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.

/pdf/severity-classification-of-repeated-isoflurane-anesthesia-in-3vl4jespwt.pdf

Severity classification of repeated isoflurane anesthesia in C57BL/6JRj mice—Assessing the degree of distress

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

/pdf/impact-of-repeated-anesthesia-with-ketamine-and-xylazine-on-36ze6whusf.pdf

Impact of repeated anesthesia with ketamine and xylazine on the well-being of C57BL/6JRj mice

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals' physiological condition, which should be considered whenever blood samples are obtained.

/pdf/physiological-and-pathological-impact-of-blood-sampling-by-4luhmdwrag.pdf

Physiological and pathological impact of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy in laboratory mice.

<ns4:p>Background Due to the lack of complexity and variety of stimuli, conventional housing conditions of laboratory mice do not allow these animals to fully express their behavioral repertoire, including manipulative and cognitive activities. Therefore, we designed mechanical puzzles, so-called lockboxes, for mice that can be provided in their home cages. We investigated the impact of the lockbox enrichment on their phenotype and affective state when compared to conventional housing and super-environmental enrichment. Methods Young adult female C57BL/6JCrl mice were examined before and after 2-month exposure to the different types of enrichment in a phenotyping test battery, including tests for trait and state anxiety-related behavior, calorimetric measurements, body weight measurements, and the analysis of stress hormone metabolite concentrations as well as sequential problem-solving abilities. At the end of the study, adrenal gland weights were determined and pathohistological evaluation was performed. For all continuous variables, the relative variability was calculated. Results We demonstrated that lockbox enrichment decreased trait anxiety-related behavior compared to conventional housing in the Grid Exploratory Paradigm. In contrast, the different types of enrichment neither influenced state anxiety-related behavior in the Open Field and Elevated Plus Maze Test nor physiological variables (i.e., bodyweight, resting metabolic rate, stress hormone metabolite concentrations, and adrenal gland weights). Lockbox enrichment improved sequential problem-solving when compared to super-environmental enrichment. Regardless of the housing condition, the relative variability increased in most variables over time, although the coefficient of variation decreased for some variables, especially in animals with access to the lockbox enrichment. A pathohistological evaluation revealed no evidence of toxicopathological effects associated with the material from which the lockbox enrichment was made. Conclusions Overall, the lockbox enrichment revealed beneficial effects on the affective state and sequential problem-solving abilities of laboratory mice. The reproducibility was demonstrated not to be compromised by lockbox enrichment, similar to super-environmental enrichment.</ns4:p> 

Lockbox enrichment facilitates manipulative and cognitive activities for mice

Due to the lack of complexity and variety of stimuli, conventional housing conditions of laboratory mice do not allow these animals to fully express their behavioral repertoire, including manipulative and cognitive activities. Therefore, we designed mechanical puzzles, so-called lockboxes, for mice that can be provided in their home cages. We investigated the impact of the lockbox enrichment on their phenotype and affective state when compared to conventional housing (CH) and super-environmental enrichment (SEE). 

Conventional bleeding of small laboratory animals is often associated with stress and injuries that can cause haematomas, inflammation and ultimately the death of animals under investigation. Here, we used faecal glucocorticoid metabolites as an indicator of stress imposed on laboratory mice (Mus musculus domesticus) when bled in three different ways: puncture of the tail vein following mechanical immobilisation; puncture of the retro-orbital plexus following chemical immobilisation; and a minimally invasive technique using blood-sucking bugs (Reduviidae, Heteroptera) without any immobilisation. We hypothesised that blood-sucking bugs provoke a lower hormonal stress response than conventional bleeding techniques because laboratory animals are not handled and because the mechanical stimulus of an insect sting is supposedly weak. Each of the 16 mice was bled using one of the three methods at a time in a random order with seven days of recovery between subsequent bleeding events. To monitor the stress hormones, we determined corticosterone metabolites in faecal samples of mice collected one day before, 8 h after and one day after the bleeding event. Concentrations of faecal glucocorticoid metabolites increased in all three treatment groups compared with baseline values. However, average concentration of stress hormone metabolites after bleeding was higher by a factor of about 1.5 when conventional bleeding techniques were applied than when bugs were used. We conclude that blood-sucking bugs may offer a gentle alternative for obtaining blood samples from small animals such as mice.

https://www.ufaw.org.uk/downloads/awj-abstracts/v22-4-voigt.pdf

Hormonal stress response of laboratory mice to conventional and minimally invasive bleeding techniques

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Hormonal stress response of laboratory mice to conventional and minimally invasive bleeding techniques