Happy animals, satisfied researchers

According to Russell and Burch, the founders of the 3Rs in animal experimentation, refinement begins at the point where there are no options to replace the use of animals with other methods or to further reduce their numbers. Refinement is defined as maximizing the welfare and minimizing the stress of laboratory animals. Several options exist to achieve this goal. Enrichment in the form of social contact in social animal species or employment material has a positive impact on animal welfare and thus on the outcome of the research. Animal-friendly handling, the use of noninvasive techniques, an effective and reliable anesthetic protocol, analgesia adapted to painfulness, and humane endpoints in animal studies are other types of refinement.¹

Implementing as many measures of refinement as possible is advocated for many reasons. Animal-friendly handling, for example, helps to reduce anxiety and stress in laboratory animals, as well as in all persons working with them. It is known that this also has a positive effect on research, as it promotes its quality and reproducibility. The existing legislation, the EU Directive 2010/63/EU, also stipulates that the method to be selected must be the one "likely to give satisfactory results and to cause the least pain, suffering or distress". According to the law, "procedures must be carried out under general or local anesthesia," and "analgesia or another appropriate method" must be used to ensure that "pain, suffering and distress are minimized." Refinement in animal studies is thus regulated by a legal standard.²

In a video posted on the NC3Rs website (https://www.nc3rs.org.uk/handling-and-training-mice-and-rats-low-stress-procedures), Therése Ahlström, an experienced technician at RISE (Research Institutes of Sweden), describes a procedure tested by her institutes for habituating laboratory animals to humans. Ms. Ahlström advises the implementation of a multi-part training program, the goal of which is to gain the animals' trust and at the same time learn to better assess them and to be able to interpret their signals. When dealing with laboratory rodents, the wearing of scents such as perfumes, noise or loud sounds, hectic movements and measures of "restraining" should always be avoided. All these things could otherwise lead to stress of the laboratory rodents, which is recognizable by a rigid body or tail, the increased depositing of urine and feces, putting on the ears, squinting the eyes, as well as vocalization and trembling. In addition, stressed animals would show defensive behavior in the form of the 5Fs. The 5Fs stand for flight, freeze, fight, faint, and fiddle about.³

Training would be beneficial from many perspectives. By habituating the animals to humans, stress and the resulting risk of injury would be reduced. Furthermore, a significant amount of time would be saved if there is less, or at best no, aversive behavior. Socialization can begin as early as three to four week old animals at the breeder's facility, or if that is not possible, at least from the time of arrival at the new facility. Even the first encounter with laboratory animals is essential and formative, he said. The actual training can take place within the acclimation period and consist of five sessions of one to two minutes each. The mice should not be lifted out of the cage by the tail during this process. Instead, objects from the cage or a tunnel can be used as an aid. Starting with the second training session, the animals can be gradually habituated to the planned actions such as various blood collection techniques or other activities while avoiding known restraining methods and to the environment. The video demonstrates the performance of various actions. Each training session should be concluded with a reward in the form of treats or, in the case of rats, petting. All training sessions should be documented to illustrate the learning process.³

In their article entitled "Impact of Refinements to Handling and Restraint Methods in Mice," Davies et al. look at the effects of different handling methods on experimental animals using a variety of measurable parameters. The use of the traditional tail restraint method is compared to the "cupping" and tunneling method. Cupping refers to picking up the mouse by hand for removal from the cage. In the tunneling method, a small tunnel is used as an aid. The alternatives of handling by means of tail fixation simplify the handling of the animals and thus lead to a reduction of anxiety and stress during the experiments but also in the course of routine activities such as transferring. The removal and fixation of the mouse by means of tail fixation could lead to the triggering of aggressive behavior. It is assumed that this type of fixation could trigger feelings that would arise during capture by a predator or a fight with a conspecific. In their study, they were able to prove that alternative and more animal-friendly methods cause less aversive and stressed behavior and thus simplify all handling of the animals.⁴

1) W.M.S. Russell and R.L. Burch, “The Principles of Humane Experimental Technique” (1992) ISBN: 0-900767-78-2. Published by UFAW, Wheathampstead, UK. 238 pages. Special Edition. Animal Welfare Institute- https://awionline.org/content/the-3rs (Zugriff 14.04.2023)
2) Hurst, J., West, R. Taming anxiety in laboratory mice. Nat Methods 7, 825–826 (2010). https://doi.org/10.1038/nmeth.1500 RL 2010/63/EU
3) NC3Rs Video: https://www.nc3rs.org.uk/handling-and-training-mice-and-rats-low-stress-procedures (Zugriff am 14.04.2023); Bengtsson, C., Eriksson, M. 2020. Handling and training of mice and rats results in calmer animals during experimental procedures. AWI Quarterly 69(2) Summer, 6-8.
4) Davies, J.R.; Purawijaya, D.A.; Bartlett, J.M.; Robinson, E.S.J. Impact of Refinements to Handling and Restraint Methods in Mice. Animals 2022, 12, 2173. https://doi.org/10.3390/ani12172173; Hurst J.L., West R.S. Taming anxiety in laboratory mice. Nat. Methods. 2010;7:825–826. doi: 10.1038/nmeth.1500; Gouveia K., Hurst J.L. Improving the practicality of using non-aversive handling methods to reduce background stress and anxiety in laboratory mice. Sci. Rep. 2019;9:20305. doi: 10.1038/s41598-019-56860-7; Litvin Y., Blanchard D.C., Pentkowski N.S., Blanchard R.J. A pinch or a lesion: A reconceptualization of biting consequences in mice. Aggress. Behav. 2007;33:545–551. doi: 10.1002/ab.20222.