Postoperative Severity Assessment in Sheep.

INTRODUCTION: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep. METHODS: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry. RESULTS: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day. CONCLUSION: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement.

RELSA-A multidimensional procedure for the comparative assessment of well-being and the quantitative determination of severity in experimental procedures.

Good science in translational research requires good animal welfare according to the principles of 3Rs. In many countries, determining animal welfare is a mandatory legal requirement, implying a categorization of animal suffering, traditionally dominated by subjective scorings. However, how such methods can be objectified and refined to compare impairments between animals, subgroups, and animal models remained unclear. Therefore, we developed the RELative Severity Assessment (RELSA) procedure to establish an evidence-based method based on quantitative outcome measures such as body weight, burrowing behavior, heart rate, heart rate variability, temperature, and activity to obtain a relative metric for severity comparisons. The RELSA procedure provided the necessary framework to get severity gradings in TM-implanted mice, yielding four distinct RELSA thresholds L1<0.27, L2<0.59, L3<0.79, and L4<3.45. We show further that severity patterns in the contributing variables are time and model-specific and use this information to obtain contextualized between animal-model and subgroup comparisons with the severity of sepsis > surgery > restraint stress > colitis. The bootstrapped 95% confidence intervals reliably show that RELSA estimates are conditionally invariant against missing information but precise in ranking the quantitative severity information to the moderate context of the transmitter-implantation model. In conclusion, we propose the RELSA as a validated tool for an objective, computational approach to comparative and quantitative severity assessment and grading. The RELSA procedure will fundamentally improve animal welfare, data quality, and reproducibility. It is also the first step toward translational risk assessment in biomedical research.

Voluntary wheel running behaviour as a tool to assess the severity in a mouse pancreatic cancer model.

Laboratory animals frequently undergo routine experimental procedures such as handling, restraining and injections. However, as a known source of stress, these procedures potentially impact study outcome and data quality. In the present study, we, therefore, performed an evidence-based severity assessment of experimental procedures used in a pancreatic cancer model including surgical tumour induction and subsequent chemotherapeutic treatment via repeated intraperitoneal injections. Cancer cell injection into the pancreas was performed during a laparotomy under general anaesthesia. After a four-day recovery phase, mice received either drug treatment (galloflavin and metformin) or the respective vehicle substances via daily intraperitoneal injections. In addition to clinical scoring, an automated home-cage monitoring system was used to assess voluntary wheel running (VWR) behaviour as an indicator of impaired well-being. After surgery, slightly elevated clinical scores and minimal body weight reductions, but significantly decreased VWR behaviour were observed. During therapy, body weight declined in response to chemotherapy, but not after vehicle substance injection, while VWR activity was decreased in both cases. VWR behaviour differed between treatment groups and revealed altered nightly activity patterns. In summary, by monitoring VWR a high impact of repeated injections on the well-being of mice was revealed and substance effects on well-being were distinguishable. However, no differences in tumour growth between treatment groups were observed. This might be due to the severity of the procedures uncovered in this study, as exaggerated stress responses are potentially confounding factors in preclinical studies. Finally, VWR was a more sensitive indicator of impairment than clinical scoring in this model.

An Approach towards Motion-Tolerant PPG-Based Algorithm for Real-Time Heart Rate Monitoring of Moving Pigs.

Animal welfare remains a very important issue in the livestock sector, but monitoring animal welfare in an objective and continuous way remains a serious challenge. Monitoring animal welfare, based upon physiological measurements instead of the audio-visual scoring of behaviour, would be a step forward. One of the obvious physiological signals related to welfare and stress is heart rate. The objective of this research was to measure heart rate (beat per minutes) in pigs with technology that soon will be affordable. Affordable heart rate monitoring is done today at large scale on humans using the Photo Plethysmography (PPG) technology. We used PPG sensors on a pig's body to test whether it allows the retrieval of a reliable heart rate signal. A continuous wavelet transform (CWT)-based algorithm is developed to decouple the cardiac pulse waves from the pig. Three different wavelets, namely second, fourth and sixth order Derivative of Gaussian (DOG), are tested. We show the results of the developed PPG-based algorithm, against electrocardiograms (ECG) as a reference measure for heart rate, and this for an anaesthetised versus a non-anaesthetised animal. We tested three different anatomical body positions (ear, leg and tail) and give results for each body position of the sensor. In summary, it can be concluded that the agreement between the PPG-based heart rate technique and the reference sensor is between 91% and 95%. In this paper, we showed the potential of using the PPG-based technology to assess the pig's heart rate.

Body weight algorithm predicts humane endpoint in an intracranial rat glioma model.

Humane endpoint determination is fundamental in animal experimentation. Despite commonly accepted endpoint criteria for intracranial tumour models (20% body weight loss and deteriorated clinical score) some animals still die before being euthanized in current research. We here systematically evaluated other measures as surrogates for a more reliable humane endpoint determination. Adult male BDIX rats (n = 119) with intracranial glioma formation after BT4Ca cell-injection were used. Clinical score and body weight were assessed daily. One subgroup (n = 14) was assessed daily for species-specific (nesting, burrowing), motor (distance, coordination) and social behaviour. Another subgroup (n = 8) was implanted with a telemetric device for monitoring heart rate (variability), temperature and activity. Body weight and clinical score of all other rats were used for training (n = 34) and validation (n = 63) of an elaborate body weight course analysis algorithm for endpoint detection. BT4Ca cell-injection reliably induced fast-growing tumours. No behavioural or physiological parameter detected deteriorations of the clinical state earlier or more reliable than clinical scoring by experienced observers. However, the body weight course analysis algorithm predicted endpoints in 97% of animals without confounding observer-dependent factors. Clinical scoring together with the novel algorithm enables highly reliable and observer-independent endpoint determination in a rodent intracranial tumour model.

Monitoring of Heart Rate and Activity Using Telemetry Allows Grading of Experimental Procedures Used in Neuroscientific Rat Models.

In animal experimentation, welfare and severity assessments of all procedures applied to animals are necessary to meet legal and ethical requirements, as well as public interests. So far, the methods suggested for this purpose are time consuming and personnel intensive. Also, evidence-based biostatistical methods for this purpose are still rare. We here tested whether the classification of heart rate (HR) and activity (Act) data monitored by telemetry in the home cage by unsupervised k-means-based class-labeling and subsequent Support Vector Machine (SVM) analysis allows severity assessment and grading of experimental procedures of different domains, including surgery, injection, behavioral testing, and routine handling for maintenance. Telemetric devices were subcutaneously implanted in young adult male Crl:CD(SD) and BDIX/UImHanZtm rats. After recovery, rats were randomly subjected to different experimental procedures, i.e., handling and cage change as routine maintenance, Rat Grimace Scale, burrowing, and social interaction for welfare assessment, as well as repeated subcutaneous injections. Thereafter, rats were either intracranially implanted with electrodes or injected with tumor cells. Directly after each procedure, HR and Act were monitored by telemetry in the home cage for 4 h. Application of k-means and SVM algorithms on the obtained data sets from baseline (as no stress), cage change (exploratory Act), and intracranial surgery (as burden) measurements computed three classes described as low HR/low Act, high HR/high Act, and high HR/low Act, respectively. Validation of the SVM model by entering data from all procedures confirmed the allocation to the high HR/low Act class (burden) after surgery, which lasted longer after subcutaneous transmitter implantation than after intracranial surgery. The majority of data points from repeated injections, behavioral testing, and maintenance handling were allocated to the low HR/low Act and high HR/high Act classes. Overall, the SVM model based on HR and Act data monitored in home cage after procedures may be useful for the classification and grading of experimental procedures of different domains.

Wheel running behaviour in group-housed female mice indicates disturbed wellbeing due to DSS colitis.

Voluntary wheel running (VWR) behaviour is a sensitive indicator of disturbed wellbeing and used for the assessment of individual experimental severity levels in laboratory mice. However, monitoring individual VWR performance usually requires single housing, which itself might have a negative effect on wellbeing. In consideration of the 3Rs principle, VWR behaviour was evaluated under group-housing conditions. To test the applicability for severity assessment, this readout was evaluated in a dextran sodium sulphate (DSS) induced colitis model. For continuous monitoring, an automated system with integrated radio-frequency identification technology was used, enabling detection of individual VWR. After a 14-day adaptation period mice demonstrated a stable running performance. Analysis during DSS treatment in combination with repeated facial vein phlebotomy and faecal sampling procedure resulted in significantly reduced VWR behaviour during the course of colitis and increased VWR during disease recovery. Mice submitted to phlebotomy and faecal sampling but no DSS treatment showed less reduced VWR but a longer-lasting recovery. Application of a cluster model discriminating individual severity levels based on VWR and body weight data revealed the highest severity level in most of the DSS-treated mice on day 7, but a considerable number of control mice also showed elevated severity levels due to sampling procedures alone. In summary, VWR sensitively indicated the course of DSS colitis severity and the impact of sample collection. Therefore, monitoring of VWR is a suitable method for the detection of disturbed wellbeing due to DSS colitis and sampling procedure in group-housed female laboratory mice.

Remote vitals monitoring in rodents using video recordings.

Laboratory animal research was always crucial for scientific breakthroughs in the fields of medicine and biology. Animal trials offer insights into various disease mechanisms, genetics, drug therapy and the effect of different external factors onto living organisms. However, conducting animal trials is highly controversial. To ensure high ethical standards, a number of directives have been adopted in the European Union, which seek to replace, reduce and refine animal trials. Hence, severity assessment plays an important role in today's laboratory animal research. Currently, severity of trials is assessed by highly rater dependent scoring systems. In this paper, we propose a method for unobtrusive, automated and contactless measurement of respiratory rate (RR) and heart rate (HR). We were able to extract RR and HR with an high agreement between our method and a contact-based reference method. The Root Mean Squared Error (RMSE) averaged 0.32 ± 0.11 breaths/min for RR and 1.28 ± 0.62 beats/min for HR in rats, respectively. In mice, the RMSE averaged 1.42 ± 0.97 breaths/min for RR and 1.36 ± 0.87 beats/min, respectively. In the future, these parameters can be used for new, objective scoring systems, which are not susceptible to inter-rater variability.

Contactless Anesthesia Monitoring in Spontanously Breathing Rodents.

Laboratory animal science plays a crucial role in medical and biological research. In the last decades, stricter regulations were enforced to safeguard laboratory animals. Following the "3Rs" guiding principles, animal trials should be replaced, reduced and refined, whenever possible.A contactless modality capable of assessing the respiratory rate (RR) and additional breath related characteristics can potentially refine anesthetic interventions in rodents by continuously monitoring their anesthetic depth. This can reduce complications and thus the number of needed animals.We were able to extract the instantaneous RR in rodents with a sum squared error (SSE) of 0.26 breaths/min from color video. A correlation of 0.9781 compared to an Electrocardiography (ECG) based reference was achieved. Furthermore, additional temporal and morphological characteristics were extracted, which are sensitive for changes in the anesthetic depth.