Refining experiments and housing conditions so as to cause the minimum possible pain and distress is one of the three principles (3Rs) on which Directive 2010/63/EU is based. In this systematic review, we aimed to identify and summarize published advances in the refinement protocols made by European Union-based research groups from 2011 to 2021, and to determine whether or not said research was supported by European or national grants. We included 48 articles, the majority of which were related to improvements in experimental procedures (37/77.1%) for mice (26/54.2%) and were written by research groups belonging to universities (36/57.1%) and from the United Kingdom (21/33.9%). More than two thirds (35/72.9%) of the studies received financial support, 26 (mostly British) at a national level and 8 at a European level. Our results indicated a clear willingness among the scientific community to improve the welfare of laboratory animals, as although funding was not always available or was not specifically granted for this purpose, studies were published nonetheless. However, in addition to institutional support based on legislation, more financial support is needed. We believe that more progress would have been made in refinement during these years if there had been more specific financial support available at both the national and European Union levels since our data suggest that countries investing in refinement have the greatest productivity in successfully publishing refinements.

In the last years, different research groups have made considerable efforts to improve the care and use of animals in research. Mice (Mus musculus) are the most widely used animal species in research in the European Union and are sociable and hierarchical creatures. During experiments, researchers tend to individualize males, but no consideration is given to whether this social isolation causes them stress. The aim of this study was, therefore, to explore whether 4 weeks of social isolation could induce changes in different physiological parameters in adult Crl:CD1(ICR) (CD1) males, which may interfere with experimental results. Body weight, blood cells, and fecal corticosterone metabolites levels were the analyzed parameters. Blood and fecal samples were collected at weeks 1 and 4 of the experimental procedure. Four weeks of single housing produced a significant time-dependent decrease in monocytes and granulocytes. Fecal corticosterone metabolite levels were higher in single-housed mice after 1 week and then normalized after 4 weeks of isolation. Body weight, red blood cells, and platelets remained unchanged in both groups during this period. We can, therefore, conclude that social isolation affects some immune and endocrine parameters, and that this should be taken into account in the interpretation of research data.

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