Acute Effects of Hypothermia and Inhalant Anesthesia on Ultrasonic Vocalizations and Neuroendocrine Markers in Neonatal Rats.

Neonatal rodents undergo anesthesia for numerous procedures and for euthanasia by anesthetic overdose. However, data regarding whether neonatal anesthesia is humane are limited. Hypothermia (cryoanesthesia) is the most commonly used anesthetic protocol for neonatal rats 10 d of age or younger. However, hypothermia has recently been restricted in several countries due to perceived painful effects, including pain on rewarming. Minimizing the potential pain and distress of neonates in research is imperative, although very challenging. Traditional validated and nonvalidated behavioral and physiologic outcome measures used for adult rats undergoing anesthesia are unsuitable for evaluating neonates. Therefore, we investigated the effects of several anesthetic methods on neonatal rats by using the innovative objective approaches of noninvasive ultrasonic vocalizations and more invasive neuroendocrine responses (i. e., serum corticosterone, norepinephrine, glucose). Our results show that hypothermia leads to heightened acute distress in neonatal rats as indicated by prolonged recovery times, increased duration of vocalizations, and elevated corticosterone levels, as compared with neonates undergoing inhalational anesthesia. We demonstrate that inhalational anesthesia is preferable to cryoanesthesia for neonatal rats, and researchers using hypothermia anesthesia should consider using inhalational anesthesia as an alternative method.

Euthanasia of Neonatal Rats and Mice using Carbon Monoxide.

Minimization of potential pain and distress of rodents undergoing euthanasia is a touchstone of veterinary clinical medicine. Evaluation of this issue in postweanling rodents has supported revisions to the AVMA (American Veterinary Medical Association) Guidelines on Euthanasia in 2020. However, relatively little information is available on humane aspects of anesthesia and euthanasia in neonatal mice and rats. These neonates are not reliably euthanized by exposure to commonly used inhalant anesthetic agents due to their physiologic adaptations to hypercapnic environments. Therefore, options such as prolonged inhalant anesthetic gas exposure, decapitation, or use of injectable anesthetics are recommended for neonates. All of these recommended methods have operational implications, ranging from reported job dissatisfaction by animal care staff to rigorous reporting requirements associated with the use of controlled substances. This lack of a euthanasia method that does not entail operational issues hampers the ability of veterinary professionals to provide appropriate guidance to scientists working with neonates. This study was designed to assess the effectiveness of carbon monoxide (CO) as an alternative euthanasia agent for mouse and rat pups on postnatal days (PND) 0 to 12. The study demonstrates that CO may be a potential alternative for preweanling mice and rats at PND6 or older but is not appropriate for neonates at PND5 or younger.

Gender of Authors in Laboratory Animal Medicine and Science in 2 Peer-Reviewed U. S. Journals.

Multiple recent surveys have examined the prevalence of female first or senior authors on publications for various scientific and medical disciplines. First and senior authorships are significant achievements for purposes of professional advancement, especially in academia. Such surveys can also provide information regarding diversity and inclusion. In this report, we present the findings of a survey performed to assess how frequently female contributors were first or senior authors in 2 of the most widely-circulated peer-reviewed journals of laboratory animal medicine and science in the United States; data were collected at 3 time points over a recent 20-y span. These data were then compared against estimated populations of potential female authors, as determined from membership rolls in the American Society of Laboratory Animal Practitioners and the American College of Laboratory Animal Medicine. Survey results suggest that female authors increased their representation as influential authors over time, in contrast to representation trends reported for other disciplines. However, whether this increase has mirrored the increase in women overall in the veterinary profession during this time span is unknown. In an era of greater attention and sensitivity to equity and inclusion, this survey is offered as a starting point for further conversation within the field of laboratory animal medicine and science.

Minimal Exposure Times for Irreversible Euthanasia with Carbon Dioxide in Mice and Rats.

When using an anesthetic overdose to euthanize laboratory rodents, a secondary method of euthanasia is recommended to ensure that the apparent death is irreversible. This secondary method usually is accomplished through the collection of tissues that are required to complete the research project. However, frequently laboratory rodents must be euthanized because they cannot be used for studies; in these cases, caretakers must perform a secondary method of euthanasia. Performing physical methods of euthanasia, even on unconscious rodents, can contribute to compassion fatigue in these persons. The current study was designed based on existing literature regarding minimal exposure times for preweanling rats and mice euthanized with carbon dioxide. The study evaluated the minimal time that adult rats and mice must remain in 100% carbon dioxide for death to be irreversible on removal. Adult rats (14 stocks and strains) and mice (more than 40 stocks and strains) were euthanized using a 50% volume per minute displacement rate of carbon dioxide for 2 min. The cages were then left undisturbed for predetermined times, ranging from 0 to almost 12 min. Upon removal from the cage, the animals were stimulated to determine whether they could be resuscitated. If an animal recovered, it was euthanized by using a physical method of euthanasia, and a duration that was 30 s longer than the previous predetermined time was assessed using other animals. The study demonstrated that exposure times of at least 3 min in carbon dioxide reliably result in irreversible euthanasia of mice but that exposure times of at least 10.5 min in carbon dioxide were required to ensure irreversible euthanasia of rats. Although an irreversible death can be attained with carbon dioxide, the use of appropriate species-specific exposure times is critical.

Effectiveness of the Glass Bead Sterilizer for Sterilizing Surgical Instruments.

Survival rodent surgery requires the use of sterile instruments for each animal, which can be challenging when performing multiple surgeries on batches of animals. Glass bead sterilizers (GBS) are widely considered to facilitate this practice by sterilizing the tips of the instruments between animals. However, other disciplines have raised questions about the efficacy of the GBS, especially when used with surgical tools that have grooves or ridges that may contain organic debris. In this study, we evaluated the efficacy of the GBS to sterilize instruments commonly used in rodent surgery by intentionally contaminating a selection of instruments with a standardized bacterial broth inoculated with Staphylococcus aureus and Escherichia coli. As expected, a simple ethanol wipe was ineffective in sterilizing instruments in all treatment groups. An ethanol wipe followed by GBS was effective in sterilizing 82.5% (99 of 120) of the instruments. Our study suggests that the GBS may not be effective for consistent sterilization of surgical instruments.

Evaluation of Carbon Dioxide Euthanasia of Female Sprague Dawley Rats Alone or With Unfamiliar Conspecifics.

Most studies evaluating methods of euthanasia to date have focused on the euthanasia of individual animals. However, larger chambers are commonly used to euthanize multiple cages of animals at once. This study evaluated the use of a commercially available system for euthanasia of 1, 2, or 4 cages containing an individual female Sprague-Dawley rat using volume per minute displacement rates (VDR/min) of either 25% or 50% of 100% carbon dioxide. Animal wellbeing was assessed based on physiologic changes (serum noradrenaline and corticosterone) and behavioral assessments (relative frequency of rearing, line crossing, and grooming). The 25% VDR/min was associated with a significantly longer time to loss of consciousness, but this was not associated with significant physiologic or behavioral changes. The 50% VDR/min treatment group was associated with significant increases in the relative frequency of movement from 1 side of the cage to the other. Increases in the relative frequency of rears were detected in the 25% VDR/min treatment group when 2 or 4 rats were in the chamber as compared with a single rat in the chamber. The absence of significant physiologic changes suggest that the behavioral changes may have been associated with the novelty of the euthanasia experience rather than with distress. The location of the cage within the chamber did not significantly affect any of the measured parameters at either 25% or 50% VDR/min. These data suggest that groups of rats euthanized in these chambers are not experiencing decreases in their welfare.

Evaluation of Treatment Options for Ulcerative Dermatitis in the P Rat.

Rotational outbred adult rats, phenotypically selected to prefer drinking alcohol ("P" rats) frequently present with selfinflicted wounds and ulcerative dermatitis, similar to that seen in C57BL/6 mice. Historically, veterinary interventions used to address this clinical condition have included triple antibiotic ointment (TABO), Columbia wound powder (CPW), nail trims, or plastic tubes that allow affected animals to hide. More recent studies have suggested that nail trims are the most successful intervention in mice, but this has not been evaluated previously in rats. In this study, we evaluated nail trims in rats and also tested whether placing a pumice stone in the cage would reduce the need for nail trims to reduce self-inflicted wounds. Our hypothesis was that interacting with the pumice stone would dull/trim the rats' nails without causing stress or illness and allow the wounds time to heal. We used 66 P rats that were assigned to 1 of 6 treatment groups (pumice stone, TABO, CWP, huts, nail trims, and an untreated control group) of 11 rats each. Rats were transferred to this study from a colony of experimentally naïve animals that had evidence of dermatitis. The wounds were photographed and measured for 12 wk at 2 wk intervals. At the end of the study, representative skin samples from the site of the wound were collected for histopathologic evaluation of inflammation. Our data showed no significant differences in the inflammation scores. The rats treated with nail trims healed significantly more often than did all of the other treatment groups. This suggests that nail trims are the most effective intervention for treating self-inflicted wounds in P rats.

Wellbeing of Mice Euthanized with Carbon Dioxide in Their Home Cage as Compared with an Induction Chamber.

The AVMA Guidelines on Euthanasia state that, to decrease potential distress of animals, the home cage should be used for the euthanasia of mice. The current study evaluated this recommendation by comparing behavioral and physiologic changes in ICR and SJL mice that were euthanized by using a 30% volume per minute displacement rate of 100% CO2 in either their home cage or an induction chamber. Blood samples were collected to assess blood glucose, serum corticosterone, and serum noradrenaline as markers of physiologic wellbeing. Behavioral assessment was performed (with emphasis on behaviors including rearing, jumping, sniffing at the gas inlet, and grooming) from the introduction of gas to the estimated time to loss of consciousness (i. e., the time period when the animal would be expected to experience pain or distress). Despite significant differences between mouse strains, no significant differences were detected in the physiologic or behavioral parameters assessed when comparing the home cage with the induction chamber. This finding suggests that- from the perspective of a mouse-either the home cage or an induction chamber can be used for induction of anesthesia with CO2 during the euthanasia procedure.

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

Background: There has been increased concern about the suitability of CO2 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 CO2; 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 CO2 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 CO2 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 CO2 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 CO2 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 CO2 gas.

The Impact of Social and Behavioral Factors on Reproducibility in Terrestrial Vertebrate Models.

The use of animal models remains critical in preclinical and translational research. The reliability of the animal models and aspects of their validity is likely key to effective translation of findings to medicine. However, despite considerable uniformity in animal models brought about by control of genetics, there remain a number of social as well as innate and acquired behavioral characteristics of laboratory animals that may impact on research outcomes. These include the effects of strain and genetics, age and development, sex, personality and affective states, and social factors largely brought about by housing and husbandry. In addition, aspects of the testing environment may also influence research findings. A number of considerations resulting from the animals' innate and acquired behavioral characteristics as well as their social structures are described. Suggestions for minimizing the impact of these factors on research are provided.

Review of Rodent Euthanasia Methods.

The optimal choice of euthanasia method for laboratory rodents depends on a number of factors, including the scientific goals of the study, the need to minimize animal pain and/or distress, applicable guidelines and laws, the training and proficiency of personnel, and the safety and emotional needs of the personnel performing the euthanasia. This manuscript aims to provide guidance to researchers so they may select the method of euthanasia that results in minimal experimental confounds, such as the creation of artifact and alteration of tissues and analytes. Specific situations addressed include euthanasia of large numbers of rodents and euthanasia of neonates. Recent literature supports the notion of significant strain-dependent differences in response to euthanasia methods such as CO2 inhalation. To assist researchers in selecting a strain-appropriate method of euthanasia, the authors present a summary of methodologies for assessing the effectiveness of euthanasia techniques, including elements and parameters for a scoring rubric to assess them.

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.

Characterization and Etiology of Swollen Muzzles in Irradiated Mice.

Several investigators performing bone marrow transplantation studies have previously reported sporadic increases in mortality that were associated with pronounced swelling in the face, head and neck of mice. Over the past few years, we and others have noted an increasing number of experiments in which mice that have received total-body irradiation (TBI) or partial-body irradiation (PBI) develop swollen muzzles, drastic thickening of the upper lip and redness, bruising and/or swelling around the nose and muzzle and sometimes over the top of the head. We refer to this rapid and extreme swelling after irradiation as swollen muzzle syndrome (SMS). The development of SMS postirradiation is associated with morbidity that occurs earlier than would be expected from the traditional hematopoietic acute radiation syndrome (H-ARS), and has impeded studies in several laboratories attempting to evaluate medical countermeasures (MCM) against radiation. However, little has been done to characterize this somewhat unpredictable radiation effect. To investigate the cause and etiology of SMS, data from three different laboratories collected over a seven-year period from 100 MCM 30-day survival studies using mice from different vendors were retrospectively analyzed to determine the time of onset, progression and incidence of SMS in male and female mice exposed to various doses of ionizing radiation. An additional study compared incidence and etiology of SMS in mice from two different vendors (identified as vendors A and B) after exposure to the LD50/30 (X rays). Mice presenting with SMS, as well as non-SMS (irradiated) control mice, were necropsied to determine microbial status of the blood, heart, spleen, liver, kidney and muzzle tissue. Only mice from vendor A (20%) developed SMS. While the number of bacterial species isolated from various tissues of SMS and non-SMS mice was not different, the number of tissues positive for bacteria was significantly greater in SMS mice. At least one tissue in 83% of SMS mice from vendor A tested positive for Streptococcus agalactiae [group B beta Streptococcus (GBS)], compared to 25% of non-SMS mice from vendor A, and 0% of non-SMS mice from vendor B. In addition, all mice from vendor A with SMS had at least one tissue with >104 CFU/g, with GBS as the predominant bacterium, compared to only 25% of non-SMS mice from vendor A, and 0% of non-SMS mice from vendor B. The incidence and magnitude of GBS growth in cultures correlated with the onset of SMS; the earliest and heaviest infections occurred in mice presenting with SMS on days 5-6 postirradiation. The majority of SMS mice (5 out of 6) had positive blood cultures, with the same bacterial strain isolated from other tissues, suggesting systemic translocation via the bloodstream. We propose that testing of mice and the identification of the microorganisms frequently associated with SMS may provide guidance for selection of antimicrobials for use by other investigators in studies evaluating potential MCM, and for the ordering, handling and care of immunodeficient mice or mice that are to be rendered immunodeficient after acute irradiation.

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.

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.

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.

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.

Cohousing Male Mice with and without Segmental Bone Defects.

Spaceflight results in bone loss like that associated with osteoporosis or decreased weight-bearing (for example, high-energy trauma such as explosive injuries and automobile accidents). Thus, the unique spaceflight laboratory on the International Space Station presents the opportunity to test bone healing agents during weightlessness. We are collaborating with NASA and the US Army to study bone healing in spaceflight. Given the unique constraints of spaceflight, study design optimization was required. Male mice were selected primarily because their femur is larger than females', allowing for more reproducible surgical outcomes. However, concern was raised regarding male mouse aggression. In addition, the original spaceflight study design included cohousing nonoperated control mice with mice that had undergone surgery to create a segmental bone defect. This strategy prompted the concern that nonoperated mice would exhibit aggressive behavior toward vulnerable operated mice. We hypothesized that operated and nonoperated male mice could be cohoused successfully when they were cagemates since birth and underwent identical anesthetic, analgesic, preoperative, and postoperative conditions. Using quantitative behavioral scoring, body weight, and organ weight analyses (Student t test and ANOVA), we found that nonoperated and operated C57BL/6 male mice could successfully be housed together. The male mice did not exhibit aggressive behavior toward cagemates, whether operated or nonoperated, and the mice did not show evidence of stress, as indicated by veterinary assessment, or change in body or proportional organ weights. These findings allowed our mission to proceed (launched February 2017) and may inform future surgical study designs, potentially increasing housing flexibility.

Bias in Rating of Rodent Distress during Anesthesia Induction for Anesthesia Compared with Euthanasia.

Selection of an appropriate method of euthanasia involves balancing the wellbeing of the animal during the procedure with the intended use of the animal after death and the physical and psychologic safety of the observer or operator. The recommended practices for anesthesia as compared with euthanasia are very disparate, despite the fact that all chemical methods of euthanasia are anesthetic overdoses. To explain this disparity, this study sought to determine whether perception bias is inherent in the discussion of euthanasia compared with anesthesia. In this study, participants viewed videorecordings of the anesthesia of either 4 rats or 4 mice, from induction to loss of consciousness. Half of the participants were told that they were observing anesthesia; the other half understood that they were observing euthanasia. Participants were asked to rate the distress of the animals by scoring escape behaviors, fear behaviors, respiratory distress, and other distress markers. For mice, the participants generally rated the distress as high when they were told that the mouse was being euthanized, as compared with the participants who were told that the mouse was being anesthetized. For rats, the effect was not as strong, and the distress was generally rated higher when participants were told they were watching anesthesia. Because the interpretation of distress showed bias in both species-even though the bias differed regarding the procedure that interpreted as distressing-this study demonstrates that laboratory animal professionals must consider the influence of potential perception bias when developing policies for euthanasia and anesthesia.