand, could not clearly discriminate between test groups. From these findings we conclude that fecal samples can be used for the non-invasive assessment of adrenocortical and female reproductive status in guinea pigs. Testosterone metabolism seems to be more complex and further investigations are needed to establish a more suitable assay.
doi:10.1016/j.ygcen.2008.03.020
PMCID: PMC2956976  PMID: 18430425
Guinea pigs; Gonadal steroids; Glucocorticoids; Feces; EIA
23.  Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system 
Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories.
Electronic supplementary material
The online version of this article (doi:10.1007/s00360-008-0330-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00360-008-0330-4
PMCID: PMC2755731  PMID: 19130060
Mouse physiology; Spaceflight; Musculoskeletal system; Stress; Behaviour; Genetically engineered animal models; Animal housing and cage
24.  Rhythmicity in Mice Selected for Extremes in Stress Reactivity: Behavioural, Endocrine and Sleep Changes Resembling Endophenotypes of Major Depression 
PLoS ONE  2009;4(1):e4325.
Background
Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, including hyper- or hypo-activity of the stress hormone system, plays a critical role in the pathophysiology of mood disorders such as major depression (MD). Further biological hallmarks of MD are disturbances in circadian rhythms and sleep architecture. Applying a translational approach, an animal model has recently been developed, focusing on the deviation in sensitivity to stressful encounters. This so-called ‘stress reactivity’ (SR) mouse model consists of three separate breeding lines selected for either high (HR), intermediate (IR), or low (LR) corticosterone increase in response to stressors.
Methodology/Principle Findings
In order to contribute to the validation of the SR mouse model, our study combined the analysis of behavioural and HPA axis rhythmicity with sleep-EEG recordings in the HR/IR/LR mouse lines. We found that hyper-responsiveness to stressors was associated with psychomotor alterations (increased locomotor activity and exploration towards the end of the resting period), resembling symptoms like restlessness, sleep continuity disturbances and early awakenings that are commonly observed in melancholic depression. Additionally, HR mice also showed neuroendocrine abnormalities similar to symptoms of MD patients such as reduced amplitude of the circadian glucocorticoid rhythm and elevated trough levels. The sleep-EEG analyses, furthermore, revealed changes in rapid eye movement (REM) and non-REM sleep as well as slow wave activity, indicative of reduced sleep efficacy and REM sleep disinhibition in HR mice.
Conclusion/Significance
Thus, we could show that by selectively breeding mice for extremes in stress reactivity, clinically relevant endophenotypes of MD can be modelled. Given the importance of rhythmicity and sleep disturbances as biomarkers of MD, both animal and clinical studies on the interaction of behavioural, neuroendocrine and sleep parameters may reveal molecular pathways that ultimately lead to the discovery of new targets for antidepressant drugs tailored to match specific pathologies within MD.
doi:10.1371/journal.pone.0004325
PMCID: PMC2627900  PMID: 19177162
25.  Spreading free-riding snow sports represent a novel serious threat for wildlife 
Stress generated by humans on wildlife by continuous development of outdoor recreational activities is of increasing concern for biodiversity conservation. Human disturbance often adds to other negative impact factors affecting the dynamics of vulnerable populations. It is not known to which extent the rapidly spreading free-riding snow sports actually elicit detrimental stress (allostatic overload) upon wildlife, nor what the potential associated fitness and survival costs are. Using a non-invasive technique, we evaluated the physiological stress response induced by free-riding snow sports on a declining bird species of Alpine ecosystems. The results of a field experiment in which radiomonitored black grouse (Tetrao tetrix) were actively flushed from their snow burrows once a day during four consecutive days showed an increase in the concentration of faecal stress hormone (corticosterone) metabolites after disturbance. A large-scale comparative analysis across the southwestern Swiss Alps indicated that birds had higher levels of these metabolites in human-disturbed versus undisturbed habitats. Disturbance by snow sport free-riders appears to elevate stress, which potentially represents a new serious threat for wildlife. The fitness and survival costs of allostatic adjustments have yet to be estimated.
doi:10.1098/rspb.2006.0434
PMCID: PMC2189568  PMID: 17341459
stress ecology; conservation biology; species protection; alpine ecosystems; human disturbance; winter snow sports

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