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Background Glucocorticoids (e.g. cortisol) are associated with variation in social behavior, and previous studies have linked baseline as well as challenge-induced glucocorticoid concentrations to dominance status. It is known that cortisol response to an acute challenge is repeatable and correlates to social behavior in males of many mammal species. However, it is unclear whether these patterns are also consistent for females. The aim of this study was to investigate whether baseline and response cortisol concentrations are repeatable in female guinea pigs (Cavia aperea f. porcellus) and whether dominance rank is stable and correlated to baseline cortisol concentration and/or cortisol responsiveness. Results Our results show that cortisol responsiveness (after 1 h: R = 0.635, 95% CI = 0.229, 0.927; after 2 h: R = 0.764, 95% CI = 0.433, 0.951) and dominance rank (R = 0.709, 95% CI = 0.316, 0.935) of females were significantly repeatable after six weeks but not correlated. Baseline cortisol was not repeatable (R = 0, 95% CI = 0, 0.690) and also did not correlate to dominance rank. Furthermore, the difference in repeatability estimates of baseline and response values was due to high within-individual variance of baseline cortisol concentration; the amount of between-individual variance was similar for baseline cortisol and the two measures of cortisol responsiveness. Conclusions Females occupying different dominance ranks did not have long-term differences in cortisol concentrations, and cortisol responsiveness does not seem to be significantly involved in the maintenance of dominance rank. Overall, this study reveals the remarkable stability of cortisol responsiveness and dominance rank in a female rodent, and it remains an open question whether the magnitude of cortisol responsiveness is adaptive in social contexts for females.

In humans, non-right-handedness is associated with a higher incidence of psychiatric disorders. Since serotonin seems to be involved in both, the development of psychiatric disorders and lateralization, the present study focuses on the effect of the serotonin transporter (5-HTT) gene on behavioral lateralization. For this, we used the 5-HTT knockout mouse model, a well-established animal model for the study of human depression and anxiety disorders. For female mice from all three 5-HTT genotypes (wild type, heterozygous, and homozygous knockout), we repeatedly observed the direction and strength of lateralization of the following four behaviors: grid climbing (GC), food-reaching in an artificial test situation (FRT), self-grooming (SG), and barrier crossing (BC), with the FRT being the standard test for assessing behavioral lateralization in mice. We found no association between behavioral lateralization and 5-HTT genotype. However, in accordance with previous findings, the strength and temporal consistency of lateralization differed between the four behaviors observed. In conclusion, since the 5-HTT genotype did not affect behavioral lateralization in mice, more research on other factors connected with behavioral lateralization and the development of symptoms of psychiatric disorders, such as environmental influences, is needed.

An uncontrolled reproduction of animals in human hands should be avoided. To meet this goal, animals are widely castrated, i.e., the gonads are completely removed. Since the gonads are the most important source of sex hormones, this is a serious intervention in the entire endocrine system of an organism. Sterilization is a much less invasive procedure. Thus, it could have advantages over castration. Therefore, the overall aim of this study was to analyze the effect of castration vs. sterilization on the release of glucocorticoids, i.e., an important indicator for welfare. Taking domestic guinea pigs as a model system, we studied baseline and response cortisol values (cortisol is the main glucocorticoid in guinea pigs) in castrated, sterilized, sham-operated and intact males and baseline values in their cohoused females. Whereas baseline values of males did not differ between the groups, castrated males showed significantly higher cortisol response levels than intact, sham-operated and sterilized males. Females housed with castrated, sterilized, sham-operated or intact males did not differ in their cortisol concentrations, neither shortly after being placed with the respective male or after being co-housed for several weeks. Overall, the results support the hypothesis that castrated males exhibited a higher cortisol responsiveness during acute challenge which could point to a generalized impaired welfare of castrated males in comparison to intact, sham-operated and sterilized males. Our results provide first evidence for a potential negative impact of castration on the animals' welfare, while at the same time pointing toward sterilization representing a less invasive, promising alternative. Therefore, the results may stimulate future research on this topic to further detect potential welfare-related side effects of castration.

Reduction of the numbers of laboratory animals is one of the three pillars of ethical animal research. Equivalently, information gain per animal should be maximized. A road towards this goal that is barely taken in current animal research is the more accurate statistical modeling of experiments. Here we show for a typical experiment (“open field test”) with outcomes that are non-normally distributed count data, how this can be implemented and what information gain is achieved. We contrast the state of the art – the use of confidence intervals based on null-hypothesis significance testing (NHST) –, with a Bayesian approach with the same underlying normal model, and a Bayesian approach with a more accurate negative binomial model. We find that the more accurate model leads to a marked improvement of knowledge gained with the experiment, especially for small sample sizes. As experimental data that violate assumptions of simple, conventional models are frequent, our findings have wider implications.

Courtship songs in mice have been investigated to understand the mechanisms and ecological relevance of vocal communication. There is evidence that courtship song characteristics vary between individuals from different genetic backgrounds, but little is known about whether individuals, even within the same strain, differ consistently from each other in the syntactical composition and complexity of their songs. In a first confirmatory experiment, we aimed to systematically identify syntactical features typical of songs from different mouse strains, by assessing the composition and complexity (i.e. entropy) of the syntax (i.e. how the different syllables are organized within a song) of male laboratory mice from four different strains (Mus musculus f. domestica: C57BL/6J, BALB/c, DBA/2 and B6D2F1). Mice were individually presented with a swab containing fresh female urine for 5 min to elicit courtship songs. The four strains differed not only in the composition but also in the complexity of their syntax. In a second experiment, we investigated repeatability over time as a marker of individuality as well as the occurrence of recurring motifs (i.e. identical sets of syllables that are repeated within a song) of BALB/c and DBA/2 mice. The same procedure as in the first experiment was followed, but in addition testing was repeated weekly over 3 weeks. In both strains, most syllable types showed moderate to high repeatability, indicating individuality in song syntax. However, a descriptive hierarchical cluster analysis indicated remarkable variability in how similar the syntaxes of songs from the same individual were over time. Furthermore, individuals varied in the expression of recurring motifs. Our study shows that not only the genetic background but also individuality can affect variability in courtship songs in mice. Together, these findings raise intriguing questions on whether different individual singing strategies exist during courtship.

Background:The amygdala is crucial for emotional cognitive processing. Affective or emotional states can bias cognitive processes, including attention, memory, and decision-making. This can result in optimistic or pessimistic behaviors that are partially driven by the activation of the amygdala. The resulting emotional cognitive bias is a common feature of anxiety and mood disorders, both of which are interactively influenced by genetic and environmental factors. It is also known that emotional cognitive biases can be influenced by environmental factors. However, little is known about the effects of genetics and/or gene-environment interactions on emotional cognitive biases. We investigated the effects of the genetic background and environmental enrichment on the transcriptional profiles of the mouse amygdala following a well-established cognitive bias test.Methods:Twenty-four female C57BL/6J and B6D2F1N mice were housed either in standard (control) conditions or in an enriched environment. After appropriate training, the cognitive bias test was performed on 19 mice that satisfactorily completed the training scheme to assess their responses to ambiguous cues. This allowed us to calculate an “optimism score” for each mouse. Subsequently, we dissected the anterior and posterior portions of the amygdala to perform RNA-sequencing for differential expression and other statistical analyses.Results:In general, we found only minor changes in the amygdala’s transcriptome associated with the levels of optimism in our mice. In contrast, we observed wide molecular effects of the genetic background in both housing environments. The C57BL/6J animals showed more transcriptional changes in response to enriched environments than the B6D2F1N mice. We also generally found more dysregulated genes in the posterior than in the anterior portion of the amygdala. Gene set overrepresentation analyses consistently implicated cellular metabolic responses and immune processes in the differences observed between mouse strains, while processes favoring neurogenesis and neurotransmission were implicated in the responses to environmental enrichment. In a correlation analysis, lipid metabolism in the anterior amygdala was suggested to influence the levels of optimism.Conclusions:Our observations underscore the importance of selecting appropriate animal models when performing molecular studies of affective conditions or emotional states, and suggest an important role of immune and stress responses in the genetic component of emotion regulation.

BackgroundDuring early phases of life, such as prenatal or early postnatal development and adolescence, an organism's phenotype can be shaped by the environmental conditions it experiences. According to the Match-Mismatch hypothesis (MMH), changes to this environment during later life stages can result in a mismatch between the individual's adaptations and the prevailing environmental conditions. Thus, negative consequences in welfare and health can occur. We aimed to test the MMH in the context of food availability, assuming adolescence as a sensitive period of adaptation.MethodsWe have previously reported a study of the physiological and behavioral effects of match and mismatch conditions of high (ad libitum) and low (90% of ad libitum intake) food availability from adolescence to early adulthood in female C57BL/6J mice (n = 62). Here, we performed RNA-sequencing of the livers of a subset of these animals (n = 16) to test the effects of match and mismatch feeding conditions on the liver transcriptome.ResultsIn general, we found no effect of the match-mismatch situations. Contrarily, the amount of food available during early adulthood (low vs. high) drove the differences we observed in final body weight and gene expression in the liver, regardless of the amount of food available to the animals during adolescence. Many of the differentially expressed genes and the corresponding biological processes found to be overrepresented overlapped, implicating common changes in various domains. These included metabolism, homeostasis, cellular responses to diverse stimuli, transport of bile acids and other molecules, cell differentiation, major urinary proteins, and immunity and inflammation.ConclusionsOur previous and present observations found no support for the MMH in the context of low vs high food availability from adolescence to early adulthood in female C57BL/6J mice. However, even small differences of approximately 10% in food availability during early adulthood resulted in physiological and molecular changes with potential beneficial implications for metabolic diseases.

We present here a unifying framework for affective phenomena: the Human Affectome. By synthesizing a large body of literature, we have converged on definitions that disambiguate the commonly used terms—affect, feeling, emotion, and mood. Based on this definitional foundation, and under the premise that affective states reflect allostatic concerns, we take a goal-directed, enactive perspective. The human affectome is comprised of allostatic features (valence, motivation, and arousal) and allostatic concerns, which differ in the amount of action required to alleviate allostatic load. Allostatic concerns often fall into three ranges: physiological (the most immediate), operational (intermediate to distal), and global. Global concerns involve summations of overall trajectory, general wellbeing, and self-identity. Within this organizational scheme, the human affectome allows vastly different scientific interests to reside within the same theoretical framework and relate to each other. We hope this framework serves as a common focal point for affective research.

Behavioural lateralization is a widespread phenomenon in the animal kingdom and can be an indicator of brain asymmetries in structure or function. It is often assessed using only one measure within the same study. Additionally, this measure is usually obtained in artificial situations, where the subjects undergo tasks requiring learning and forced performance of a specific action. Combining different measures of lateralization seems to be particularly promising, as artificially tested and spontaneously displayed behaviours may cover different aspects of behavioural lateralization. Therefore, the aim of the present study was to provide a comprehensive characterization of behavioural lateralization, using the laboratory mouse, Mus musculus f. domestica, as a model system. We examined lateralization using four behavioural measures, namely food reaching in an artificial test situation (the reference, most established, test) and three spontaneous behaviours (grid climbing, self-grooming and barrier crossing). We investigated their temporal consistency, assessed the association of the three spontaneous behaviours with the established food-reaching test and explored possible influences of genetic background and sex. We found differences in the distributions of lateralization across these behaviours, with fewer mice showing laterality in spontaneous behaviours. All behaviours showed high temporal consistency for the direction of laterality, while only food reaching did so for the strength of laterality. Furthermore, laterality of the spontaneous behaviours was unrelated to laterality of the food-reaching test. Lastly, strain affected behavioural lateralization, with C57BL/6J mice being more strongly lateralized than CD-1 mice for some behaviours, while sex did not affect it. Therefore, we conclude that behavioural lateralization is affected not only by genetic background, but also by the complexity of a given task. To obtain a comprehensive picture of behavioural lateralization, several measures from different contexts thus need to be included in a single study.

Cancer-induced bone pain (CIBP) is a common and devastating symptom with limited treatment options in patients, significantly affecting their quality of life. The use of rodent models is the most common approach to uncovering the mechanisms underlying CIBP; however, the translation of results to the clinic may be hindered because the assessment of pain-related behavior is often based exclusively on reflexive-based methods, which are only partially indicative of relevant pain in patients. To improve the accuracy and strength of the preclinical, experimental model of CIBP in rodents, we used a battery of multimodal behavioral tests that were also aimed at identifying rodent-specific behavioral components by using a home-cage monitoring assay (HCM). Rats of all sexes received an injection with either heat-deactivated (sham-group) or potent mammary gland carcinoma Walker 256 cells into the tibia. By integrating multimodal datasets, we assessed pain-related behavioral trajectories of the CIBP-phenotype, including evoked and non-evoked based assays and HCM. Using principal component analysis (PCA), we discovered sex-specific differences in establishing the CIBP-phenotype, which occurred earlier (and differently) in males. Additionally, HCM phenotyping revealed the occurrence of sensory-affective states manifested by mechanical hypersensitivity in sham when housed with a tumor-bearing cagemate (CIBP) of the same sex. This multimodal battery allows for an in-depth characterization of the CIBP-phenotype under social aspects in rats. The detailed, sex-specific, and rat-specific social phenotyping of CIBP enabled by PCA provides the basis for mechanism-driven studies to ensure robustness and generalizability of results and provide information for targeted drug development in the future.

Discussions of reproducibility are casting doubts on the credibility of experimental outcomes in the life sciences. Although experimental evolution is not typically included in these discussions, this field is also subject to low reproducibility, partly because of the inherent contingencies affecting the evolutionary process. A received view in experimental studies more generally is that standardization (i.e., rigorous homogenization of experimental conditions) is a solution to some issues of significance and internal validity. However, this solution hides several difficulties, including a reduction of external validity and reproducibility. After explaining the meaning of these two notions in the context of experimental evolution, we import from the fields of animal research and ecology and suggests that systematic heterogenization of experimental factors could prove a promising alternative. We also incorporate into our analysis some philosophical reflections on the nature and diversity of research objectives in experimental evolution.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions 1–4. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment 5. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between rearing facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multi-center study, where inbred mice from the same breeding stock were reared in five different facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that common variation among rearing facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organization. We detected changes in chromatin organization in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity and regulation of behavior. Our findings demonstrate that common environmental differences between rearing facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. We expect our findings to stimulate further research into the mechanisms and drivers of these epigenetic changes mediated by the laboratory environment. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and a need to account for the animals’ environmental background in study design to produce robust and replicable findings.

Translational neuroscience bridges insights from specific mechanisms in rodents to complex functions in humans and is key to advance our general understanding of central nervous function. A prime example of translational research is the study of cross-species mechanisms that underlie responding to learned threats, by employing Pavlovian fear conditioning protocols in rodents and humans. Hitherto, evidence for (and critique of) these cross-species comparisons in fear conditioning research was based on theoretical viewpoints. Here, we provide a perspective to substantiate these theoretical concepts with empirical considerations of cross-species methodology. This meta-research perspective is expected to foster cross-species comparability and reproducibility to ultimately facilitate successful transfer of results from basic science into clinical applications.

Background Glucocorticoids (e.g. cortisol) are associated with variation in social behavior, and previous studies have linked baseline as well as challenge-induced glucocorticoid concentrations to dominance status. It is known that cortisol response to an acute challenge is repeatable and correlates to social behavior in males of many mammal species. However, it is unclear whether these patterns are also consistent for females. The aim of this study was to investigate whether baseline and response cortisol concentrations are repeatable in female guinea pigs (Cavia aperea f. porcellus) and whether dominance rank is stable and correlated to baseline cortisol concentration and/or cortisol responsiveness. Results Our results show that cortisol responsiveness (after 1 h: R = 0.635, 95% CI = 0.229, 0.927; after 2 h: R = 0.764, 95% CI = 0.433, 0.951) and dominance rank (R = 0.709, 95% CI = 0.316, 0.935) of females were significantly repeatable after six weeks but not correlated. Baseline cortisol was not repeatable (R = 0, 95% CI = 0, 0.690) and also did not correlate to dominance rank. Furthermore, the difference in repeatability estimates of baseline and response values was due to high within-individual variance of baseline cortisol concentration; the amount of between-individual variance was similar for baseline cortisol and the two measures of cortisol responsiveness. Conclusions Females occupying different dominance ranks did not have long-term differences in cortisol concentrations, and cortisol responsiveness does not seem to be significantly involved in the maintenance of dominance rank. Overall, this study reveals the remarkable stability of cortisol responsiveness and dominance rank in a female rodent, and it remains an open question whether the magnitude of cortisol responsiveness is adaptive in social contexts for females. Supplementary Information The online version contains supplementary material available at 10.1186/s12983-021-00449-2.

The basic question as to whether male laboratory mice should be singly or group housed represents a major animal welfare concern within current laboratory animal legislation and husbandry. To better understand the behavioural and physiological mechanisms underlying this issue, we conducted two longitudinal experiments using C57BL/6J mice. In the first experiment (N = 32), we explored social behaviour of pair housed males from weaning to adulthood. We took weekly measures of agonistic, socio-exploratory and affiliative behaviours within two different contexts, i.e. in the undisturbed home cage and immediately after cage cleaning. In the second experiment (N = 36), we investigated whether separation of male pairs into single housing at different ages (35, 56 or 77 days of age) affected welfare-related measures such as faecal corticosterone metabolites (FCMs) and anxiety-like behaviours. In the first experiment we found that levels of agonistic behaviour were higher after cage cleaning than in the undisturbed cage as expected, but did not significantly change with age in either context. Instead, affiliative behaviour increased with age in the undisturbed home cage. In the second experiment, social separation did not affect levels of FCMs or anxiety-like behaviours at any age point. Taken together, this study shows that pair housed male mice can maintain low levels of aggression across a long period of their life and perform increasing levels of sociopositive behaviours which may serve to promote stable social relations. At the same time, our results suggest that male mice can quickly adapt to separation into single housing at different ages, from adolescence to adulthood. These findings are in line with the behavioural ecology of wild male mice, which suggests that both solitary and group living represent two alternative strategies.

Cognitive judgement bias tests have become important new tools for the assessment of animal emotions. They allow for the inference of an animal's emotional state based on ambiguous cue interpretations. As mice are the predominantly used animal model for cognitive and behavioural neuroscience, research in this field would considerably benefit from the development of suitable judgement bias tests for this species. Against this background, we aimed to implement two different active choice cognitive judgement bias paradigms for mice in a methodological study. For this purpose, two experiments were conducted: in experiment I, an automated, vision-based touchscreen technique was applied, allowing for the direct translation of tasks from rodents to humans and vice versa. Experiment II comprised a task relying on more ecologically relevant cues in form of tunnels of different lengths. While the touchscreen task was characterized by automation-related advantages such as the possibility to present many trials per session and a high convenience for the experimenter, the tunnel task was learned faster by the mice. In both tests, however, the response to the trained and ambiguous conditions resulted in a graded curve, the basic requirement for proving task validity. Thus, both the translational touchscreen task as well as the ecologically more relevant tunnel task could successfully be implemented and provide new tools for the future assessment of cognitive judgement biases in mice.

Spontaneous limb preferences exist in numerous species. To investigate the underlying mechanisms of these preferences, different methods, such as training, have been developed to shift preferences artificially. However, studies that systematically examine the effects of shifting preferences on behaviour and physiology are largely missing. Therefore, the aim of this study was to assess the impact of shifting paw preferences via training on spontaneous home cage behaviour, as well as anxiety-like behaviour and exploratory locomotion (Elevated plus maze test, Dark light test, Open field test, Free exploration test), learning performance (Labyrinth-maze) and stress hormones (fecal corticosterone metabolites) in laboratory mice (Mus musculus f. domestica). For this, we assessed spontaneous paw preferences of C57BL/6J females (N

Individuals differ in the way they perceive the world. From human psychological research, it is known that these differences become particularly evident in ambiguous situations: while some individuals interpret ambiguous information pessimistically, others bias their inter-pretations in a more optimistic way, referred to as cognitive judgement bias (CJB). CJBs have also been studied in non-human animals as tools for the assessment of affective states. However, the ecological and evolutionary relevance of CJB has so far been overlooked. We here aimed to transfer the concept of CJB to behavioural ecology. More specifically, we investigated the causes of differences in CJB in mice, focusing on both genetic and environmental factors. Furthermore, we assessed whether individual differences in CJB are repeatable over time, addressing the question whether “optimistic” and “pessimistic” decision styles, respectively, may represent stable traits. Thus, two strains of mice (C57BL/6J and B6D2F1N) were housed in two different environmental conditions: “scarce” or “complex”. While mice living in the “scarce environment” experienced standard housing conditions, those living in the “complex environment” had regular access to a super-enriched “playground”. To calculate the repeatability of “optimistic” and “pessimistic” decision styles, we assessed CJB four times across the course of seven weeks. Moreover, we assessed anxiety-like behaviour to detect potential differences in the effects of genetic or environmental factors on CJB and anxiety. While the selected genotypes and environments influenced some aspects of anxiety-like behaviour, no influence on CJB could be detected, indicating that CJB and anxiety might represent distinct systems. Remarkably, CJB was moderately repeatable, suggesting that decision-making under ambiguity constitutes a relatively stable trait and might even be considered an aspect of animal personality.

Courtship songs in mice have been investigated to understand the mechanisms and ecological relevance of vocal communication. There is evidence that courtship song characteristics vary between different genotypes, but little is known on whether individuals, even within the same genotype, differ from each other in the composition, complexity, and temporal consistency of their songs. In a first study, we aimed to systematically identify song features typical of different genotypes, by assessing the composition and complexity (i.e., entropy) of the syllabic sequences of male laboratory mice from four different strains (Mus musculus f. domestica: C57BL/6J, BALB/c, DBA/2 and B6D2F1). Mice were individually presented with a swab containing fresh female urine for 5 minutes to elicit courtship songs. The four strains differed not only in the composition but also in the complexity of their syllabic sequences. In a second study, we investigated within-strain individual differences in temporal consistency and recurring motifs (i.e., identical sets of syllables that are repeated within a song), using BALB/c and DBA/2 mice. The same procedure as in the first study was followed, but in addition testing was repeated weekly over three weeks. Both strains showed some level of individual temporal consistency; BALB/c in the overall amount of emitted vocalisations and DBA/2 in the expression of specific syllable types. However, hierarchical cluster analysis revealed remarkable individual variability in how consistent song characteristics were over time. Furthermore, recurring motifs were expressed at varying levels depending on the individual. Taken together, not only genotype but also individuality can affect variability in courtship songs in mice, suggesting the existence of different courtship strategies (e.g., higher song consistency to facilitate individual identification) related to varying levels of behavioural plasticity.HIGHLIGHTSCourtship songs in mice can serve as a model to study vocal communicationWe explore how genotype and individuality affect courtship songs’ characteristicsGenotypes differ in composition and also in complexity of syllabic sequencesWe find remarkable individual variability in how consistent songs are over timeResults suggest the existence of variation in male courting behaviour

The use of millions of mice in scientific studies worldwide emphasises the continuing need for a reduction of sample sizes, however, not at the expense of scientific validity. Split-plot designs have been suggested to enhance statistical power while allowing a reduction of animal numbers in comparison to traditional experimental designs. Recently, a promising approach of a split-plot design has been implemented and proven useful using mixed-strain housing of at least three different mouse strains. However, the impact of co-housing different strains of mice in one cage on animal welfare has still to be defined. This study aimed at comparing the effects of mixed-strain and same-strain housing of female C57BL/6J and DBA/2N mice on welfare and behaviour in two experimental phases. In a first phase, mice were housed in either mixed- or same-strain pairs. Home cage behaviour, activity rhythm, body weight, and faecal corticosterone metabolites were assessed. Furthermore, tests for anxiety-like and exploratory behaviour as well as spatial learning were performed. In a second phase, sociability was investigated in newly formed mixed-strain quartets. Mixed-strain housing did not induce alterations in anxiety, locomotion, learning, stereotypic behaviour, and stress hormone levels. However, changes in social behaviours and activity rhythm were observed. Increased agonistic and decreased socio-positive behaviours might point towards mild impacts on welfare in C57BL/6J mice under co-housing conditions. Altogether, scientific research may greatly benefit from co-housing mice of different strains within the same cages (e.g. for the realisation of a split-plot design), provided that strains are carefully selected for compatibility.

During early phases of life, an organism's phenotype can be shaped by the environmental conditions which it experiences. If the conditions change subsequently, the mismatch between the environment in early and later life could have negative effects on the individual's health and welfare. The aim of this study was to systematically test the predictions of this Match-Mismatch hypothesis in laboratory mice. Therefore, female C57BL/6 J mice were exposed to matching or mismatching combinations of low and high food availability in adolescence and early adulthood. A comprehensive analysis of various physiological and behavioral parameters was conducted. No indication of a mismatch effect was found, which might be attributed to the specific ecology of mice. Alternatively, food availability might cause a shaping of the phenotype only during the prenatal or early postnatal development. However, various effects of low vs high food availability were found regarding the individuals’ physiology and, to a small extent, their behavior. Low food availability caused higher concentrations of fecal corticosterone metabolites, as well as higher liver and lower spleen weights, suggesting an adaptation of the metabolism to this situation.

The accepted norm in most laboratories around the globe is feeding laboratory mice an ad libitum diet, although several health impairments are well-established. In contrast, reducing the animals' body weight by feeding them less food once per day (referred to as 24 h schedule) has been shown to enhance life span and reduce disease susceptibility. Against this background, this study aimed at systematically investigating the effects of different feeding routines. Therefore, three feeding routines were compared to the standard ad libitum feeding and effects on body weight development and welfare were investigated in male C57BL/6J mice. In particular, a 24 h schedule group, an AUTO group, characterized by an automated supply of small pieces of food all over the day, and a 4 h removal group, characterized by daily removal of food for 4 h, were studied. While the removal of food for 4 h per day did not lead to a reduction of body weight, and hence is unlikely to prevent negative effects of overfeeding, both the 24 h schedule group and the AUTO group led to the aspired body weight reduction. In the AUTO group, however, higher levels of corticosterone metabolites and stereotypies were observed, implying a rather negative impact on welfare. By contrast, no distinct negative effects of a 24 h schedule were found. Studies like this underline the general need for evidence-based severity assessments of any procedure involving living animals.

Phenotypic plasticity allows individuals to adjust traits to the environment. Whether long-term adjustments of the phenotype occur during later life stages is largely unknown. To address this question, we examined whether hormonal phenotypes that are shaped by the environment during adolescence can still be reshaped in full adulthood. For this, guinea pig males were either housed in mixed-sex colonies or in heterosexual pairs. In adulthood, males were individually transferred to pair housing with a female. This way, a social niche transition was induced in colony-housed males, but not in pair-housed males. Before transfer, corresponding to findings in adolescence, adult colony-housed males showed significantly higher baseline testosterone levels and lower cortisol responsiveness than pair-housed males. One month after transfer, the hormonal phenotype of colony-housed males was changed towards that of pair-housed males: animals showed comparable baseline testosterone levels and cortisol responsiveness was significantly increased in colony-housed males. This endocrine readjustment builds the basis for an adaptive behavioural tactic in the new social situation. Thus, an adaptive change of the behavioural phenotype may still occur in adulthood via modification of underlying mechanisms. This suggests a greater role for developmental plasticity in later life stages than is commonly presumed.

The start of actual breeding in male social mammals can occur long after individuals attain sexual maturity. Mainly prevented from reproduction by older and dominant males, young males often queue until strong enough to compete for favorable social positions and, in this way, to obtain access to females. However, to what extent maturing males also apply tactics to reproduce before this time is largely unknown. Therefore, the aim of the present study was to elucidate male socio-sexual development from onset of sexual maturity through first mating success until the achievement of a stable social position in a complex social environment. For this purpose, guinea pigs were used as a model system and reproductive success of males living in large mixed-sex colonies was assessed during their first year of life. As a reference, males in a mixed-sex pair situation were examined. Pair-housed males reproduced for the first time around the onset of sexual maturity whereas colony-housed males did so much later in life and with a considerably higher variance. In colonies, reproductive success was significantly affected by dominance status. Dominance itself was age-dependent, with older males having significantly higher dominance ranks than younger males. Surprisingly, both younger and older colony-housed males attained substantial reproductive success of comparable amounts. Thus, younger males reproduced irrespective of queuing and already before reaching a high social status. This mating success of maturing males was most likely achieved via several reproductive tactics which were flexibly applied with the onset of sexual maturity. The period of socio-sexual development before a stable social position is established may, therefore, be a time during which male mammals use flexible behavioral tactics to achieve reproductive success more frequently than commonly is presumed. In addition, the findings strongly indicate that high behavioral plasticity exists well beyond sexual maturity.

According to current guidelines on animal experiments, a prospective assessment of the severity of each procedure is mandatory. However, so far, the classification of procedures into different severity categories mainly relies on theoretic considerations, since it is not entirely clear which of the various procedures compromise the welfare of animals, or, to what extent. Against this background, a systematic empirical investigation of the impact of each procedure, including behavioral testing, seems essential. Therefore, the present study was designed to elucidate the effects of repeated versus single testing on mouse welfare, using one of the most commonly used paradigms for behavioral phenotyping in behavioral neuroscience, the open-field test. In an independent groups design, laboratory mice ( Mus musculus f. domestica) experienced either repeated, single, or no open-field testing – procedures that are assigned to different severity categories. Interestingly, testing experiences did not affect fecal corticosterone metabolites, body weights, elevated plus-maze or home cage behavior differentially. Thus, with respect to the assessed endocrinological, physical, and behavioral outcome measures, no signs of compromised welfare could be detected in mice that were tested in the open-field repeatedly, once, or, not at all. These findings challenge current classification guidelines and may, furthermore, stimulate systematic research on the severity of single procedures involving living animals.

The scientific literature is full of articles discussing poor reproducibility of findings from animal experiments as well as failures to translate results from preclinical animal studies to clinical trials in humans. Critics even go so far as to talk about a "reproducibility crisis" in the life sciences, a novel headword that increasingly finds its way into numerous high-impact journals. Viewed from a cynical perspective, Fett's law of the lab "Never replicate a successful experiment" has thus taken on a completely new meaning. So far, poor reproducibility and translational failures in animal experimentation have mostly been attributed to biased animal data, methodological pitfalls, current publication ethics and animal welfare constraints. More recently, the concept of standardization has also been identified as a potential source of these problems. By reducing within-experiment variation, rigorous standardization regimes limit the inference to the specific experimental conditions. In this way, however, individual phenotypic plasticity is largely neglected, resulting in statistically significant but possibly irrelevant findings that are not reproducible under slightly different conditions. By contrast, systematic heterogenization has been proposed as a concept to improve representativeness of study populations, contributing to improved external validity and hence improved reproducibility. While some first heterogenization studies are indeed very promising, it is still not clear how this approach can be transferred into practice in a logistically feasible and effective way. Thus, further research is needed to explore different heterogenization strategies as well as alternative routes toward better reproducibility in animal experimentation.

In humans, the short allele of a common polymorphism in the serotonin transporter (5-HTT) gene is associated with a higher risk to develop depression and anxiety disorders. Furthermore, individuals carrying this allele are characterized by negative judgment biases, as they tend to interpret ambiguous information in a more pessimistic way. 5-HTT knockout mice, lacking the 5-HTT gene either homo- or heterozygously, provide a widely used model organism for the study of symptoms related to human anxiety disorders. In the present study, we aimed to prove the anxiety-like phenotype of the 5-HTT mouse model, and to investigate whether 5-HTT genotype also causes differences in judgment bias. While our results confirm that homozygous 5-HTT knockout mice display highest levels of anxiety-like behavior, it was decreased in heterozygous mice. Against our expectations, we did not detect differences in the animals' judgment bias. These results indicate that at least in mice the association between 5-HTT genotype and judgment bias is not straightforward and that other factors, including multiple genes as well as environmental influences, are implicated in the modulation of judgment biases. More research is needed to gain further insights into their function as potential endophenotypes for psychopathology.

The ongoing debate on the reproducibility crisis in the life sciences highlights the need for a rethinking of current methodologies. Since the trend towards ever more standardised experiments is at risk of causing highly idiosyncratic results, an alternative approach has been suggested to improve the robustness of findings, particularly from animal experiments. This concept, referred to as “systematic heterogenisation”, postulates increased external validity and hence, improved reproducibility by introducing variation systematically into a single experiment. However, the implementation of this concept in practice requires the identification of suitable heterogenisation factors. Here we show that the time of day at which experiments are conducted has a significant impact on the reproducibility of behavioural differences between two mouse strains, C57BL/6J and DBA/2N. Specifically, we found remarkably varying strain effects on anxiety, exploration, and learning, depending on the testing time, i.e. morning, noon or afternoon. In a follow-up simulation approach, we demonstrate that the systematic inclusion of two different testing times significantly improved reproducibility between replicate experiments. Our results emphasise the potential of time as an effective and easy-to-handle heterogenisation factor for single-laboratory studies. Its systematic variation likely improves reproducibility of research findings and hence contributes to a fundamental issue of experimental design and conduct in laboratory animal science.

The neurotransmitter serotonin plays a key role in the control of aggressive behaviour. While so far most studies have investigated variation in serotonin levels, a recently created tryptophan hydroxylase 2 (Tph2) knockout mouse model allows studying effects of complete brain serotonin deficiency. First studies revealed increased aggressiveness in homozygous Tph2 knockout mice in the context of a resident-intruder paradigm. Focussing on females, this study aimed to elucidate effects of serotonin deficiency on aggressive and non-aggressive social behaviours not in a test situation but a natural setting. For this purpose, female Tph2 wildtype (n = 40) and homozygous knockout mice (n = 40) were housed with a same-sex conspecific of either the same or the other genotype in large terraria. The main findings were: knockout females displayed untypically high levels of aggressive behaviour even after several days of co-housing. Notably, in response to aggressive knockout partners, they showed increased levels of defensive behaviours. While most studies on aggression in rodents have focussed on males, this study suggests a significant involvement of serotonin also in the control of female aggression. Future research will show, whether the observed behavioural effects are directly caused by the lack of serotonin or by potential compensatory mechanisms.

The time of dominance rank acquisition is a crucial phase in male life history that often affects reproductive success and hence fitness. Hormones such as testosterone and glucocorticoids can influence as well as be affected by this process. At the same time, hormone concentrations can show large individual variation. The extent to which such variation is repeatable, particularly in dynamic social settings, is a question of current interest. The aim of the present study was therefore to investigate how dominance rank and individual differences contribute to variance in hormone concentrations during male rank acquisition in a complex social environment. For this purpose, dominance rank as well as baseline testosterone, baseline cortisol, and cortisol responsiveness after exposure to a novel environment were determined in colony-housed guinea pig males from late adolescence through adulthood. Hormone-dominance relationships and repeatability of hormone measures beyond their relation to rank were assessed. There was a significant positive relationship between baseline testosterone and rank, but this link became weaker with increasing age. Baseline cortisol or cortisol responsiveness, in contrast, were not significantly related to dominance. Notably, all three endocrine parameters were significantly repeatable independent of dominance rank from late adolescence through adulthood. Baseline testosterone and cortisol responsiveness showed a significantly higher repeatability than baseline cortisol. This suggests that testosterone titres and cortisol responsiveness represent stable individual attributes even under complex social conditions.

Automated touchscreen techniques find increasing application for the assessment of cognitive function in rodents. However, hardly anything is known about the potential impact of touchscreen-based training and testing procedures on the animals under investigation. Addressing this question appears particularly important in light of the long and intensive training phases required for most of the operant tasks. Against this background, we here investigated the influence of regular touchscreen training on hormones and behaviour of mice. Faecal corticosterone metabolites (FCMs), reflecting corticosterone levels around the time of treatment, were significantly increased in touchscreen-trained mice, even one week after the training phase was already terminated. Such an effect was not detected on baseline FCMs. Thus, regular touchscreen training can be assumed to cause long-term effects on hypothalamus-pituitary-adrenal axis activity. Furthermore, anxiety-like behaviour was increased in touchscreen-trained mice two weeks after the end of the training phase. Traditionally, this would be interpreted as a negative influence of the training procedure on the animals’ affective state. Yet, we also provide two alternative explanations, taking the possibility into account that touchscreen training might have enriching properties.

Based on the concept that experience-dependent memory may be biased by affective state at the time of learning, the affective bias test represents a novel approach for the assessment of emotions in animals. The task uses a within-subjects study design, where animals encounter two independent discrimination tasks on separate test days and learn that digging in one out of two substrates is rewarded with food. While one pair of substrates is associated with an affective manipulation, the other functions as a neutral control. Potential affective biases are then quantified by a preference test in which both initially rewarded substrates are tested against each other. Since the test was originally developed for the work with rats, the aims of the present study were (1) to translate the paradigm from rats to mice, and (2) to investigate the impact of an environmental enrichment on affective state in male C57BL/6J mice. For this reason, two control groups were included in the study that served to validate the overall testing procedure. One control group experienced equal absolute reward values for both rewarded substrates, assuming that this would prevent a substrate preference. In the second control group, we tripled the reward for one substrate, hypothesizing that mice would bias their choice towards this higher rewarded substrate in the preference test. Indeed, animals of the first control group did not show any substrate preference, while subjects of the second control group preferred the substrate associated with the high reward. Thus, mice were able to learn the association between absolute reward value and respective substrate, indicating that the affective bias test has the potential to work in mice. Besides these control groups, a third group was implemented in the study: As for the first control group, the absolute reward value was the same for both rewarded substrates, but in addition to that, affective state at the time of learning was modified for one substrate by subjecting mice to a highly enriched cage directly after testing. Since enrichment is known to influence the behaviour and physiology of rodents mostly in a positive way, we expected to observe a preference for the substrate associated with the enriched cage. However, in contrast to our expectations, mice did not show any substrate preference, suggesting that the additional enrichment provided in the enriched cage was probably not sufficient to induce measurable changes in an affective state.

Besides being recognized as a potential welfare indicator, play behaviour has long been considered to have immediate and/or long-term benefits. In particular, it has been suggested that in play animals learn to cope physically and emotionally with unexpected events. Given that the propensity to play varies greatly between conspecific individuals, such interindividual variability in playfulness may be associated with differences in the animals' future behaviour, a prediction that has rarely been tested. To investigate whether different levels of playfulness in juvenility indeed coincide with behavioural differences in later life, 30 female C57BL/6J mice were subjected to the following series of behavioural observations and tests: (1) quantification of juvenile play behaviour; (2) behavioural testing in paradigms that assess anxiety-like behaviour and exploratory locomotion in an unfamiliar environment; and (3) observation of spontaneous behaviour in the familiar home cage environment. Surprisingly, a high level of juvenile playfulness was predictive of high levels of state anxiety and low levels of exploratory locomotion in later life. While this relationship existed already in adolescence, it became even more prominent in adulthood. By contrast, no substantial differences between playful and less playful mice were found with respect to home cage behaviour. While these findings may reflect better coping abilities in novel and dangerous environments in those mice that played the most during juvenility, they may also argue for the existence of different types of mice. Thus, despite genetic homogeneity and identical housing environments, preferences for either local or global use of space were observed that indicate the emergence of individuality. Concerning animal welfare, our findings suggest that play may constitute a plausible welfare indicator at the population level, but is probably less meaningful for the individual.

Phenotypic plasticity allows individuals to adjust traits to the environment. Whether long-term adjustments of the phenotype occur during later life stages is largely unknown. To address this question, we examined whether hormonal phenotypes that are shaped by the environment during adolescence can still be reshaped in full adulthood. For this, guinea pig males were either housed in mixed-sex colonies or in heterosexual pairs. In adulthood, males were individually transferred to pair housing with a female. This way, a social niche transition was induced in colony-housed males, but not in pair-housed males. Before transfer, corresponding to findings in adolescence, adult colony-housed males showed significantly higher baseline testosterone levels and lower cortisol responsiveness than pair-housed males. One month after transfer, the hormonal phenotype of colony-housed males was changed towards that of pair-housed males: animals showed comparable baseline testosterone levels and cortisol responsiveness was significantly increased in colony-housed males. This endocrine readjustment builds the basis for an adaptive behavioural tactic in the new social situation. Thus, an adaptive change of the behavioural phenotype may still occur in adulthood via modification of underlying mechanisms. This suggests a greater role for developmental plasticity in later life stages than is commonly presumed.

Recently, a discussion about the reproducibility of results from behavioural phenotyping experiments has emerged. A huge emphasis has therefore been put on the identification of those factors that might limit the reproducibility of behavioural data. As a comprehensive phenotypic characterisation can involve testing of the same animal repeatedly over a specific time period, the aim of the present study was to systematically investigate effects of two potentially confounding variables, age of the animals and test experience. For this purpose, the behaviour of 48 male C57BL/6 J mice of two different ages (9 vs. 13 weeks) was assessed in a battery of common behavioural tests measuring anxiety-like and exploratory behaviour (Elevated Plus Maze, Dark-Light test, Open Field test, Novel Cage test). While half of the mice of each age group was naive to the test battery, the other half had experienced the same tests before. Besides main effects of both age and test experience on anxiety-like and exploratory behaviour, the analysis also revealed profound interactions between these factors. More precisely, an effect of age was apparent in experienced but not in naive mice. Furthermore, the effect of previous test experience was more pronounced in older than in younger mice. These findings clearly demonstrate that experimental factors, such as age and test experience, can influence behavioural data not just additively, but also in a complex, interactive way. To provide robust and reproducible results, it is thus fundamental to consider such factors systematically in the study design.

The scientific community is increasingly concerned with cases of published “discoveries” that are not replicated in further studies. The field of mouse behavioral phenotyping was one of the first to raise this concern, and to relate it to other complicated methodological issues: the complex interaction between genotype and environment; the definitions of behavioral constructs; and the use of the mouse as a model animal for human health and disease mechanisms. In January 2015, researchers from various disciplines including genetics, behavior genetics, neuroscience, ethology, statistics and bioinformatics gathered in Tel Aviv University to discuss these issues. The general consent presented here was that the issue is prevalent and of concern, and should be addressed at the statistical, methodological and policy levels, but is not so severe as to call into question the validity and the usefulness of model organisms as a whole. Well-organized community efforts, coupled with improved data and metadata sharing, were agreed by all to have a key role to play in identifying specific problems and promoting effective solutions. As replicability is related to validity and may also affect generalizability and translation of findings, the implications of the present discussion reach far beyond the issue of replicability of mouse phenotypes but may be highly relevant throughout biomedical research.

Social experiences can have profound effects on an individual's level of anxiety. While various studies have addressed consequences of experiences of a specific type, e.g., social defeat, a recent study in mice investigated the impact of combinations of adverse and beneficial social experiences. Quite surprisingly, mice exposed to benefits during early life phases followed by escapable adversity in adulthood displayed lowest levels of anxiety, even compared to individuals having experienced throughout beneficial conditions. The present study aimed to elucidate whether this phenomenon is restricted to these specific life phases or whether it also exists when all these experiences are made in full adulthood. For this purpose, we compared anxiety-like behavior and stress response of adult male mice exposed to escapable social defeat following beneficial social experiences to that of mice exposed to either throughout adverse or throughout beneficial conditions. More precisely, we performed three established behavioral paradigms measuring anxiety-like behavior and assessed corticosterone metabolites non-invasively via feces sampling. Interestingly, we found no effects of social experience on anxiety-like behavior. In contrast to that, the animals' stress hormone levels were profoundly affected by current social conditions: escapable social defeat (adverse condition) led to an increase in corticosterone metabolite concentrations, whereas living with a female (beneficial condition) led to a decrease. Thus, on the one hand this study suggests the importance of the timing of social experience for affecting an individual's level of anxiety. On the other hand, it demonstrates that anxiety and stress hormone levels can be affected separately by social experience during adulthood.

Over the past years, certain “vulnerability genes” have been identified that play a key role in the development of mood and anxiety disorders. In particular, a low-expressing variant of the human serotonin transporter (5-HTT) gene has been described that renders individuals more susceptible to adverse experience and hence to the development of psychiatric diseases. However, some authors have recently argued that lower 5-HTT expression not only increases vulnerability to adverse experiences, but also enhances susceptibility to beneficial experiences, thus promoting phenotypic plasticity. The aim of the present study was to assess the effects of 5-HTT expression on susceptibility to beneficial experience in a hypothesis-driven experimental approach. Using a well-established rodent model for the human polymorphism, male heterozygous 5-HTT knockout (HET) and 5-HTT wildtype (WT) mice were either provided with the beneficial experience of cohabitation with a female (mating experience) or kept as naive controls in single-housing conditions. Following the experimental treatment, they were tested for their anxiety-like behaviour and exploratory locomotion in three widely used behavioural tests. Interestingly, while cohabitation reduced anxiety-like behaviour and increased exploratory locomotion in the open field test in HET mice, it did not affect WT mice, pointing to a genotype-dependent susceptibility to the beneficial experience. Thus, our results might support the view of the low expressing version of the 5-HTT gene as a “plasticity” rather than a “vulnerability” variant.

Behavioural performance of mice - group comparison between the tests rounds