Evaluating rearing behaviour as a model-specific pain indicator in mouse osteotomy models.

To assess pain in mouse models of bone fractures, currently applied assessment batteries use combinations of clinical signs with spontaneous behaviours and model-specific behaviours, including walking and weight-bearing behaviour. Rearing behaviour - an upright position on the hindlimbs - has a motivational and an ambulatory component. Thus, rearing behaviour might have the potential to be an indicator for model-specific pain in mouse fracture models. To date, the assessment of rearing behaviour in bone fracture models using mice is only scarcely described. In this study, we aimed to determine whether the duration of rearing behaviour is affected by osteotomy of the femur in male and female C57BL/6N mice with external fixation (rigid vs. flexible) and could be an additional sign for model-specific pain, such as the presence of limping. Rearing duration was significantly decreased after osteotomy in male and female mice at 24 h, 48 h and 72 h, but was not affected by anaesthesia/analgesia alone. In male mice, the relative rearing duration increased over 72 h (both fixations) and at 10 days in the rigid fixation group but remained significantly lower in the flexible fixation group. In contrast, in female mice, no increase in rearing duration was observed within 72 h and at 10 days post-osteotomy, independent of the fixation. We did not identify any association between relative rearing time and presence or absence of limping. In summary, our results do not provide sufficient evidence that altered rearing behaviour might be an indicative sign for pain in this model.

Rumination Detection in Sheep: A Systematic Review of Sensor-Based Approaches.

The use of sensors to analyze behavior in sheep has gained increasing attention in scientific research. This systematic review aims to provide an overview of the sensors developed and used to detect rumination behavior in sheep in scientific research. Moreover, this overview provides details of the sensors that are currently commercially available and describes their suitability for sheep based on the information provided in the literature found. Furthermore, this overview lists the best sensor performances in terms of achieved accuracy, sensitivity, precision, and specificity in rumination detection, detailing, when applicable, the sensor position and epoch settings that were used to achieve the best results. Challenges and areas for future research and development are also identified. A search strategy was implemented in the databases PubMed, Web of Science, and Livivo, yielding a total of 935 articles. After reviewing the summaries of 57 articles remaining following filtration (exclusion) of repeated and unsuitable articles, 17 articles fully met the pre-established criteria (peer-reviewed; published between 2012 and 2023 in English or German; with a particular focus on sensors detecting rumination in sheep) and were included in this review. The guidelines outlined in the PRISMA 2020 methodology were followed. The results indicate that sensor-based systems have been utilized to monitor and analyze rumination behavior, among other behaviors. Notably, none of the sensors identified in this review were specifically designed for sheep. In order to meet the specific needs of sheep, a customized sensor solution is necessary. Additionally, further investigation of the optimal sensor position and epoch settings is necessary. Implications: The utilization of such sensors has significant implications for improving sheep welfare and enhancing our knowledge of their behavior in various contexts.

Opportunities to Improve Animal Welfare during Transport and Slaughter of Cattle and Pigs through Staff Training-Results of a Delphi Survey.

To improve animal welfare for cattle and pigs during transport and at slaughter, online training modules for all staff including employees in the lairage pen, the slaughter line as well as animal welfare officers are developed at Freie Universität Berlin, Germany. Before starting the development of these modules, an expert elicitation survey using a modified Delphi approach was performed to identify action points considered most relevant for animal welfare during transport and slaughter, and as having the potential for improvement through training. In total, 49 participating experts rated predetermined action points of each step in the transport and slaughter process in two survey rounds. The rating scale included numbers 0 (='not relevant', respectively 'no possibility of improvement') to 10 (='very relevant', respectively 'very high possibility of improvement'). None of the action points were rated with a median score of less than 5. Assessment of fitness for transport, unloading at the abattoir, handling at stunning and exsanguination were amongst the highest rated action points, and were therefore selected to develop online training modules. The Delphi approach was seen as a valuable method to include external expertise to select the most relevant action points for the development of online training modules.

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.

A systematic review of the development and application of home cage monitoring in laboratory mice and rats.

BACKGROUND: Traditionally, in biomedical animal research, laboratory rodents are individually examined in test apparatuses outside of their home cages at selected time points. However, the outcome of such tests can be influenced by various factors and valuable information may be missed when the animals are only monitored for short periods. These issues can be overcome by longitudinally monitoring mice and rats in their home cages. To shed light on the development of home cage monitoring (HCM) and the current state-of-the-art, a systematic review was carried out on 521 publications retrieved through PubMed and Web of Science. RESULTS: Both the absolute (~ × 26) and relative (~ × 7) number of HCM-related publications increased from 1974 to 2020. There was a clear bias towards males and individually housed animals, but during the past decade (2011-2020), an increasing number of studies used both sexes and group housing. In most studies, animals were kept for short (up to 4 weeks) time periods in the HCM systems; intermediate time periods (4-12 weeks) increased in frequency in the years between 2011 and 2020. Before the 2000s, HCM techniques were predominantly applied for less than 12 h, while 24-h measurements have been more frequent since the 2000s. The systematic review demonstrated that manual monitoring is decreasing in relation to automatic techniques but still relevant. Until (and including) the 1990s, most techniques were applied manually but have been progressively replaced by automation since the 2000s. Independent of the year of publication, the main behavioral parameters measured were locomotor activity, feeding, and social behaviors; the main physiological parameters were heart rate and electrocardiography. External appearance-related parameters were rarely examined in the home cages. Due to technological progress and application of artificial intelligence, more refined and detailed behavioral parameters have been investigated in the home cage more recently. CONCLUSIONS: Over the period covered in this study, techniques for HCM of mice and rats have improved considerably. This development is ongoing and further progress as well as validation of HCM systems will extend the applications to allow for continuous, longitudinal, non-invasive monitoring of an increasing range of parameters in group-housed small rodents in their home cages.

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.

A buprenorphine depot formulation provides effective sustained post-surgical analgesia for 72 h in mouse femoral fracture models.

Adequate pain management is essential for ethical and scientific reasons in animal experiments and should completely cover the period of expected pain without the need for frequent re-application. However, current depot formulations of Buprenorphine are only available in the USA and have limited duration of action. Recently, a new microparticulate Buprenorphine formulation (BUP-Depot) for sustained release has been developed as a potential future alternative to standard formulations available in Europe. Pharmacokinetics indicate a possible effectiveness for about 72 h. Here, we investigated whether the administration of the BUP-Depot ensures continuous and sufficient analgesia in two mouse fracture models (femoral osteotomy) and could, therefore, serve as a potent alternative to the application of Tramadol via the drinking water. Both protocols were examined for analgesic effectiveness, side effects on experimental readout, and effects on fracture healing outcomes in male and female C57BL/6N mice. The BUP-Depot provided effective analgesia for 72 h, comparable to the effectiveness of Tramadol in the drinking water. Fracture healing outcome was not different between analgesic regimes. The availability of a Buprenorphine depot formulation for rodents in Europe would be a beneficial addition for extended pain relief in mice, thereby increasing animal welfare.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Impact of Veterinary Herd Health Management on German Dairy Farms: Effect of Participation on Farm Performance.

German dairy farming has intensified markedly in recent years, and the demand for Veterinary Herd Health Management (VHHM) is rising. To protect farms from epidemics, ensure food safety, and prevent developing of antibiotic resistance, VHHM has been anchored in EU law since April 2021. Via an online survey, distributed by different farmers' organizations, dairy farmers were asked about the cooperation with their veterinarian. The aim was to evaluate farm performance as a function of participation in VHHM. From 216 analyzed questionnaires, 106 respondents participated in VHHM. Results showed that farmers who make use of VHHM and consult their veterinarian in decision-making frequently have the highest 305-day milk yield (305dMY), the lowest bulk tank somatic cell counts, and the lowest age at first calving (AFC). However, these farmers tended to have higher replacement rates and a higher mortality of cows in the period up to 60 days in milk (MORT60DIM). Furthermore, respondents who defined VHHM as "evaluation of herd data, strategic planning" had the highest 305dMY compared with those who defined VHHM through one of the different options given ("pregnancy checks and support in reproduction"/"problem solving"). In the multifactorial regression model, VHHM participating farms had a 660-kg higher 305dMY and 1 month less in AFC, compared with farms not participating in VHHM. However, within the VHHM participants, no association between VHHM practices and performance parameters was found. Further research is needed, to find out if tailored advice of the VHHM approach may show effect herein.

Creating Veterinary Public Health Online Cases by Students for Students.

Online-based processing of case reports is often used and well accepted in veterinary medical education. However, lecturers usually develop cases from their own point of view, without input from students. In order to give students the chance to create online cases for students, an elective course Creative Workshop Case Creation, was held three times between 2017 and 2019 at the Faculty of Veterinary Medicine, Freie Universität Berlin. During this course, students created cases based on animal welfare and epizootics issues through a problem-based blended learning approach. In this approach, students worked on an assigned veterinary public health problem and actively solved it in small groups in class and then used the issue as the basis to create cases for their fellow students. The cases were implemented in interdisciplinary lectures, which are mandatory for every student in semesters six to eight. After taking these classes, fellow students evaluated one of these cases, specifically, on animal welfare and another one on epizootics. Evaluations showed these cases were received well. Moreover, we received excellent feedback from students participating in the elective course, and working with a proactive and motivated group of six students throughout the course was a very productive experience. The course made it possible to create cases that are more accurately tailored to the needs of students. The students' good ideas and preparatory work also saved time in the preparation of cases for lecturers.

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.

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.

Comparison of Two Different Feather Sampling Methods to Measure Corticosterone in Wild Greater Flamingos (Phoenicopterus roseus) and Wild Mallards (Anas platyrhynchos).

This research project had the aim to validate the possible alternative and less-painful sampling method of cutting feathers close to the skin instead of plucking them for subsequent feather corticosterone analysis, confirming recently-published results for other species in captivity. Analyzing CORTf is often used in animal welfare studies in combination with behavioral monitoring. The background of this idea was to act in the sense of animal welfare and reduce the burden of animal studies according to the 3-R-Principle (Replacement, Reduction, and Refinement) by refining procedures. To confirm the hypothesis that the sampling method itself has no influence on CORTf levels measured, plucked and cut samples of the respective bird were collected. Birds of two wild species were used: the Mallard (Anas platyrhynchos) and the Greater Flamingo (Phoenicopterus roseus). The CORTf was measured by using an enzyme-linked immunosorbent assay (ELISA). The determined values were inspected for their mean values, standard deviation (SD), and average differences. Afterwards, the CORTf levels of both species were compared, according to the sampling method, with the concordance correlation coefficient (CCC). In the Bland-Altman (BA) plot the differences of the methods were displayed against the mean values. Additionally, sex, as a possible factor influencing CORTf, was analyzed using the Mann-Whitney U test. The values of CCC showed poor agreement in the comparability of the two methods, whereas the concordance of the BA plot was decent. The average differences between the methods were marginal for both species (Mallards: -0.16 pg/mm, Flamingos -0.13 pg/mm). In summary, all anomalies or differences between the methods were negligible. Therefore, the alternative sampling method seems to be as suitable as the common standard method. No significant difference was found between females and males. Nevertheless, our results suggest that CORTf should not be interpreted in just considering the values themselves, but the results they should be analyzed in the context of a wider set of parameters. Hence, further studies are encouraged to create a larger data pool.

Feather Corticosterone Measurements and Behavioral Observations in the Great White Pelican (Pelecanus onocrotalus) Living under Different Flight Restraint Conditions in German Zoos.

The pinioning of birds was previously one of the most-accepted forms of mutilation in zoos. Despite a lack of knowledge on the effects of deflighting procedures with regard to the well-being of deflighted birds, pelicans are often reversibly deflighted by feather-clipping to keep them in open enclosures, including those with ponds without netting. In the present study, we focused on the welfare implications of flight restraint on one of the most commonly kept types of birds in German zoos, the great white pelican. A combination of behavioral observations and feather corticosterone concentrations (CORTf) of pelicans with different deflighting statuses (i.e., irreversibly deflighted, reversibly deflighted, and airworthy) was used to evaluate the effects of deflighting status on pelican welfare. We observed 215 individuals in 21 different German zoos. The pelicans lived in differently designed exhibits. An ethogram for these species was developed and their behavior was evaluated by scan sampling. Feather samples from 182 individuals were collected to determine if different deflighting conditions influenced the CORTf and therefore stress levels. The hypothesis was that the CORTf values of airworthy pelicans differ from those of deflighted pelicans. Tendencies with regard to the flight status groups were found. Conversely, reversibly deflighted pelicans had higher CORTf levels than irreversible deflighted and airworthy pelicans. Tendencies with regard to CORTf values and the group size of the kept pelicans were observed. The CORTf values were lower in groups consisting of more than 10 animals. In addition, the frequency of fluttering behavior was positively associated with CORTf values. Pelicans that frequently showed fluttering had higher CORTf values. Therefore, fluttering behavior might be considered a sign of stress levels in pelicans. This study is one of the first important steps in assessing the impact of deflighting procedures on the welfare of great white pelicans kept in zoos.

Evaluating Horse Owner Expertise and Professional Use of Auxiliary Reins during Horse Riding.

Auxiliary reins are commonly used for the training of riders and horses as well as in daily training. They are often criticized when used incorrectly, as they will not help and can harm the horse by causing overwork, accidents, and injuries, which harm the horse in the long term. They also often conceal causal rider problems while trying to achieve quick success. The aim of this paper was to investigate, with an online horse-owner questionnaire, which and how often auxiliary reins were used and whether they were used appropriately. Only participants who were currently using auxiliary reins were selected. Consequently, 823 participants were questioned, of which 362 were currently using auxiliary reins at least every two weeks. Auxiliary reins were mainly used according to their discipline: the running side rein was the most popular when working from the ground and the sliding ring martingale was the most popular for ridden equestrian activities. Most of the test subjects only attached the auxiliary reins after the warm-up phase, but half of the participants did not change them during the entire training session. Most participants (75%) could at least identify what the correct head position of the horse should look like. However, there were still too many (50%) who adjusted their horse too tightly and did not change anything at that time despite the related breathing problems. The study found that most participants used the reins responsibly, but there is still a need for clarification and information relating to the functions of the different auxiliary reins among horse owners.

Breeding and Maintenance of Immunodeficient Mouse Lines under SPF Conditions-A Call for Individualized Severity Analyses and Approval Procedures.

In the EU, the breeding of genetically modified laboratory animals is, by definition, an animal experiment if the offspring may experience pain, suffering, or harm. In order to determine the actual burden of genetically modified mice, established methods are available. However, the breeding of immunodeficient mice is considered an experiment requiring a permit, even if no pain, suffering or harm is observed under scientifically required defined hygienic housing conditions, as determined by established methods of severity assessment. This seems contradictory and leads to uncertainty among scientists. With this commentary, we would like to shed light on this topic from different perspectives and propose a solution in terms of individualized severity assessment and approval procedures.

Alternatives in Education-Rat and Mouse Simulators Evaluated from Course Trainers' and Supervisors' Perspective.

Simulators allow the inexperienced to practice their skills prior to exercise on live animals. Therefore, they bear great potential in overcoming the dilemma between the present demand for high-quality practical training involving live animals whilst implementing the 3R principle according to the Directive 2010/63/EU. Currently, one mouse and six rat simulators are commercially available. As data on their impact are lacking, this project aimed at providing an overview of the awareness, implementation, and methodical and practical satisfaction provided by 35 course trainers and supervisors of laboratory animal training courses for mice and rats regarding the simulators available. Although simulators facilitate training of relevant techniques and relatively high awareness of them seemed to be present, their implementation is currently very low, possibly due to lack of meeting the respondents' demands. Thus, this study revealed the overall approval of simulator training and general demand for user-optimized, realistic, and financially affordable simulators and, hence, indicates a strong impulse for new developments strengthening the 3Rs as a benefit to all animals used in research.

Keeping Stallions in Groups-Species-Appropriate or Relevant to Animal Welfare?

This literature review was aimed at analyzing whether stallion husbandry in groups is possible and desirable or poses risks. This was determined on the basis of different studies in order to be able to give practical recommendations from the viewpoint of animal welfare. Consequently, 50 different sources were analyzed, as well as observations of an experiment of the Swiss National Stud on the subject of change from single-stallion to group husbandry and its influence on animal welfare. The results revealed that stallion husbandry in groups is possible but still rarely practiced. It was found that 6% of stallions in 2003, more than 11% in 2012, and nearly 23% of the stallions in 2015 were kept in groups. Furthermore, studies showed that the still widespread individual husbandry of stallions has a negative impact on psyche and body health. Almost half of all stallions showed undesirable patterns of behavior, mostly stallions in individual housing. In addition, many of the latter stallions had problems with their respiratory, digestive, and musculoskeletal systems, which improved when the husbandry conditions of the horses were changed, with the exception of the problems with the digestive system. Conversion into group husbandry is possible, as revealed by an experiment by the Swiss National Stud with a socialization of active breeding stallions outside the breeding season. Therefore, the widespread fear of serious injuries for stallions housed in groups was refuted and the aggressive behavior of the stallions decreased rapidly. Success rates for group husbandry are influenced by the individual character of the stallion, previous experience of the stallion, changes in the group, qualification and management of the farm, and organization of the group housing and husbandry system. This enables species-appropriate husbandry in groups while also considering animal welfare without stress, disadvantages, and serious injuries for stallions.