Laboratory Animals

This article provides recommendations for the care of laboratory zebrafish (Danio rerio) as part of the further implementation of Annex A to the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes, EU Commission Recommendation 2007/526/EC and the fulfilment of Article 33 of EU Directive 2010/63, both concerning the housing and care of experimental animals. The recommendations provide guidance on best practices and ranges of husbandry parameters within which zebrafish welfare, as well as reproducibility of experimental procedures, are assured.

Husbandry procedures found today in zebrafish facilities are numerous. While the vast majority of these practices are perfectly acceptable in terms of zebrafish physiology and welfare, the reproducibility of experimental results could be improved by further standardisation of husbandry procedures and exchange of husbandry information between laboratories. Standardisation protocols providing ranges of husbandry parameters are likely to be more successful and appropriate than the implementation of a set of fixed guidance values neglecting the empirically successful daily routines of many facilities and will better reflect the wide range of environmental parameters that characterise the natural habitats occupied by zebrafish.

A joint working group on zebrafish housing and husbandry recommendations, with members of the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the Federation of European Laboratory Animal Science Associations (FELASA) has been given a mandate to provide guidelines based on a FELASA list of parameters, ‘Terms of Reference’.

Driven by the longer lifespans of humans, particularly in Westernised societies, and the need to know more about ‘healthy ageing’, ageing mice are being used increasingly in scientific research. Many departments and institutes involved with ageing research have developed their own systems to determine intervention points for potential refinements and to identify humane end points. Several good systems are in use, but variations between them could contribute to poor reproducibility of the science achieved. Working with scientific and regulatory communities in the UK, we have reviewed the clinical signs observed in ageing mice and developed recommendations for enhanced monitoring, behaviour assessment, husbandry and veterinary interventions. We advocate that the default time point for enhanced monitoring should be 15 months of age, unless prior information is available. Importantly, the enhanced monitoring should cause no additional harms to the animals. Where a mouse strain is well characterised, the onset of age-related enhanced monitoring may be modified based on knowledge of the onset of an expected age-related clinical sign. In progeroid models where ageing is accelerated, enhanced monitoring may need to be brought forward. Information on the background strain must be considered, as it influences the onset of age-related clinical signs. The range of ageing models currently used means that there will be no ‘one-size fits all’ solution. Increased awareness of the issues will lead to more refined and consistent husbandry of ageing mice, and application of humane end points will help to reduce the numbers of animals maintained for longer than is scientifically justified.

Hygienic monitoring of laboratory rodents has focused more and more on the analysis of environmental sample material by quantitative polymerase chain reaction (qPCR) assays. This approach requires profound knowledge of specific genetic sequences of the agents to be monitored and the assays need to be permanently adapted to take the latest research into account. [Pasteurella] pneumotropica was recently reclassified into the new genus Rodentibacter, with Rodentibacter (R.) pneumotropicus and R. heylii as the most commonly detected species in laboratory mouse colonies. This study aimed at the development of a specific qPCR assay for the simultaneous detection of both agents.

A novel primer probe set, based on detection of the specific virulence factor‚ ‘inclusion body protein A’ gene (ibpA), was confirmed by testing the assay on currently described Rodentibacter type species and other Pasteurellaceae. Furthermore, it was validated within four different barrier units and results were compared with the cultural analysis of sentinel mice.

The assay was suitable to specifically detect R. pneumotropicus and R. heylii and discriminate them from other murine Rodentibacter spp. In addition, it revealed high sensitivity for the detection of both agents in environmental sampling material including exhaust air dust in individually ventilated cage systems. Altogether, higher pathogen prevalence was detected via qPCR of environmental samples compared with cultural diagnostics of sentinel mice.

This study describes a qPCR assay for the simultaneous detection of R. pneumotropicus and R. heylii. This assay was demonstrated to be beneficial during routine health monitoring, especially with regard to environmental sampling strategies.

Severity assessment in biomedical research is required by the European authorities. Therefore, a variety of score sheets are available. The first score sheets were designed and introduced by Morton and Griffith (M&G) in 1985, to assess pain and distress in animals. Score sheets are an important part of the 3R principles to evaluate the degree of severity in different studies. Here, we used a modified score sheet from M&G for severity assessment of 12 Aachen minipigs after partial liver resection for safety testing of a novel synthetic sealant (VIVO-107). The control group was treated with the clinical standard fibrin. Estimation of recovery status of both groups was performed from the day of surgery to postoperative day 7 using a score sheet. Included parameters were blood loss during the surgical procedure, general state, spontaneous behaviour and clinical results. Values from 0 to 20 were graded for each category and resulted in the degree of strain (DS) from DS0 to DS4. An increasing DS indicated higher severity. Suitability of the implemented score sheet was evaluated. Higher score points were documented almost exclusively as an outcome of the clinical results, influenced mainly by increased temperature in the fibrin treated control group, whereas, spontaneous behaviour had only slight influence and general state had no influence. The average score seven days after surgery was <2. The laparotomy, where the partial liver resection is a part, is rated as moderate severity in the EU Directive 2010/63, while the assessment done in the present study hints to a mild severity of the model in our hands.

The use of juvenile conventional pigs as a preclinical animal model to perform pharmacokinetic (PK), pharmacodynamic (PD) and safety studies for the paediatric population is increasing. Repetitive oral administration of drugs to juvenile pigs is however challenging. A representative method which can be used from birth till adulthood is necessary. The current study presents the placement and use of a gastrostomy button in pigs with a weight ranging from 2.4 to 161 kg. The surgical placement was performed via a laparotomic procedure on, each time, 12 pigs (six male, six female) of 1 week, 4 weeks, 8 weeks and 6–7 months old. For every age category, eight pigs were part of a PK study with a non-steroidal anti-inflammatory drug (NSAID) and four pigs served as a control group. No severe complications were observed during surgery. The button remained functional for 10 days in 40 out of 48 pigs. No significant differences in body temperature or white blood cell count were observed during the trial. Three control pigs showed signs of inflammation indicating a NSAID might be warranted. Autopsy revealed minimal signs of major inflammation in the abdominal cavity or the stomach. A limited number of pigs showed mucosal inflammation, ulcers or abscesses in the stomach or around the fistula. These results indicate that the laparotomic placement of a gastrostomy button might be considered safe and easy in growing pigs to perform repetitive oral dosing preclinical studies. However, the method is not advised in pigs weighing more than 100 kg.

Plasma lipid and glucose levels are important parameters for evaluating the onset and development of metabolic and cardiovascular diseases. In clinical and experimental studies of humans or mice, fasting is often required before testing plasma lipid and glucose levels. The rabbit is a valuable animal model for cardiovascular disease research. However, whether fasting is necessary for measuring plasma lipid and glucose levels in rabbits remains unclear. In the current study, 12 healthy Japanese white rabbits (males weighing 2.5–3.0 kg) were randomly divided into a chow diet group (n = 6) and a high cholesterol diet group (n = 6). They were fed either a standard chow diet or a chow diet supplemented with 0.5% cholesterol and 3% corn oil for 12 weeks. After 12 weeks, the plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol and glucose were measured before and after various fasting durations (8, 12, 16, 20 and 24 h). The results showed that there were no significant differences in lipid levels between the fasting and non-fasting samples, whereas glucose levels were lower after 8 h of fasting than in the absence of fasting. Moreover, the glucose levels were restored to normal after 8 h of refeeding. These results indicate that fasting does not affect plasma lipid values in rabbits but that fasting is important for determining the glucose level in rabbits. These findings may be helpful for future rabbit experiments and beneficial for animal welfare.

Anolis lizards have served as important research models in fields ranging from evolution and ecology to physiology and biomechanics. However, anoles are also emerging as important models for studies of embryo development and tissue regeneration. The increased use of anoles in the laboratory has produced a need to establish effective methods of anesthesia, both for routine veterinary procedures and for research procedures. Therefore, we tested the efficacy of different anesthetic treatments in adult female Anolis sagrei. Alfaxalone, dexmedetomidine, hydromorphone, ketamine and tribromoethanol were administered subcutaneously (SC), either alone or combined at varying doses in a total of 64 female anoles. Drug induction time, duration, anesthesia level and adverse effects were assessed. Differences in anesthesia level were observed depending on injection site and drug combination. Alfaxalone/dexmedetomidine and tribromoethanol/dexmedetomidine were the most effective drug combinations for inducing a surgical plane of anesthesia in anoles. Brown anoles injected SC with alfaxalone (30 mg/kg) plus dexmedetomidine (0.1 mg/kg) or with tribromoethanol (400 mg/kg) plus dexmedetomidine (0.1 mg/kg) experienced mean durations of surgical anesthesia levels of 31.2 ± 5.3 and 87.5 ± 19.8 min with full recovery after another 10.9 ± 2.9 and 46.2 ± 41.8 min, respectively. Hydromorphone given with alfaxalone/dexmedetomidine resulted in deep anesthesia with respiratory depression, while ketamine/hydromorphone/dexmedetomidine produced only light to moderate sedation. We determined that alfaxalone/dexmedetomidine or tribromoethanol/dexmedetomidine combinations were sufficient to maintain a lizard under general anesthesia for coeliotomy. This study represents a significant step towards understanding the effects of anesthetic agents in anole lizards and will benefit both veterinary care and research on these animals.