Interpreting Neuroendocrine Hormones, Corticosterone, and Blood Glucose to Assess the Wellbeing of Anesthetized Rats during Euthanasia

Current recommendations for assessing animal wellbeing during euthanasia suggest that measuring neuroendocrine hormones-such as ACTH, noradrenaline, and adrenaline-is preferable to measuring corticosterone and blood glucose because of the sensitivity of neuroendocrine hormones to the acute stress associated with rapid methods of euthanasia. However, theseneuroendocrine hormones can be stimulated in ways that confound interpretation of welfare assessment in euthanasia studies.Although this property does not negate the usefulness of neuroendocrine hormones as tools of assessment, it is importantto differentiate the stress associated with the induction of anesthesia before the loss of consciousness (an animal wellbeingconcern) with the physiologic responses that occur after the loss of consciousness (not an animal wellbeing concern). In thisstudy, rats were anesthetized by using a ketamine-xylazine combination. Once the rats achieved a surgical plane of anesthesia,they were exposed to O2, CO2, or isoflurane, followed by terminal blood collection to assess concentrations of ACTH,noradrenaline, corticosterone, and blood glucose. Compared with animals exposed to O2 or isoflurane, rats exposed to CO2had significant increases in their serum concentrations of ACTH and noradrenaline, but blood glucose and corticosteronedid not differ between groups. These findings indicate that noradrenaline and ACTH should be used with caution to assessanimal wellbeing when the method of euthanasia might confound that assessment

Home Cage Compared with Induction Chamber for Euthanasia of Laboratory Rats

This study compared behavioral and physiologic changes in Sprague-Dawley and Brown Norway rats that were euthanizedby using a 30% volume displacement rate of CO2 in either their home cage or an induction chamber; rats euthanized in thehome cage were hypothesized to demonstrate a higher level of animal wellbeing. No significant differences were detectedin the physiologic responses to home cage versus induction chamber euthanasia groups. A few strain-related behavioraldifferences occurred. The number of digs per second was higher in Brown Norway compared with Sprague-Dawley rats when in the home cage, where a digging substrate was present. Rearing frequency was higher in both Brown Norway and Sprague-Dawley rats in the induction chamber compared with the home cage. This study demonstrated that although strainspecific differences were associated with the process of euthanasia, there were no significant differences between the treatment groups of home cage compared with induction chamber. This finding suggests that-from the perspective of a rat-either the home cage or an induction chamber can be used for euthanasia, with likely extension of this conclusion to use of either method to the induction of anesthesia

Wellbeing of Alcohol-preferring Rats Euthanized with Carbon Dioxide at Very Low and Low Volume Displacement Rates

The 2013 AVMA Guidelines on Euthanasia recommend the use of very-low or low flow rates of 100% carbon dioxide to euthanize small rodents. Although inhalation of high concentrations of carbon dioxide are generally recognized as painful in humans, whether the use of these low-flow methods of euthanasia increase potential distress for rats is unclear. This study compared physiologic and behavioral markers of animal wellbeing for rats euthanized by using 10% volume displacement per minute (VD/min), 30% VD/min, and 70% VD/min of 100% carbon dioxide. Rats were recorded during euthanasia for subsequent behavioral scoring, and blood samples were taken after euthanasia for assessment of blood glucose and serum corticosterone levels. In this study, rats euthanized with 10% or 30% VD/min of 100% carbon dioxide demonstrated increases in various behaviors, such as rearing and standing, concurrent with increases in serum corticosterone. Rats euthanized with 70% VD/min of 100% carbon dioxide did not exhibit these changes. The results suggest that a euthanasia method of 70% VD/min of 100% carbon dioxide may minimize potential pain and distress and thus be more humane for rats, as compared with very-low- and low-flow methods of carbon dioxide euthanasia

Effects of Music Enrichment on Individually Housed Male New Zealand White Rabbits

The beneficial effect of music has been demonstrated in many species. Although commercially available music CDs intended for use with rabbits are available, these enrichments have not been critically evaluated to determine whether they reduce distress. In this study, we used an aging colony of male rabbits to evaluate how the provision of music enrichment affected the wellbeing of the colony. After 6 mo of music enrichment, the rabbits in the colony demonstrated a significant decrease in fecal cortisol, suggesting that their stress was reduced. Six months after removal of the music enrichment, the rabbits demonstrated a significant increase in fecal cortisol and the heterophil:lymphocyte ratio, suggesting that they werestressed. These findings suggest that music enrichment with a commercially available music discs for rabbits can be used toimprove the wellbeing of animals used in biomedical research

Effects of Various Commercially Available Enrichment Options on Handling and Chronic Stress Markers in Female ICR Mice

Although social housing of mice generally is preferred, mice must be individually housed in some situations. In these cases, enhanced attention to environmental enrichment is encouraged, but few studies assess the wellbeing of mice provided various enrichments. In this study, we used female ICR mice to evaluate enrichment strategies that encouraged natural behaviors including foraging, exercise, sheltering, and socialization. After 3 mo of exposure to the assigned enrichment strategy, wellbeing was assessed by evaluating behavioral and physiologic differences between groups. The results suggested that the use of red-tinted igloos may decrease markers of mouse wellbeing. However, none of the selected strategies yielded measures of wellbeing indicating improvement as compared to individually housed mice with no enrichment (negative control). Furthermore, measures were not significantly different between paired mice and individually housed mice with no enrichment

Evaluation of Low versus High Volume per Minute Displacement CO₂ Methods of Euthanasia in the Induction and Duration of Panic-Associated Behavior and Physiology

Current recommendations for the use of CO ₂ as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO ₂ that are associated with pain. However, exposing rats to CO ₂ , concentrations as low as 7% CO ₂ are reported to cause distress and 10%-20% CO ₂ induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO ₂ concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO ₂ that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO ₂ for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO ₂ exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO ₂ also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO ₂ infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO ₂ and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO ₂ , while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal

Development of a step-down method for altering male C57BL/6 mouse housing density and hierarchical structure: Preparations for spaceflight studies

This study was initiated as a component of a larger undertaking designed to study bone healing in microgravity aboard the International Space Station (ISS). Spaceflight experimentation introduces multiple challenges not seen in ground studies, especially with regard to physical space, limited resources, and inability to easily reproduce results. Together, these can lead to diminished statistical power and increased risk of failure. It is because of the limited space, and need for improved statistical power by increasing sample size over historical numbers, NASA studies involving mice require housing mice at densities higher than recommended in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). All previous NASA missions in which mice were co-housed, involved female mice; however, in our spaceflight studies examining bone healing, male mice are required for optimal experimentation. Additionally, the logistics associated with spaceflight hardware and our study design necessitated variation of density and cohort make up during the experiment. This required the development of a new method to successfully co-house male mice while varying mouse density and hierarchical structure. For this experiment, male mice in an experimental housing schematic of variable density (Spaceflight Correlate) analogous to previously established NASA spaceflight studies was compared to a standard ground based housing schematic (Normal Density Controls) throughout the experimental timeline. We hypothesized that mice in the Spaceflight Correlate group would show no significant difference in activity, aggression, or stress when compared to Normal Density Controls. Activity and aggression were assessed using a novel activity scoring system (based on prior literature, validated in-house) and stress was assessed via body weights, organ weights, and veterinary assessment. No significant differences were detected between the Spaceflight Correlate group and the Normal Density Controls in activity, aggression, body weight, or organ weight, which was confirmed by veterinary assessments. Completion of this study allowed for clearance by NASA of our bone healing experiments aboard the ISS, and our experiment was successfully launched February 19, 2017 on SpaceX CRS-10

Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats : A Systematic Review

Background: There has been increased concern about the suitability of CO as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO; and the availability of better alternatives. These discussions have been useful in providing new information, but have resulted in significant confusion regarding the acceptability of CO for rodent euthanasia. In some cases, researchers and veterinarians have become uncertain as to which techniques to recommend or use for euthanasia of laboratory mice and rats. Methods: The International Association of Colleges of Laboratory Animal Medicine (IACLAM) convened a taskforce to examine the evidence for adverse welfare indicators in laboratory rats and mice undergoing CO euthanasia using a SYRCLE-registered systematic review protocol. Of 3,772 papers identified through a database search (PubMed, Web of Science, CAB Direct, Agricola, and grey literature) from 1900 to 2017, 37 studies were identified for detailed review (some including more than one species or age group), including 15 in adult mice, 21 in adult rats, and 5 in neonates of both species. Experiments or reports were excluded if they only assessed parameters other than those directly affecting animal welfare during CO induction and/or euthanasia. Results: Study design and outcome measures were highly variable and there was an unclear to high risk of bias in many of the published studies. Changes in the outcome measures evaluated were inconsistent or poorly differentiated. It is likely that repeated exposures to carbon dioxide inhalation are aversive to adult rats and mice, based on avoidance behavior studies; however, this effect is largely indistinguishable from aversion induced by repeated exposures to other inhalant anesthetic gasses. Conclusion: There is insufficient evidence to permit an unbiased assessment of the effect of CO inhalation during euthanasia on welfare indicators in laboratory mice and rats. Additional well-designed, unbiased, and adequately powered studies are needed to accurately assess the welfare of laboratory mice and rats undergoing euthanasia via CO gas

Impact of Optimized Breastfeeding on the Costs of Necrotizing Enterocolitis in Extremely Low Birthweight Infants

To estimate risk of NEC for ELBW infants as a function of preterm formula and maternal milk (MM) intake and calculate the impact of suboptimal feeding on NEC incidence and costs