Current Status of the Zebrafish Euthanasia and Humane Endpoint in the Republic of Korea and Guideline Suggestion from Nationwide Expert Elicitation: A Model for Other Countries.

Since its introduction as a model organism in the 1980s, the use of zebrafish (Danio rerio) in research has expanded worldwide. Despite its now widespread use in research, guidelines to safeguard the ethical treatment of zebrafish, particularly with regard to euthanasia and humane endpoint practices, remain inadequate. One well-recognized example is the use of excess tricaine methanesulfonate (MS-222) as a means to euthanize zebrafish, regardless of life stage. In this study, through nationwide expert elicitation, we provide a detailed account of zebrafish research practices within the Republic of Korea and the challenges of implementing appropriate methods for euthanasia as a humane endpoint, with many opting for hypothermic shock. We report a local expert consensus for establishing national guidelines to improve zebrafish welfare and good research practice. Suggestions and recommendations for national guidelines were offered. Taken together, our findings raise awareness broadly among zebrafish research practitioners in the field, offer an accurate account of the welfare and treatment of zebrafish in research within the Republic of Korea, and advocate for the development and implementation of national guidelines. As such, our study is useful as a model to adopt the expert elicitation approach to investigate, quantify, and address welfare concerns in zebrafish research, and to establish best practice guidelines.

Efficacy of a novel cervical dislocation tool for humane euthanasia of broilers and broiler breeders.

Euthanasia is an essential task performed daily on commercial poultry farms around the world to safeguard animal welfare. Manual cervical dislocation (MCD) is the most common euthanasia method but can be challenging to perform given the physical strength required to implement this technique. Therefore, the objective of this study was to evaluate the efficacy of a novel cervical dislocation tool (NCDT) compared to MCD. A total of 60 Ross 308 chickens (6-wk old) and 60 Ross 706 parent stock breeders (21-wk old) were enrolled in the study. Birds were sexed, blocked by body weight, and allocated to 1 of 2 treatments: 1) MCD and 2) NCDT. Immediately following euthanasia application, insensibility, and death were monitored. Once death was confirmed, gross evaluation, radiograph, and macroscopic/microscopic scoring were performed. Both euthanasia methods were 100% effective in achieving insensibility followed by cardiac and respiratory arrest in both age groups. In 6-wk-old broilers, there were no differences in insensibility measures or location and severity of the dislocation site by treatment. The NCDT treatment group showed an increased frequency of fractures located at the tooth-like process that projects from the cranial aspect of the centrum of the axis (dens) but had no impact on bird insensibility. For parent stock, differences in nictitating membrane reflex (NMR) and laceration scores for birds euthanized with NCDT were found and likely associated with additional force exerted with the tool. The NCDT is a promising replacement for MCD and future work should address the development of free and accessible training materials for on-farm use.

Anesthesia and humane euthanasia methods for king worm larvae (Zophobas morio).

A growing spotlight on insect welfare as research subjects, fodder for vertebrate animals, and patients for veterinarians highlights the need to establish humane euthanasia methods for these species. An insect of increasing commercial importance is the king worm (Zophobas morio), a type of darkling beetle larvae. The initial objective of this study was to determine if prolonged exposure to isoflurane would lengthen anesthetic recovery and lead to euthanasia. Larvae were exposed to isoflurane for 10 min, 40 min, 70 min, 190 min, 13 h, and 24 h (n = 10 for each treatment duration group), and time to recovery was subsequently monitored in room air. The second objective was to determine effectiveness of secondary euthanasia methods: injectable potassium chloride at 1 mg/g body weight (n = 5) and 3 mg/g body weight (n = 5) doses; immersion in 200 proof ethanol (n = 10), 10% buffered formalin (n = 10), and water (n = 10); and freezing at -18°C (n = 10). Prolonged exposure to isoflurane led to extended recovery times, but 100% mortality was not achieved. Submersion in ethanol and injection of potassium chloride at a 3 mg/g body weight dose resulted in 100% mortality. If these larvae are to be used as future food sources, a humane euthanasia method that preserves the ability to be consumed is required.

Swine industry stakeholders' perception on the use of water-based foam as an emergency mass depopulation method.

The U.S. pork supply chain is vulnerable to various internal and external threats and in need of prompt, comprehensive response plans. Under urgent circumstances, for example in the case of foreign disease incursions, swine farms will have to perform on-farm animal depopulation to prevent disease spread. Several animal depopulation methods including water-based foam (WBF) have been proposed and are under evaluation for feasibility in the field. However, the psychological/emotional impacts of applying depopulation methods for personnel managing and carrying on the tasks are not currently well understood. Thus, this study aimed to investigate WBF as an alternative for depopulation compared to existing methods approved by the American Veterinary Medical Association. Swine industry stakeholders were invited to voluntarily observe a WBF depopulation trial and to provide their self-reported perspectives before and after the observation. A survey was designed to explore key areas on expected and perceived method effectiveness, efficiency, and animal welfare considerations, as well as to evaluate short-term post-observation psychological impacts. Among 42 recruited stakeholders, 31.0% (13/42) were educators/researchers followed by animal health officials (26.2%, 11/42) and veterinarians (23.8%, 10/42), with an average of 11.7 ± 12.6 (n = 39) years of work experience. After the trial, respondents' positive perception of WBF depopulation increased specifically regarding the animal loading process being less stressful than restrained in-barn depopulation options (P = 0.003) and by the observation of fewer swine escape attempts and vocalizations than expected (P < 0.001). Respondents' positive perception of WBF also increased regarding to the time required to fill the trailer with foam, to stop hearing animal vocalization, and stop hearing animal movement, as the observed trial times were faster than their pre-observation estimates (P < 0.001). Additionally, 79.5% (31/39) of respondents agreed that the rapid destruction of animal populations had priority over animal welfare under urgent scenarios. Minor post-traumatic stress disorder-like (PTSD-like) symptoms from the observed trials were reported (26.7%, 4/15 respondents) one month after the observation. This study showed that the WBF depopulation process was perceived positively by swine stakeholders and may have limited short-term psychological impacts on personnel involved in animal depopulation.

POTASSIUM CHLORIDE ADMINISTERED VIA FOUR ROUTES FOR EUTHANASIA OF ANESTHETIZED GOLDFISH (CARASSIUS AURATUS).

Immersion overdose in tricaine methanesulfonate (MS-222) is ineffective for euthanasia of adult goldfish (Carassius auratus), and investigation of alternative techniques is warranted. This study evaluated potassium chloride (KCl) administered via four routes for goldfish euthanasia. Thirty clinically healthy adult goldfish (17.2-41.9 g) were anesthetized via immersion in buffered MS-222 (300 mg/L) for 5 min beyond anesthetic induction. Fish were then randomly administered one of five treatments: KCl (333 mg/mL, 4.5 mmol/mL, 4.5 mEq/mL) via intracardiac injection at 10 mEq/kg (IC), intracoelomic injection (ICe) or bilateral topical delivery over the gill filaments (T) at 100 mEq/kg, or 90-min immersion at 4,500 mEq/L (saturated solution) dissolved in induction solution (water [W]), or no treatment (control [C]). Following treatment, all fish were moved to anesthetic-free freshwater. Serial heart rates were collected via Doppler device until sound cessation or recovery. Median (range) time to perform treatments was 5.3 (1.2-8.0) min, 3 (3-3) s, and 10 (10-10) s in IC, ICe, and T, respectively. Doppler cessation occurred in 6/6, 6/6, 6/6, 6/6, and 0/6 fish in median (range) times of 3 (0-210), 18 (10-45), 118 (90-390), and 150 (60-240) min in IC, ICe, T, W, and C, respectively; this was significantly different between groups (P = 0.002). Following or during treatment, 1/6, 2/6, 6/6, and 4/6 fish in IC, ICe, T, and W, respectively, exhibited intermittent transient jerking movements. Median (range) time to recovery in C was 5.5 (3.5-6.5) min. All administration routes were effective, but time to Doppler cessation varied and transient movements were noted. Preliminary follow-up research determined that return to MS-222 induction solution following ICe KCl treatment abolished movements. ICe KCl at 100 mEq/kg was technically simple and resulted in Doppler sound cessation in a clinically relevant time frame in 6/6 anesthetized goldfish. Return to MS-222 immersion following KCl administration is recommended.

Intracardiac Induced Ventricular Fibrillation for the Euthanasia of Sheep.

Euthanasia is the humane termination of an animal's life and an important consideration for scientists, veterinarians, regulators, and others contemplating investigations involving animals. Techniques for euthanasia must induce the most rapid, painless, and distress-free death possible. This study investigated the effectiveness of direct current induction of ventricular fibrillation for the euthanasia of sheep after a primary study in which artifacts or chemical contamination from injectable euthanasia agents were undesirable. Female crossbred adult sheep (Ovis aries; n = 12) under deep isoflurane general anesthesia were instrumented with electrophysiology catheters to induce ventricular fibrillation for euthanasia. Data regarding invasive arterial blood pressure, expired airway gases, limb lead electrocardiograms, and pulse oximetry were collected and assessed just prior to, immediately after, and at 5, 10, 15, and 20min after energy delivery. In all animals, a single 10-s application of 9V of direct current to the right ventricular endocardium via the electrophysiology catheter induced persistent ventricular fibrillation. Arterial blood pressure (mean ± 1 SD) immediately after fibrillation induction was 22.9±4.5mmHg, with negligible difference between systolic and diastolic pressures. The lack of differential pressure continued through the end of the monitoring period. Arterial blood pressure reached an initial nadir at 1??0.5min after fibrillation induction, peaked (40.8±11.1mmHg) due to a vasoconstrictive reflex at 3min after induction, and returned to a static uniform pressure (20.4±17.8mmHg) with mildly increased variability due to reflexive diaphragmatic contractions at 10min after induction. The use of 9V direct current for the induction of ventricular fibrillation via an electrophysiology catheter is a reliable method of euthanasia in sheep.

Investigation of Potassium Chloride for Euthanasia of Anesthetized African Clawed Frogs (Xenopus laevis).

Euthanasia is frequently performed in amphibians, but techniques are currently limited in number and variable in effectiveness. The current study examined the use of potassium chloride (KCl) for euthanasia of anesthetized African clawed frogs (Xenopus laevis). Twenty adult, female African clawed frogs were anesthetized by immersion in buffered tricaine methanesulfonate (MS-222) for 5 min beyond loss of righting reflex. Frogs were then randomly assigned to receive one of 4 treatments: KCl via intracardiac injection (10 mEq/kg; IC, n = 5), intracoelomic injection (100 mEq/kg; ICe, n = 5), immersion (4,500 mEq/L; IMS, n = 5), or no treatment (C, n = 5). After treatment, serial heart rate was measured via Doppler device until either the loss of Doppler sounds, a 60-min endpoint (IC, ICe, IMS), or recovery (C). Times to loss of righting reflex, loss of Doppler sounds, and/or recovery were recorded. Plasma potassium concentrations were measured immediately after Doppler sound cessation in frogs in IC (n = 1), ICe (n = 2), and IMS (n = 5). Injection failure occurred in 1 IC frog, and 1 Ice frog regained spontaneous movement 4 min after treatment administration. Data from these 2 frogs were not included in statistical analysis. Doppler sound cessation occurred in 4 of 4, 4 of 4, 0 of 5, and 0 of 5 frogs in IC, ICe, IMS, and C, respectively. Median (range) times to Doppler sound cessation in IC and ICe were 6 (0 to 16) s and 18 (10 to 25) min, respectively. Plasma potassium concentration was greater than 9.0 mmol/L in sampled frogs. Intracardiac KCl at 10 mEq/kg and intracoelomic KCl at 100 mEq/kg were effective for euthanasia of anesthetized African clawed frogs. Return to MS-222 solution after KCl administration may be warranted to prevent unintended, premature anesthetic recovery prior to death.

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.

Striving for humane deaths for laboratory mice: hypobaric hypoxia provides a potential alternative to carbon dioxide exposure.

Killing is often an unavoidable and necessary procedure for laboratory mice involved in scientific research, and providing a humane death is vital for public acceptance. Exposure to carbon dioxide (CO2) gas is the most widely used methodology despite well proven welfare concerns. Consequently, the continued use of CO2 and its globally permitted status in legislation and guidelines presents an ethical dilemma for users. We investigated whether killing with hypobaric hypoxia via gradual decompression was associated with better welfare outcomes for killing laboratory mice. We compared the spontaneous behaviour of mice exposed to CO2, decompression or sham conditions, and used analgesic or anxiolytic interventions to determine their relative welfare impact. Gradual decompression resulted in longer times to unconsciousness and death and the pharmacological interventions support the notion of a minimally negative animal experience, while providing further evidence for pain and anxiety associated with exposure to CO2. Decompression resulted in moderate ear haemorrhage, but our welfare assessment suggests this may happen when mice are unconscious. Hence, gradual decompression could be the basis of significant refinement for killing laboratory mice. Future work should corroborate behaviour with neurobiological markers of loss of consciousness to verify the conscious phase of concern for animal welfare.

Intrathecal mepivacaine after general anesthesia is an effective method of equine euthanasia when compared to intravenous pentobarbital.

OBJECTIVES: This study aims to assess intrathecal mepivacaine for euthanasia in anesthetized horses and compare it to a traditional euthanasia method using a single intravenous injection of pentobarbital in sedated horses. ANIMALS: Client-owned horses and horses requiring euthanasia due to involvement in concurrent research projects were used. Horses were randomly assigned to 1 of 2 groups: intrathecal mepivacaine after anesthesia or intravenous pentobarbital after sedation. All horses had normal vital parameters and no signs of infectious disease at the time of euthanasia. PROCEDURES: The intrathecal mepivacaine group was anesthetized before the intrathecal injection of mepivacaine. The pentobarbital group was sedated, concurrently anesthetized and euthanized using intravenous pentobarbital, then received an intrathecal saline (0.9% NaCl) solution injection to a blind observer. Both groups were sedated with detomidine and the time from sedation to the cessation of vital parameters (respirations, pulse, corneal reflex, and ECG) was recorded. Euthanasias were recorded for review by a blinded anesthesiologist, using an independent scale to assess the quality of sedation, anesthesia induction, and lateral recumbency. RESULTS: Time from detomidine administration to cessation of each vital parameter was significantly longer in the intrathecal mepivacaine group. There was no statistically significant difference in qualitative scores between groups for sedation or induction, but lateral recumbency was subjectively superior in the anesthetized intrathecal mepivacaine group. CLINICAL RELEVANCE: Intrathecal mepivacaine provided a safe, effective, alternative method of euthanasia to intravenous pentobarbital and addresses concerns about barbiturate availability. This study also informs practitioners of what to expect (ie, longer cessation of vital parameters) when using the intrathecal mepivacaine method.

Survey of euthanasia methods used in neotropical chiropterans in São Paulo State, Brazil: critical analyses and proposal for standardization of bat euthanasia methods / evantamento dos métodos de eutanásia utilizados em quirópteros neotropicais no estado de São Paulo, Brasil: análise crítica e proposta de padronização de métodos de eutanásia em morcegos

Zoonotic Surveillance Divisions (ZSDs) rescue and euthanize bats in contact with humans. The euthanasia procedure should be conducted safely for the animal and caretaker and minimal stress for the animal is important. The objectives of this study were: i. evaluate the methods of euthanasia used by ZSDs in the State of São Paulo; ii. compare the methods with national and international guidelines for animal euthanasia practices; iii. assess the methodologies considering taxonomy and eating habits of the main bat species, and iv. propose standardization of euthanasia procedures. Sixty-five ZSDs locations received an online questionnaire or were contacted by telephone and 33 ZSDs (50.8%) responded and are distributed in 11 mesoregions in the state to remain anonymous. The euthanasia methods were divided into chemical (injectable or inhalation), physical, or mixed methods. Bat specimens (n = 550) were identified and classified to evaluate the main genera found in the state. The location of the ZSD, species, eating habits, and the method of euthanasia used were analyzed. The specimens by bat families were Molossidae (n = 340), Phyllostomidae (n = 171), and Vespertilionidae (n = 39). Chemical methods were used in 25 ZSDs (75.75%), physical in 5 (15.15%), and mixed in 3 (9.09%). There is no uniformity or standardization in bat euthanasia methods used by ZSDs, although most are based on acceptable chemical methods. It was proposed an algorithm to assist the veterinarian in choosing the method of euthanasia for bats that will allow standardizing euthanasia procedures for this species, considering physiological differences, and respecting technical, bioethical, and animal welfare guidelines.(AU)

As Divisões de Vigilância de Zoonoses (DVZs) coletam e eutanasiam morcegos que tiveram contato com humanos. O procedimento de eutanásia precisa ocorrer de modo seguro para o animal e para o manipulador, sendo importante garantir o mínimo de estresse ao animal. Os objetivos deste estudo foram: i. avaliar os métodos de eutanásia praticados nas DVZ do estado de São Paulo; ii. comparar esses métodos com os guias nacionais e internacionais de práticas de eutanásia animal; iii. avaliar as metodologias aplicadas considerando a taxonomia e os hábitos alimentares das principais espécies de ocorrência de morcegos; e iv. propor a padronização dos procedimentos de eutanásia. Sessenta e cinco DVZs receberam o questionário online ou foram contatadas por telefone, dessas, trinta e três DVZs (50,8%) que responderam ao questionário foram incluídas no estudo e distribuídas em 11 mesorregiões para garantir anonimato. Os métodos de eutanásia reportados foram divididos em métodos químicos (injetáveis; inalatórios), físicos ou mistos. Os espécimes de morcegos (n = 550) foram identificados e classificados para avaliar os principais gêneros encontrados no estado. A localização da DVZ, as espécies, os hábitos alimentares e o método de eutanásia utilizado foram analisados. Os espécimes por família de espécies de morcegos foram Molossidae (n = 340), Phyllostomidae (n = 171) e Vespertilionidae (n = 39). Métodos químicos foram utilizados em 25 DVZs (75,75%), físicos em 5 (15,15%) e mistos em 3 (9,09%). Não há uniformidade ou padronização nos métodos de eutanásia de morcegos, embora a maioria seja baseada em métodos químicos aceitáveis. É proposto um algoritmo para auxiliar o veterinário na escolha do método de eutanásia, que permite a padronização desses procedimentos para os morcegos, considerando as diferenças fisiológicas e respeitando as diretrizes técnicas, bioéticas e de bem-estar animal.(AU)

Evaluation of mechanical cervical dislocation, captive bolt, carbon dioxide, and electrical methods for individual on-farm euthanasia of broiler breeders.

Efficacious euthanasia by applying manual cervical dislocation can be difficult on large and mature poultry. The challenge with using manual cervical dislocation is that the strength required to hold heavy poultry and swiftly apply cervical dislocation can be physically impossible for most people. Therefore, alternative methods of euthanasia are needed for mature and large poultry. Mechanical cervical dislocation using the Koechner Euthanizing Device (KED), captive bolt using the Turkey Euthanasia Device (TED), carbon dioxide (CO2), and electrical euthanasia were evaluated for use on 65-wk-old broiler breeders at flock termination. Following application of each method, physiological reflexes including the eye nictitating membrane reflex, mouth gaping, and body movement, broken skin, blood loss, kill success, time to cessation of heartbeat, and blood plasma corticosterone levels were assessed. Birds euthanized using the KED had longer response durations for eye nictitating membrane (91 s) and reflexive mouth gaping (161 s) compared to TED, CO2, and electrical euthanasia (0-7 s). Body movement durations were also longer for KED (214 s) and TED (209 s) than for CO2 and electrical euthanasia (0-8 s). The highest percentages of broken skin (93%) and blood loss (96%) were observed for TED, followed by KED (71%, 68%), then CO2 (0%, 6%) and electrical euthanasia (0%, 3%). No significant differences (P = 0.1781) were observed for kill success rates with 98% for KED, 100% for TED, 97% for CO2, and 100% for electrical euthanasia at 4-min. Time to heartbeat cessation did not differ between KED (659 s), TED (427 s), or CO2 (583 s) euthanasia methods. No heartbeat was detected following electrical euthanasia. Blood plasma corticosterone levels did not differ between preeuthanasia or posteuthanasia from any of the methods applied. Based on these results each euthanasia method is acceptable for use with broiler breeders.

Propofol Immersion As a Euthanasia Method for Adult Zebrafish (Danio Rerio).

The exponential rise of the zebrafish (Danio rerio) as a model organism in biomedical research has far outstripped our un- derstanding of basic husbandry and welfare for this species. As a case in point, here we investigate the efficacy and welfare impact of different euthanasia methods for zebrafish. Not only is a humane death central to welfare and the 3Rs, but stress during euthanasia can change scientific outcomes. However, the most frequently used methods of euthanasia have multiple shortcomings with regard to animal welfare and human safety. In this study, we propose the use of propofol for immersion euthanasia of adult zebrafish. Propofol has been known to rapidly induce anesthesia in many species, including zebrafish, but its efficacy as a euthanasia agent for zebrafish has not fully been explored. In this study, adult zebrafish were euthanized by immersion on one of 5 different preparations: ice bath, 250 ppm MS222, 600 ppm lidocaine hydrochloride, 100 ppm propofol, or 150 ppm propofol for 20 or 30 min. Display of aversive behaviors, time to loss of righting reflex, time to cessation of opercular movement, and time to recovery after transfer to clean tank water were assessed and recorded. Propofol at both concentrations induced loss of righting reflex and loss of opercular movement more quickly than did MS222 or lidocaine hydrochloride and caused no display of aversive behaviors as seen with ice bath or lidocaine exposure. However, fish exposed to propofol at either concentration for 20 min sometimes recovered, whereas a 30-min exposure was sufficient for euthanasia of all fish tested. These findings suggest that exposure to propofol for a duration of at least 30 min quickly and effectively euthanizes adult zebrafish without compromising end-of-life welfare.

Reliability of water-based medium-expansion foam as a depopulation method for nursery pigs and cull sows.

Animal disease preparedness plans including depopulation guidelines are paramount to prevent the spread of emerging infectious diseases but difficult to implement for swine under field conditions. However, water-based foam (WBF) is currently an approved and successfully deployed depopulation methodology in poultry. Therefore, the reliability of WBF as a depopulation method and the effectiveness and irreversibility of consciousness and consequential mortality in pigs of different ages was assessed across two trials. Trial 1 investigated the time to loss of consciousness and cessation of cardiac activity in nursery pigs (n = 72) at six different foam immersion time points (2.5, 5, 7.5, 10, 12.5 and 15 min) when placed in a 1.47 m3 (1.2 × 1.2 × 1.02 m, length × width × height) plastic bulk container. One pig per replicate was implanted with an ECG bio-logger. Irreversible loss of consciousness was observed after a 5-min immersion. The average (SD) time to development of a fatal arrhythmia from the initiation of the foam application was 7.3 min (1.82 s). Trial 2 aimed to validate the findings from Trial 1 in 75 larger cull sows across three replicates (n = 25). Sows were loaded into a 41-m3 sealed trailer (12.2 × 1.5 × 2.24 m), immersed in WBF and left undisturbed for 5 min post foam-filling completion. Six pigs in each replicate were implanted with an ECG bio-logger. A 5-min dwell time resulted in irreversible loss of consciousness and subsequent mortality in all cull sows. The average time (SD) to cessation of movement and fatal arrhythmia post foam-filling completion was 2.2 min (34.8 s) and 8.7 min (138.0 s), respectively. While a 5-min immersion in WBF induced irreversible loss of consciousness and death in both trials, a 7.5-min dwell time followed by observation for confirmation of death post WBF removal would be advisable for pigs of all sizes.

Anesthesia Overdose Versus Rapid Cooling for Euthanasia of Adult Zebrafish.

The rapid increase in zebrafish use needs to be accompanied by research into the refinement of procedures. The European (EU) Directive lists three possible euthanasia methods for fish: anesthetic overdose, electrical stunning, and concussion. However, for small fish such as zebrafish, concussion and electrical stunning are difficult to perform, leaving anesthetic overdose as the most used method. Our aim was to test the efficacy and side effects of anesthesia overdose using different anesthetics and the rapid cooling method to euthanize adult zebrafish. Adult mixed-sex AB zebrafish were randomly assigned to: 250 mg/L MS222; 20 mg/L propofol +100 mg/L lidocaine; 6 mg/L etomidate; 50 mg/L clove oil; and rapid cooling (water at 2°C-4°C). Two minutes after opercular movement ceased, animals were transferred into clean water for 20 min and recovery assessed, or decapitated and used for biochemical analysis of the gills, muscle, liver, and brain; for the histological analysis of the gills and muscle; or for the assessment of cortisol levels. No animal recovered; rapid cooling was the quickest and etomidate overdose was the slowest method to cease the opercular movements. There were no major differences between euthanasia methods regarding the biochemical or histological data. Cortisol levels were higher in the rapid cooling group, but only when compared with the propofol/lidocaine group. The use of a physical method of euthanasia, such as rapid cooling, is essential when chemicals, such as anesthetics, may interfere with postmortem analyses. Although anesthetic overdose can be used without major effects on the analyses conducted in this work, rapid cooling can be another option with the advantage of being simple to administer, easily available, affordable, and very quick; this decreases the potential duration of suffering, being more humane. Therefore, a change in EU legislation should be considered to include additional humane options for euthanasia, such as rapid cooling, for zebrafish and other small tropical fish.

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.

Technical Note: Validation of the effectiveness of electric stunning for euthanasia of mature swine (Sus scrofa domesticus).

Electrocution and the use of a penetrating captive bolt gun (PCBG) are both acceptable methods of euthanasia for market weight swine. Research has demonstrated that a PCBG is effective in both growing and mature swine. Given limited to no published research base on electrocution in mature swine, the objectives of the present study were to evaluate the efficacy of a two-stage (head only followed by head to heart, 10 s contact for each) mobile electric stunner (E-STUN, Hubert HAAS TBG 96N) and to assess euthanasia outcomes when comparing E-STUN with the frontal placement of a heavy-duty PCBG (Jarvis, In-line Cylinder Style) when applied to heavy-weight (>200 kg) mature boars and sows. Effectiveness of the E-STUN and PCBG was evaluated first in unconscious anesthetized mature swine (n = 7 boars and sows per treatment; average weight 282 ± 48 kg, n = 28) to reduce the risk of failure in a conscious animal and then in conscious mature swine (n = 3 boars and sows per treatment; average weight 282 ± 63 kg, n = 12). Data from both stages were combined for analyses. Treatment efficacy was defined as any pig that achieved cardiac and respiratory arrest within 10 min after treatment application. A three-point traumatic brain injury score (0 = normal; 1 = some abnormalities; and 2 = grossly abnormal, unrecognizable) was used to evaluate six neuroanatomical structures (cerebral cortex, cerebellum, hypothalamus, thalamus, pons, and brain stem), and the presence of intracranial hemorrhage was also noted. All animals were immediately rendered insensible with E-STUN and PCBG, and no difference was noted between treatments for the detection of corneal reflex following treatment application (P = 0.11). Rhythmic breathing was absent following the administration of either E-STUN or PCBG. When evaluating the time to last heartbeat, there was a significant interaction between sex and treatment. Boars euthanized via E-STUN had a 346.8-s decrease in time to last heartbeat compared with boars euthanized via PCBG (P < 0.001), and females euthanized via E-STUN had a 479.3-s decrease in time to last heartbeat compared with females euthanized via PCBG (P < 0.001). Intracranial hemorrhage was common for both methods, and visible disruption of neural tissue was evident due to the physical nature of the PCBG. This study demonstrated that a mobile E-STUN system is as effective as a heavy-duty PCBG in inducing insensibility and death and shows promise as an alternative method for euthanizing mature pigs on-farm.

Euthanasia is a moral obligation of all individuals working in the swine industry. A majority of acceptable methods have been validated for market weight pigs, while less attention has focused on heavy-weight mature boars and sows. The objectives of the current study were to evaluate the effectiveness of a mobile electric stunner (E-STUN) as a method of humane euthanasia in heavy-weight mature boars and sows and to assess the outcomes when compared with a penetrating captive bolt gun (PCBG) method. The efficacy of the treatment was defined as any pig that achieved cardiac and respiratory arrest within 10 min after treatment application. The amount of traumatic brain injury was evaluated across the brain, and the presence of intracranial hemorrhage was also noted. All animals were immediately rendered insensible, and rhythmic breathing was absent following either treatment application. Boars and sows had a decreased time to last heartbeat with the E-STUN when compared with the PCBG method. Intracranial hemorrhage was common for both methods, and visible disruption of brain tissue was evident due to the physical nature of the PCBG. This study demonstrated that a mobile E-STUN is as effective as a PCBG for humane euthanasia of heavy-weight mature swine.

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.

Intrahepatic Injection of Sodium Pentobarbital as an Alternative to Intraperitoneal Injection for the Euthanasia of Rats (Rattus norvegicus).

The most commonly accepted method of rat euthanasia in North America is intraperitoneal injection of sodium pentobarbital (PB). However, misinjection can occur, and intraperitoneal PB may cause pain and distress. The objective of this study was to test an alternative method of euthanasia: intrahepatic injection of PB. A pilot study was conducted to develop a method of intrahepatic injections (evaluated using CT scans and test injections), followed by a full study comparing intraperitoneal (n = 14) and intrahepatic PB injections (n = 66) in adult rats. Full study outcomes were: 1) time from injection to loss of right- ing reflex (LORR), 2) time from injection to cessation of heartbeat (CHB), 3) number of failed euthanasia attempts, and 4) confirmation of successful intrahepatic injection or misinjection via necropsy. All injections were performed by a veterinary student. CT revealed that intrahepatic injections were feasible. Times (median [range]) to LORR and CHB were faster after successful intrahepatic injections (LORR, 3 s [1 to 5 s]; CHB, 8 s [2 to 242 s]) than after intraperitoneal injections (LORR, 89.5 s [73 to 110 s], CHB: 284.5 s [237 to 423 s]). The misinjection rate was higher with intrahepatic injections (59%) than with intraperitoneal injections (29%), but intrahepatic misinjection still resulted in fast and successful euthanasia (LORR, 29 s [1 to 96 s]; CHB, 216 s [12 to 330 s]), with the injectate distributed between the intraperitoneal and intrahepatic locations. The number of failed euthanasia attempts with intrahepatic injections was low (n = 2). Intrahepatic injections show potential as an alternative to intraperitoneal injections for rat euthanasia.

Analysis of Volatile Compounds in Sea Bass (Lateolabrax japonicus) Resulting from Different Slaughter Methods Using Electronic-Nose (E-Nose) and Gas Chromatography-Ion Mobility Spectrometry.

Sea bass (Lateolabrax japonicus) is known for its unique flavor and high nutritional value. In this study, the influence of slaughter methods on the volatile compounds (VOCs) in sea bass was investigated using electronic nose (E-nose) technology and gas chromatography-ion mobility spectrometry (GC-IMS). VOCs in raw and cooked sea bass resulting from different slaughter methods were effectively distinguished using both techniques. Aldehydes, ketones, and alcohols were associated with the basic flavor of sea bass, whereas esters, organic acids, and furans enriched the aroma. In raw sea bass, the fishy odor was the strongest in the HSD group (head shot control death), followed by that in the IFD (ice faint to death) and BDS (bloodletting to death) groups. The VOC content increased and stabilized after steaming, enhancing pleasant odors such as fatty and fruity aromas. In cooked sea bass, the content of diacetyl and ethanol was the highest in the EAD group (eugenol anesthesia to death), which may be a residue of eugenol, imparting a distinct irritating chemical odor. Furthermore, abundant (E)-2-octenal, 2-heptanone, benzaldehyde, and esters in the BDS group imparted a strong, pleasant aroma. The findings indicate that heart puncture and bloodletting is the preferred slaughter method to maintain sea bass quality, providing new insights into the volatile changes in sea bass induced by different slaughter methods.