Laboratory Animals, Ahead of Print.
For neonatal pups, parenteral anaesthesia is said to be not reliable as low doses induce no anaesthesia whereas high doses render high mortality rates. In this work we have adapted parenteral anaesthesia procedures approved for pups >7 days of age, to anaesthetize neonatal animals (postnatal days 3–4; P3–P4) for keeping them immobile for a long period. In our first experiment we analysed the behaviour of P3–P4 mouse pups for 70 min after intraperitoneal administration of low (37.5/3.75 mg/kg) or high (50/5) doses of a ketamine/xylazine anaesthetic mixture, both in the low range as compared with dosages employed in adults. Pups became immobile in ≈7 min and remained immobile for ≈45 min, irrespective of the age and dose of anaesthesia, younger pups (P3) being apparently more sensitive to the dosage. In the second experiment, we studied the response of P3 pups to mildly nociceptive stimulations, performed with a 4.0 g von Frey filament applied to the dorsal aspect of their paws. These stimuli elicited reaction in 100% of the cases in non-anaesthetized pups. The results indicate that the high dose significantly reduced responses as compared with the low dose of anaesthesia. With the low dose, <40% of the pups were unresponsive to nociceptive stimulation, whereas the high dose resulted in 50–60% of the animals not responding. Mortality was low irrespective of age or dose, suggesting that doses can be further increased if needed for invasive experimental procedures.

Laboratory Animals, Ahead of Print.
Staphylococcus aureus nasal carriage is considered a risk factor for infections, and the development of nasal decolonization strategies is highly relevant. Despite they are not naturally colonized by Staphylococcus, mice are a good model for S. aureus nasal colonization. Murine models are easy to manipulate, and inter-laboratory reproducibility makes them suitable for nasal colonization studies. Strategies using bioluminescent bacteria allow for the monitoring of infection over time without the need to sacrifice animals for bacterial quantification. In this study, we evaluated S. aureus nasal colonization in three mouse strains (BALB/c, C57BL/6, and Swiss Webster) using a bioluminescent strain (SAP231). In vitro, a visible Bioluminescent Signal Emission (BLSE) was observed until 106 bacteria and detected by IVIS® imaging system up to 104 cells. Animals were inoculated with one or two doses of approximately 109 colony-forming units (CFU) of SAP231. Swiss Webster mice showed the longest colonization time, with some animals presenting BLSE for up to 140 h. In addition, BLSE was higher in this strain. BALB/c and C57BL/6 strains showed consistent BLSE results for 48 h. BLSE intensity was higher in Swiss Webster inoculated with both doses. Three different positions for image capture were evaluated, with better results for the lateral and ventrodorsal positions. After the loss of BLSE, bacterial quantification was performed, and Swiss Webster mice presented more bacteria in the nasal cavity (approximately 105 CFU) than the other strains. Our results demonstrate that bioluminescent S. aureus allow monitoring of nasal colonization and estimation of the bacterial burden present in live animals until 48 h.

Laboratory Animals, Volume 58, Issue 1, Page 95-95, February 2024.

Laboratory Animals, Volume 58, Issue 1, Page 93-93, February 2024.

Laboratory Animals, Ahead of Print.
Animal welfare has evolved during the past decades to improve not only the quality of life of laboratory rodents but also the quality and reproducibility of scientific investigations. Bibliometric analysis has become an important tool to complete the current knowledge with academic databases. Our objective was to investigate whether scientific research on cannibalism/infanticide is connected with maternal aggression towards the offspring in laboratory rodents. To carry out our research, we performed a specific search for published articles on each concept. Results were analyzed in the open-source environment RStudio with the package Bibliometrix. We obtained 253 and 134 articles for the first search (cannibalism/infanticide) and the second search (maternal aggression towards the pups) respectively. We observed that the interest in infanticide/cannibalism started in the 1950s, while researchers started showing interest in maternal aggression towards the pups 30 years later. Our analyses indicated that maternal aggression had better citations in scientific literature. In addition, although our results showed some common features (e.g. oxytocin or medial preoptic area in the brain), we observed a gap between cannibalism/infanticide and maternal aggression towards the pups with only 14 published articles in common for both the searches. Therefore, we recommend researchers to combine both concepts in further investigations in the context of cannibalism for better dissemination and higher impact in laboratory rodents’ welfare research.

Laboratory Animals, Ahead of Print.
Catheterisation of the urinary bladder is needed in many types of human disease models in pigs. Based on our extensive experience with the pig as an infection model, we here demonstrate an approach of catheterising domestic pigs (40 attempts) and Göttingen minipigs (10 attempts) using a blinded method, that is, without speculums or videoscopes to visualise the urethral opening. The procedure was tested on control animals and pigs with experimental Escherichia coli urinary tract infection (UTI) to assess the potential influence of this condition on procedural outcome. Lastly, we performed cystoscopy in three animals to visualise the route to the urethra and to localise potential anatomical obstacles. All domestic pigs were catheterised successfully in an average of 2 minutes and 23 seconds, and this was not influenced by UTI (p  =  0.06) or bladder urine content at the time of catheterisation (p = 0.32). All Göttingen minipigs were successfully catheterised in an average of 4 minutes and 27 seconds. We conclude that blinded catheterisation is a fast and reliable approach that can be performed in pigs with or without UTI with minimal risk of trauma or contamination.

Laboratory Animals, Ahead of Print.
The housing conditions of laboratory mice must be strictly controlled in order to reduce the impact of pathophysiological changes that affect animal health and welfare, possibly resulting in increased variability within experimental results. One way to improve the activity and survival of laboratory mice is to provide nesting material. The objective of this study was to determine if nest-building quality could be used to detect changes in murine mating behaviour in a rodent facility under controlled conditions. Nesting scores of 847 cages with monogamous pairs from three different genetic backgrounds (129, B6 and BALB/c) of both sexes were correlated with 18 predefined variables. The effects on nest quality were evaluated using descriptive data analysis, correspondence analysis and ordinal logistic model fitting. The results showed a strong relationship between nest quality and nest position. Humidity, genetic background, cage change and the number and age of pups in the cage affected the nest-building scores. The most important indicators were cage change and relative humidity, both of which exerted significant negative effects on nest-building quality. Even though the criteria were well defined, the observer could still influence nest score appraisal. However, in a long-term observational study, observers could improve their assessment by training and acquiring greater experience in score assignment. Nest-building scores are easy to assess in the cage, with little discomfort to the animal. Moreover, the nest score is a valid indicator of the health and well-being of laboratory mice and can provide valuable support in the management of animal facilities.

Laboratory Animals, Ahead of Print.
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.

Laboratory Animals, Ahead of Print.
Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe.

Laboratory Animals, Ahead of Print.
Non-alcoholic fatty liver disease (NAFLD) and subsequent steatohepatitis (NASH) is the most common cause of liver disease and liver transplantation in humans. Affecting millions of patients worldwide, diagnosis relies on a biopsy, not without risk to the patient, and emphasises the need for improved diagnostic measures to determine and monitor disease progression. Despite intensive research, approved pharmacological treatment modalities are few, underlining that animal models with increased translational validity are important to advance preclinical drug development. This study validates the applicability of computed tomography (CT) as a non-invasive diagnostic tool for the assessment of liver steatosis in a guinea pig model of NAFLD/NASH. Guinea pigs with induced NAFLD or NASH were compared to healthy controls at two separate time points: week 16, serving as baseline measure, and week 25 to monitor disease progression over time. The animals were subsequently euthanised, and samples were collected to confirm disease stage. The data showed a strong negative correlation between liver triglycerides and Hounsfield unit (HU) values (R2 = 0.8157; p < 0.0001). A significant difference in histopathological scoring and HU values between grade 0 and more advanced stages of steatosis was recorded (p < 0.001), although the degree of liver fibrosis could not be accurately evaluated by differences in HU. In conclusion, the present study validates CT scanning for the determination of hepatic steatosis in guinea pigs, and it strongly supports the technique as a relevant non-invasive diagnostic tool in this species.