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Context-dependent biological variation presents a unique challenge to the reproducibility of results in experimental animal research, because organisms’ responses to experimental treatments can vary with both genotype and environmental conditions. In March 2019, experts in animal biology, experimental design and statistics convened in Blonay, Switzerland, to discuss strategies addressing this challenge. In contrast to the current gold standard of rigorous standardization in experimental animal research, we recommend the use of systematic heterogenization of study samples and conditions by actively incorporating biological variation into study design through diversifying study samples and conditions. Here we provide the scientific rationale for this approach in the hope that researchers, regulators, funders and editors can embrace this paradigm shift. We also present a road map towards better practices in view of improving the reproducibility of animal research.In this Perspective, Hanno Würbel and colleagues argue that a disregard for incorporating biological variation in study design is an important cause of poor reproducibility in animal research. They put the case for the use of systematic heterogenization of study samples and conditions in studies to improve reproducibility.

Single-laboratory studies conducted under highly standardized conditions are the gold standard in preclinical animal research. Using simulations based on 440 preclinical studies across 13 different interventions in animal models of stroke, myocardial infarction, and breast cancer, we compared the accuracy of effect size estimates between single-laboratory and multi-laboratory study designs. Single-laboratory studies generally failed to predict effect size accurately, and larger sample sizes rendered effect size estimates even less accurate. By contrast, multi-laboratory designs including as few as 2 to 4 laboratories increased coverage probability by up to 42 percentage points without a need for larger sample sizes. These findings demonstrate that within-study standardization is a major cause of poor reproducibility. More representative study samples are required to improve the external validity and reproducibility of preclinical animal research and to prevent wasting animals and resources for inconclusive research.

“We demand rigidly defined areas of doubt and uncertainty!” —D. Adams

Social organization is a key aspect of animal ecology, closely interlinked with all aspects of animal behaviour. The structure of animal assemblages is highly diverse, both within and between species. The complexity and variety of social systems and the dynamic nature of interactions and dependencies between members of social groups have long been major obstacles for developing operational characterizations of social organization. Here, social network analysis, a set of statistical tools rooted in graph theory, suggests itself as a potential solution for this problem, by offering quantitative measures for various aspects of social relationships. In this review I will first introduce network analysis as a tool to characterize the social organization of animal groups and population and, then, focus on the application of this method for epidemiological modelling, specifically the prediction of spreading patterns of pathogens in livestock and its potential for informing targeted surveillance and planning of intervention measures.

Poor reproducibility is considered a serious problem in laboratory animal research, with important scientific, economic, and ethical implications. One possible source of conflicting findings in laboratory animal research are environmental differences between animal facilities combined with rigorous environmental standardization within studies. Due to phenotypic plasticity, study-specific differences in environmental conditions during development can induce differences in the animals’ responsiveness to experimental treatments, thereby contributing to poor reproducibility of experimental results. Here, we studied how variation in weaning age (14–30 days) and housing conditions (single versus group housing) affects the phenotype of SWISS mice as measured by a range of behavioral and physiological outcome variables. Weaning age, housing conditions, and their interaction had little effect on the development of stereotypies, as well as on body weight, glucocorticoid metabolite concentrations, and behavior in the elevated plus-maze and open field test. These results are surprising and partly in conflict with previously published findings, especially with respect to the effects of early weaning. Our results thus question the external validity of previous findings and call for further research to identify the sources of variation between replicate studies and study designs that produce robust and reproducible experimental results.

Position and flap phasing between birds in formation flight indicate aerodynamic benefit. Phasing maximizes aerodynamics in bird formations Some bird species have refined their flying skills to the extent that they can fly in near-perfect V, but why they adopt such a tactic has been a matter of conjecture. One suggestion is that by flying in a V the birds are able to minimize energy costs, and now an analysis of data captured from free-flying migratory northern bald ibises suggests that there are energetic benefits. The data also reveal a sophisticated and dynamic process of in-flight control. Birds in the V phase their wing-beats to path-match, allowing a trailing bird to exploit the aerodynamic upwash from the bird in front. A bird flying directly behind, however, flaps with opposite phasing in order to minimize the detrimental downwash from the leader's wings. All this must require the birds to have developed a range of phasing strategies to cope with the dynamic wakes produced by flapping wings. Many species travel in highly organized groups 1 , 2 , 3 . The most quoted function of these configurations is to reduce energy expenditure and enhance locomotor performance of individuals in the assemblage 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 . The distinctive V formation of bird flocks has long intrigued researchers and continues to attract both scientific and popular attention 4 , 7 , 9 , 10 , 11 , 12 , 13 , 14 . The well-held belief is that such aggregations give an energetic benefit for those birds that are flying behind and to one side of another bird through using the regions of upwash generated by the wings of the preceding bird 4 , 7 , 9 , 10 , 11 , although a definitive account of the aerodynamic implications of these formations has remained elusive. Here we show that individuals of northern bald ibises ( Geronticus eremita ) flying in a V flock position themselves in aerodynamically optimum positions, in that they agree with theoretical aerodynamic predictions. Furthermore, we demonstrate that birds show wingtip path coherence when flying in V positions, flapping spatially in phase and thus enabling upwash capture to be maximized throughout the entire flap cycle. In contrast, when birds fly immediately behind another bird—in a streamwise position—there is no wingtip path coherence; the wing-beats are in spatial anti-phase. This could potentially reduce the adverse effects of downwash for the following bird. These aerodynamic accomplishments were previously not thought possible for birds because of the complex flight dynamics and sensory feedback that would be required to perform such a feat 12 , 14 . We conclude that the intricate mechanisms involved in V formation flight indicate awareness of the spatial wake structures of nearby flock-mates, and remarkable ability either to sense or predict it. We suggest that birds in V formation have phasing strategies to cope with the dynamic wakes produced by flapping wings.

Home cage aggression in group-housed male mice is a major welfare concern and may compromise animal research. Conventional cages prevent flight or retreat from sight, increasing the risk that agonistic encounters will result in injury. Moreover, depending on social rank, mice vary in their phenotype, and these effects seem highly variable and dependent on the social context. Interventions that reduce aggression, therefore, may reduce not only injuries and stress, but also variability between cage mates. Here we housed male mice (Balb/c and SWISS, group sizes of three and five) with or without partial cage dividers for two months. Mice were inspected for wounding weekly and home cages were recorded during housing and after 6h isolation housing, to assess aggression and assign individual social ranks. Fecal boli and fur were collected to quantify steroid levels. We found no evidence that the provision of cage dividers improves the welfare of group housed male mice; The prevalence of injuries and steroid levels was similar between the two housing conditions and aggression was reduced only in Balb/c strain. However, mice housed with cage dividers developed less despotic hierarchies and had more stable social ranks. We also found a relationship between hormone levels and social rank depending on housing type. Therefore, addition of cage dividers may play a role in stabilizing social ranks and modulating the activation of hypothalamic–pituitary–adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes, thus reducing phenotypic variability between mice of different ranks.

Understanding the consequences of losing individuals from wild populations is a current and pressing issue, yet how such loss influences the social behaviour of the remaining animals is largely unexplored. Through combining the automated tracking of winter flocks of over 500 wild great tits (Parus major) with removal experiments, we assessed how individuals' social network positions responded to the loss of their social associates. We found that the extent of flockmate loss that individuals experienced correlated positively with subsequent increases in the number of their social associations, the average strength of their bonds and their overall connectedness within the social network (defined as summed edge weights). Increased social connectivity was not driven by general disturbance or changes in foraging behaviour, but by modifications to fine-scale social network connections in response to losing their associates. Therefore, the reduction in social connectedness expected by individual loss may be mitigated by increases in social associations between remaining individuals. Given that these findings demonstrate rapid adjustment of social network associations in response to the loss of previous social ties, future research should examine the generality of the compensatory adjustment of social relations in ways that maintain the structure of social organization.

Reproducibility in biomedical research, and more specifically in preclinical animal research, has been seriously questioned. Several cases of spectacular failures to replicate findings published in the primary scientific literature have led to a perceived reproducibility crisis. Diverse threats to reproducibility have been proposed, including lack of scientific rigour, low statistical power, publication bias, analytical flexibility and fraud. An important aspect that is generally overlooked is the lack of external validity caused by rigorous standardization of both the animals and the environment. Here, we argue that a reaction norm approach to phenotypic variation, acknowledging gene-by-environment interactions, can help us seeing reproducibility of animal experiments in a new light. We illustrate how dominating environmental effects can affect inference and effect size estimates of studies and how elimination of dominant factors through standardization affects the nature of the expected phenotype variation through the reaction norms of small effect. Finally, we discuss the consequences of reaction norms of small effect for statistical analysis, specifically for random effect latent variable models and the random lab model.

Significance Cooperation in animals is an enigma because it contravenes the basic notion that evolution favors selfish genes that promote only their own well-being. Bird migration in organized V-shaped or echelon formations constitutes such a cooperation dilemma. We show that juvenile Northern bald ibis ( Geronticus eremita ) cooperate by taking turns and precisely matching times they spend in the advantageous trailing position and in the disadvantageous front position. This time matching is done on a pairwise level. Furthermore, we found evidence that the animals' propensity to reciprocate in leading has a substantial influence on the size and cohesion of the flight formations. This study shows that direct reciprocation can enable cooperation between animals in a natural context. One conspicuous feature of several larger bird species is their annual migration in V-shaped or echelon formation. When birds are flying in these formations, energy savings can be achieved by using the aerodynamic up-wash produced by the preceding bird. As the leading bird in a formation cannot profit from this up-wash, a social dilemma arises around the question of who is going to fly in front? To investigate how this dilemma is solved, we studied the flight behavior of a flock of juvenile Northern bald ibis ( Geronticus eremita ) during a human-guided autumn migration. We could show that the amount of time a bird is leading a formation is strongly correlated with the time it can itself profit from flying in the wake of another bird. On the dyadic level, birds match the time they spend in the wake of each other by frequent pairwise switches of the leading position. Taken together, these results suggest that bald ibis cooperate by directly taking turns in leading a formation. On the proximate level, we propose that it is mainly the high number of iterations and the immediacy of reciprocation opportunities that favor direct reciprocation. Finally, we found evidence that the animals' propensity to reciprocate in leading has a substantial influence on the size and cohesion of the flight formations.