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.
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.
The behaviour shown by an animal at any one time is the consolidated output of its behavioural control mechanism. Moreover, what animals “want” is viewed as (the most) important component for ...individual welfare. Accordingly, studying the motivation of animals helps understanding basic mechanisms and welfare related needs. However, studying wants of animals is notoriously difficult and many previous studies on the preferences of animals have been restricted in the sense that only two choice options were presented in an artificial test environment. Here, an extended approach, the “Small World” is presented, in which the choices of animals between eight ecologically relevant resources can be observed in a long‐term test to reach conclusions with respect to everyday moment‐to‐moment decisions. In this sense, the system offers a quasi‐natural environment. The approach was tested in three experiments with observations of individual female rats, small groups of female rats (Long Evans, Rattus norvegicus) and small groups of female chickens (Lohmann Brown, Gallus gallus domesticus). The animals oriented themselves quickly in the system and it was possible to collect multifaceted data on the use of the resources. These data included the faecal and urinary markings in the Small World cages, the daily frequency and duration of visits to these cages, the sequential analyses of the choices for and decisions among the resources, and the synchrony of the animals in the groups. Given the richness of these data and the lack of a stress response in the tested animals, the use of the Small World approach seems to be highly promising as an extension to previously used procedures. To further improve the approach and more directly reflect the subjective value of the different resources from the animals' point of view, the distances between the resources in a quasi‐natural landscape shall be manipulated in future studies.
In three pilot studies, rats and hens were studied in a Small World system. This approach allows the investigation of everyday moment‐to‐moment decisions under experimentally manipulable but ecologically meaningful conditions. These decisions are relevant for basic understanding proximate behavioural control mechanisms and behavioural needs in relation to questions of animal welfare.
Within the scope of the 3Rs of Russel and Burch, the number of laboratory animals can be reduced by repeated use of an animal. This strategy only becomes relevant, if the total amount of pain, ...distress or harm the individual animal experiences does not exceed the severity of a single manipulation. For example, when using imaging techniques, an animal can be examined several times during a study, but it has to be anesthetized each time imaging is performed. The severity of anesthesia is thought to be mild according to the Directive 2010/63/EU. However, the Directive does not differentiate between single and repeated anesthesia, although repeated anesthesia may have a greater impact on well-being. Hence, we compared the impact of single and repeated anesthesia (six times at an interval of three to four days) by injection of ketamine and xylazine (KX) on the well-being of adult female and male C57BL/6JRj mice. After anesthesia, well-being of mice was assessed according to a protocol for systematic assessment of well-being including nesting, the Mouse Grimace Scale (MGS), a test for trait anxiety, home cage activity, and the rotarod test for motor activity, food intake, and body weight, as well as corticosterone (metabolite) analysis. Repeated anesthesia increased the MGS in mice of both sexes and caused short-term effects on well-being of female mice in the immediate post-anesthetic period, indicated by longer lasting effects on trait anxiety-related behavior. However, corticosterone metabolite concentrations suggested that mice habituated to the stress induced by repeated KX administration. Hence, the mildly negative effects on well-being of repeated KX anesthesia do not seem to accumulate over time using the respective regimen. However, further observations for severity classification are warranted in order to more specifically determine the duration of mild distress and trait anxiety.
A high prevalence of deviations and fractures of the keel bone is a widespread welfare problem in laying hens. The aim of this study was to experimentally investigate this multifactorial problem ...throughout the laying period and to compare the prevalence and severity in different layer lines and different housing systems. High performing white (WLA) and brown (BLA) pure bred layer lines and low performing white (R11, G11) and brown layer lines (L68) were kept in both single cages and a floor housing system. A total of 97 hens (19 or 20 from each line, respectively) were repeatedly radiographed in the 35th, 51st and 72nd week of age. Fracture prevalence increased with age (p<0.001). The proportion of deviated keel bone area increased only for caged BLA, WLA and R11 hens (p<0.05) and was significantly higher for caged WLA and R11 hens compared to floor-housed WLA and R11 hens in the 72nd week of age (p<0.05). In the 72nd week of age hens in the floor housing system showed significantly more fractures than hens kept in cages (p<0.05). Prevalence of keel bone deviations was significantly higher in the white layer line R11 but significantly lower in the white layer line G11 compared to both brown layer lines and WLA (p<0.05). Brown layers showed significantly more fractures than white layers (p<0.05) in the 51st and 72nd week of age. Within the brown layers there was a significantly lower prevalence of deviations (p<0.05) and fractures (p<0.05) in the low performing (L68) compared to the high performing line (BLA). Our results show a different development of keel bone damage in caged compared to floor-housed hens under experimental conditions. Additionally, they indicate genetic effects on keel bone damage.
Laboratory male mice are often housed individually due to aggressive behavior or experimental requirements, though social isolation can cause welfare issues. As a strategy to refine housing of male ...mice, we introduce the separated pair housing system. A perforated transparent wall divides the cage into two compartments and allows olfactory, acoustic, and visual communication between the two mice but prevents fighting and injuries. Long-term effects of separated pair housing on well-being and distress of adult male C57BL/6JRj mice were investigated and compared with both single- and group-housed mice. Behavioral analysis after eight weeks in three different housing systems revealed no differences in burrowing performance, social interaction, anxiety, and stress hormone concentrations. However, pair-housed mice built more complex nests compared to single-housed mice and the nest position suggested that pair-housed mice preferred the close proximity to their cage mates. Moreover, pair-housed mice showed less locomotor activity compared to group- and single-housed mice. Body weight was higher in group-housed mice. All in all, no unambiguous long-term beneficial effects of pair housing on the well-being were found. However, the findings emphasized that effects of the housing systems on behavioral, physical, and biochemical parameters must be considered in the design of animal experimental studies.
To maintain and foster the welfare of laboratory mice, tools that reliably measure the current state of the animals are applied in clinical assessment. One of these is the Mouse Grimace Scale (MGS), ...a coding system for facial expression analysis. Since there are concerns about the objectivity of the MGS, we further investigated its reliability. Four observers (two experienced and two inexperienced in use of the MGS) scored 188 images of 33 female and 31 male C57BL/6JRj mice. Images were generated prior to, 150 min, and two days after ketamine/xylazine anesthesia. The intraclass correlations coefficient (ICC = 0.851) indicated good agreement on total MGS scores between all observers when all three time points were included in the analysis. However, interrater reliability was higher in the early post-anesthetic period (ICC = 0.799) than at baseline (ICC = 0.556) and on day 2 after anesthesia (ICC = 0.329). The best agreement was achieved for orbital tightening, and the poorest agreement for nose and cheek bulge, depending on the observers’ experience levels. In general, experienced observers produced scores of higher consistency when compared to inexperienced. Against this background, we critically discuss factors that potentially influence the reliability of MGS scoring.
Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even ...catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing.
PDF
