Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques

Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17β-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine

Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice—Assessing the degree of distress

According to the EU Directive 2010/63, the severity of a procedure has to be classified as mild, moderate or severe. General anesthesia is thought to be mild, but the Directive does not differentiate between single and repeated anesthesia. Therefore, we investigated the impact of repeated administration of isoflurane, the most commonly used inhalation anesthetic, on the well-being of adult C57BL/6JRj mice, in comparison to single administrations and to untreated animals, when applied six times for 45 min at an interval of 3–4 days. For the animals anesthetized, excitations, phases of anesthesia, and vital parameters were monitored. Well-being after anesthesia was assessed using a behavioral test battery including luxury behavior like burrowing and nest building behavior, the Mouse Grimace Scale (MGS), the free exploratory paradigm for anxiety-related behavior, home cage activity and the rotarod test for activity, as well as food intake and body weight. Additionally, hair corticosterone and fecal corticosterone metabolites were measured. Our results show that nest building behavior, home cage activity, body weight, and corticosterone concentrations were not influenced by anesthesia, whereas changes in burrowing behavior, the MGS, food intake, and the free exploratory behavior indicated that the well-being of the mice was more affected by repeated than single isoflurane anesthesia. This effect depended on the sex of the animals, with female mice being more susceptible than male mice. However, repeated isoflurane anesthesia caused only short-term mild distress and impairment of well-being, mainly in the immediate postanesthetic period. Well- being stabilized at 8 days after the last anesthesia, at the latest. Therefore, we conclude that when using our anesthesia protocol, the severity of both single and repeated isoflurane anesthesia in C57BL/6JRj mice can be classified as mild. However, within the mild severity category, repeated isoflurane anesthesia ranks higher than single isoflurane anesthesia. Additionally, our results imply that male and female mice can differently perceive the severity of a procedure

The implementation of tunnel handling in a mouse breeding facility revealed strain-specific behavioural responses

As a step towards implementing non-aversive handling techniques at a big mouse breeding facility in Germany, tunnel handling was introduced in a breeding unit comprising three inbred mouse strains. To assess whether tunnel handling would be feasible for the animal technicians in their everyday work and beneficial for the mice when being handled during weekly cage change only, the behaviour of tunnel- and tail-handled animals of both sexes was examined before, during and after the handling events over a period of nine weeks. Moreover, the time expenditure was compared between both handling techniques. It was possible to use the tunnel in all three mouse strains. However, the impact of the handling techniques on the behavioural parameters investigated in the present study were strain-specific. All behavioural parameters suggested that NZW mice benefited the most from tunnel handling. The results obtained from Hello Kitty and WNK mice were ambiguous, which may suggest that a brief handling session during the cage clean may have not been sufficient to habituate them to the process of handling. It took the animal technicians approximately 3 seconds longer per mouse when using a tunnel. The strain-specific results should encourage researchers to share their experiences with non-aversive handling techniques in different mouse strains, for example, along with their research articles

Towards a fully automated surveillance of well-being status in laboratory mice using deep learning: Starting with facial expression analysis

Assessing the well-being of an animal is hindered by the limitations of efficient communication between humans and animals. Instead of direct communication, a variety of parameters are employed to evaluate the well-being of an animal. Especially in the field of biomedical research, scientifically sound tools to assess pain, suffering, and distress for experimental animals are highly demanded due to ethical and legal reasons. For mice, the most commonly used laboratory animals, a valuable tool is the Mouse Grimace Scale (MGS), a coding system for facial expressions of pain in mice. We aim to develop a fully automated system for the surveillance of post-surgical and post-anesthetic effects in mice. Our work introduces a semi-automated pipeline as a first step towards this goal. A new data set of images of black-furred laboratory mice that were moving freely is used and provided. Images were obtained after anesthesia (with isoflurane or ketamine/xylazine combination) and surgery (castration). We deploy two pre-trained state of the art deep convolutional neural network (CNN) architectures (ResNet50 and InceptionV3) and compare to a third CNN architecture without pre-training. Depending on the particular treatment, we achieve an accuracy of up to 99% for the recognition of the absence or presence of post-surgical and/or post-anesthetic effects on the facial expression

Anatomical Evaluation of Rat and Mouse Simulators for Laboratory Animal Science Courses

According to the European Directive 63/2010/EU, education and training involving living rats and mice are classified as an animal experiment and demands the implementation of the 3Rs. Therefore, as a method of refinement, rat and mouse simulators were developed to serve as an initial training device for various techniques, prior to working on living animals. Nevertheless, little is known about the implementation, anatomical correctness, learning efficiency and practical suitability of these simulators. With this in mind, a collaborative research project called “SimulRATor” was initiated to systematically evaluate the existing rat and mouse simulators in a multi-perspective approach. The objective of the study presented here was to identify the anatomical strengths and weaknesses of the available rat and mouse simulators and to determine anatomical requirements for a new anatomically correct rat simulator, specifically adapted to the needs of Laboratory Animal Science (LAS) training courses. Consequently, experts of Veterinary Anatomy and LAS evaluated the anatomy of all currently available rat and mouse simulators. The evaluation showed that compared to the anatomy of living rats and mice, the tails were perceived as the most anatomically realistic body part, followed by the general exterior and the limbs. The heads were rated as the least favored body part

Challenges and advanced concepts for the assessment of learning and memory function in mice

The mechanisms underlying the formation and retrieval of memories are still an active area of research and discussion. Manifold models have been proposed and refined over the years, with most assuming a dichotomy between memory processes involving non-conscious and conscious mechanisms. Despite our incomplete understanding of the underlying mechanisms, tests of memory and learning count among the most performed behavioral experiments. Here, we will discuss available protocols for testing learning and memory using the example of the most prevalent animal species in research, the laboratory mouse. A wide range of protocols has been developed in mice to test, e.g., object recognition, spatial learning, procedural memory, sequential problem solving, operant- and fear conditioning, and social recognition. Those assays are carried out with individual subjects in apparatuses such as arenas and mazes, which allow for a high degree of standardization across laboratories and straightforward data interpretation but are not without caveats and limitations. In animal research, there is growing concern about the translatability of study results and animal welfare, leading to novel approaches beyond established protocols. Here, we present some of the more recent developments and more advanced concepts in learning and memory testing, such as multi-step sequential lockboxes, assays involving groups of animals, as well as home cage-based assays supported by automated tracking solutions; and weight their potential and limitations against those of established paradigms. Shifting the focus of learning tests from the classical experimental chamber to settings which are more natural for rodents comes with a new set of challenges for behavioral researchers, but also offers the opportunity to understand memory formation and retrieval in a more conclusive way than has been attainable with conventional test protocols. We predict and embrace an increase in studies relying on methods involving a higher degree of automatization, more naturalistic- and home cage-based experimental setting as well as more integrated learning tasks in the future. We are confident these trends are suited to alleviate the burden on animal subjects and improve study designs in memory research

Alternatives in Education - Evaluation of Rat Simulators in Laboratory Animal Training Courses from Participants' Perspective

In laboratory animal science (LAS) education and training, five simulators are available for exercises on handling and routine procedures on the rat, which is—beside mice—the most commonly used species in LAS. Since these simulators may have high potential in protecting laboratory rats, the aim of this study is to investigate the simulators’ impact on the 3R (replace, reduce, refine) principle in LAS education and training. Therefore, the simulators were evaluated by 332 course participants in 27 different LAS courses via a practical simulator training workshop and a paper-based two-part questionnaire—both integrated in the official LAS course schedule. The results showed a high positive resonance for simulator training and it was considered especially useful for the inexperienced. However, the current simulators may not completely replace exercises on live animals and improvements regarding more realistic simulators are demanded. In accordance with literature data on simulator-use also in other fields of education, more research on simulators and new developments are needed, particularly with the aim for a broad implementation in LAS education and training benefiting all 3Rs

Alternatives in Education—Rat and Mouse Simulators Evaluated from Course Trainers’ and Supervisors’ Perspective

Simulators for training in laboratory animal science bear great potential to overcome the dilemma between the present demand for high-quality practical training involving live animals whilst implementing the “3R principle” (Replace, Reduce, Refine) according to the Directive 2010/63/EU. Currently, one mouse and six rat simulators are available, but only few data on them exist. To advance simulator-based training, an online survey for course trainers and supervisors of laboratory animal training courses focusing mice and rats was conducted, as these groups are most aware of its implementation due to applying alternative education and training methods regularly. This study reflects the current awareness, implementation, and satisfaction concerning methodical and practical criteria of the simulators including the requirements for a new development of 35 course trainers and supervisors who completed a German online survey conducted between May 2018 and June 2019. Although the study revealed a high awareness of existing simulators, their implementation is rather low, perhaps due to them not meeting certain demands. Generally, an approval of simulator-based training and a demand for user-optimized, realistic, financially affordable, and robust rat and mouse simulators were indicated, which may strongly benefit the 3Rs and animals in all experimental areas.Peer Reviewe