Comparison of sublingual, facial and retro-bulbar blood sampling in mice in relation to animal welfare and blood quality.

INTRODUCTION: Repeated blood sampling is a common procedure in laboratory mice, but at present it is unknown which technique has the least impact on the animals when large or repeated blood samples are required. Retro-bulbar sinus puncture is a reliable technique but has been shown to cause many changes in the animals, why sublingual and facial vein puncture have been suggested as suitable alternatives. This study investigated 1) which of the three blood sampling techniques had the least impact on nest building activity, level of faecal corticosterone metabolites, body weight, fur status, and macroscopic changes, 2) whether the blood sampling techniques gave rise to variation in blood quality between blood samples, and 3) whether sublingual and facial vein puncture should be performed with or without anaesthesia in female C57BL/6 mice. METHOD: Three hundred and sixty C57BL/6 female mice divided into five batches were included in the study and randomized to a short (blood sampling on Day 8, 9 and 10) or a long protocol (blood sampling on Day 8, 15 and 22). Each protocol consisted of six identical groups: sublingual vein puncture (SVP), sublingual vein puncture in isoflurane (SVPiso), facial vein puncture (FVP), facial vein puncture in isoflurane (FVPiso), retro-bulbar sinus puncture (RBP), and a control group (CONTROL) with only scruffing being performed. At baseline (Day 2) nest building activity (NBA) was assessed and faecal pellets collected for evaluation of faecal corticosterone metabolites (FCM). The day after each blood sampling day NBA and FCM were reassessed. RESULTS AND CONCLUSION: None of the blood sampling techniques proved to be superior to the others in any of the measured parameters. Finally, sublingual and facial vein puncture performed under anaesthesia gave rise to variation in the quality of the blood. A refinement of all three techniques are therefore warranted.

Effect of Repeated Exposure to Isoflurane on Nest Building and Burrowing in Mice.

Nest building and burrowing are highly motivated natural behaviors in rodents, and changes in these behaviors can serve as welfare assessment tools. In this study, we investigated: 1) the limits of agreement between 2 observers for a refined scoring method for nest-building behavior; 2) the effect of repeated exposure to 15 min of isoflurane on nest-building behavior; 3) the effect of 24 h of grid-floor housing, repeated exposure to 15 min isoflurane, and daily intraperitoneal injection of 0.2 mL 0.9% isotonic saline for 3 d on burrowing behavior; and 4) the effect of exposure to grid-floor housing, isoflurane, and intraperitoneal injections on fecal corticosterone metabolites, body weight, fur status, and sucrose preference in mice. SPF C57BL/6NTac female mice (n = 27) were included in the study and were assessed first for burrowing behavior, followed by 2 wk of rest and then for nesting behavior. The refined scoring method for nest-building activity had good inter observer agreement. According to this method, a single exposure to anesthesia with isoflurane led to a decrease in nest-building activity and sucrose preference; a second exposure to anesthesia with isoflurane had no effect on nest building. Neither grid-floor housing nor repeated exposure to isoflurane anesthesia had any effect on burrowing behavior in mice. In contrast, intraperitoneal injections increased burrowing behavior. In conclusion, a refined scoring method for nest-building activity test that we developed for this study proved to be objective and sensitive to the effect of an initial exposure to anesthesia with isoflurane.

Burrowing and nest building activity in mice after exposure to grid floor, isoflurane or ip injections.

Burrowing and nest building are examples of highly motivated innate behaviours in rodents. Assessments based on these behaviours can be used as non-invasive indicators of pain, distress and suffering. In this study, we investigated the effect of three stressful procedures, namely grid floor housing for 24 h, 15 min of isoflurane anaesthesia and an intraperitoneal saline injection daily for three days, on nest building and burrowing in female C57BL/6NTac mice. For burrowing, we also investigated the effect of the presence of a shelter and nesting material (standard home cage enrichment) versus no shelter and nesting material, and whether the test can be performed during normal working hours. Alongside this, we investigated the effect of grid floor, isoflurane anaesthesia and intraperitoneal injections on stress hormone level, body weight, fur status and sucrose preference. The burrowing test was found to be sensitive to 24 h grid floor housing when no shelter but only a cardboard tube was present during testing. The mice burrowed a mean of 21 g less after grid floor housing (P < .01). This change was accompanied by decreased sucrose preference (P < .01) and body weight (P < .01). 15 min of anaesthesia with isoflurane induced changes in the nest building activity test. After exposure to isoflurane the mice built less complex nests (P = .04). This was accompanied by a decrease in sucrose preference (P < .01), a decrease in body weight (P < .01), and elevated stress hormone levels (P < .01). One daily intraperitoneal injection of saline for three days did not result in changes in nest building activity (P > .01). We also found that the mice burrowed equal amount during normal working hours and prior to the dark phase, indicating that the burrowing test can be performed during normal working hours instead of prior to the dark phase (P = .62).

Eye, body or tail? Thermography as a measure of stress in mice.

Infrared thermography has been suggested as a non-invasive, objective tool to evaluate animal welfare. In this study, we investigated: 1) how body temperature, measured through thermal imaging, is affected by different mild stressors frequently experienced by laboratory mice; 2) which methodology to use for assessing temperature variations with infrared thermography; 3) whether the chosen stressors cause anxiety in mice. Eighty C57BL/6 male mice were included in the study. The mice were allocated to either a control group or one of three groups being subjected to a mild stressor once daily for 4 days: 1) anaesthesia with isoflurane for 10 min; 2) handling by scruffing; 3) intraperitoneal injection of 0.2 ml 0.9% saline. On all four intervention days, thermal images were obtained in all groups and all animals were assessed for fur status and body weight. On day five, all animals were tested in the elevated-plus-maze for 5 min. From the thermal images, the maximum eye temperature, the maximum tail base temperature and the average body temperature were obtained. Ten minutes of anaesthesia with isoflurane led to a decrease in maximum eye temperature, average body temperature and maximum tail base temperature. The animals recovered from this drop in temperature within 10 min. No drop in temperature was seen after scruffing or intraperitoneal injection of saline. Based on the number of missing values, intra-rater and inter-rater agreement, the average body temperature was found most ideal for measuring body temperature variations in mice. Finally, the elevated plus maze did not reveal any differences in anxiety between the groups and the body weight did not decrease at any time point during the study.