Laboratory Mice Euthanasia: Speed Death and Animal Welfare

The laboratory animal science, an important scientific and technological development area in all countries, act, in summary, five sub-areas: Education, bioethics, legislation and biosafety in laboratory animals; Animal welfare and bio-models behavior; Technology for breeding animals in the house facilities; Manipulation and experimentation in biomodels and Building and management of the lab animal house facilities. A difficult and widely debated question is how to euthanize laboratory animals and maintain animal welfare during the respective procedure. Projects, studies and meetings clearly demonstrate the advantages and disadvantages of each methodology or procedure and each substance administered. However, despite all efforts, so far, due to the complexity of the topic, there is still no consensus in the scientific community of the ideal euthanasia method for the humanitarian finalization of the laboratory animals [1-3].


Introduction
The laboratory animal science, an important scientific and technological development area in all countries, act, in summary, five sub-areas: Education, bioethics, legislation and biosafety in laboratory animals; Animal welfare and bio-models behavior; Technology for breeding animals in the house facilities; Manipulation and experimentation in biomodels and Building and management of the lab animal house facilities. A difficult and widely debated question is how to euthanize laboratory animals and maintain animal welfare during the respective procedure. Projects, studies and meetings clearly demonstrate the advantages and disadvantages of each methodology or procedure and each substance administered. However, despite all efforts, so far, due to the complexity of the topic, there is still no consensus in the scientific community of the ideal euthanasia method for the humanitarian finalization of the laboratory animals [1][2][3]. Inhaled anesthetics and 4) Decapitation [9][10][11][12][13][14][15][16][17][18][19][20]. nlm.nih.gov/pubmed -in January, 2020), showed that mouse as the most biomodel used in biomedical preclinical assays. We found a total of the 1.723,604 published manuscripts. When associated (Boolean Term -AND) terms between Mouse AND Euthanasia, only 756 studies are related to this important subject. Then, the investigation and study about euthanasia mouse lab methodologies and your welfare is restricted to only 0.04% of the published manuscripts [16].
Among the most used methods, carbon dioxide (CO 2 ) is the one that most raises questions, debates and studies when compared with the other methods, 45 manuscripts related to the use of CO 2 ; 20 for cervical dislocation; 10 for the use of injectable anesthetics; 15 for inhaled anesthetics and only 7 when the method is decapitation (National Center for Biotechnology Information, U.S. National Library of Medicine -https://www.ncbi.nlm.nih.gov/pubmed/ -January 2020). According to Boivin et al. [3] despite the greater use and study on CO 2 , this author considers that an alternative and more efficient method for euthanasia in small rodents in animal house facilities must still be identified [3,14]. The AVMA (American Veterinary Medical Association) Panel on Euthanasia recommends the use of CO 2 with 10% to 30% exchange of chamber gas [19]. The Canadian Council for the Care of Laboratory Animals in its guidelines on the euthanasia of animals recommended the use of anesthetics before euthanasia with CO 2 (CCAC, 2017). Notwithstanding the detailed review by Boivin et al., demonstrate adverse effects of the use of CO 2 in mouse and to disagree scientifically with several methodologies suggested by societies related to laboratory animals, also, by the Scientific Meeting in Newcastle (Hawkins et al. 2006)at two points we come together with the critical analysis of this author: i) It must be considered that all euthanasia procedures currently available can be considered as stressors and promote greater or lesser degree of distress and suffering to animals and ii) Whichever method is used, including CO 2 , one should try to reduce the time of the euthanasia process as much as possible, thus avoiding the adverse effects of euthanasia methods to a minimum and elevated welfare [3].
Propose to add more data to this controversial subject and in accordance with CONCEA Euthanasia Normative (associated with biometric analysis and the worldwide incidence of most used methodologies) we elaborated this study suggesting new procedure for the Swiss Webster Outbred Stock mice (male and adult) euthanasia. Based on this premise, the objectives of our work were: i) Investigation of the use of inhalation agents in the process of euthanasia of mice based on the calculation and evaluation of flow, noise and saturation of the chamber with different types of injector valves [20][21][22][23][24][25][26][27]; ii) Comparison of the effects and death dynamics through etological, cardiological, neurological, immunological, biochemical and behavioral systems between inhaled and injectable methods [28][29][30][31]; iii) The use of the combination of anesthetic induction with Isoflurane and CO 2 flow; iv) Financial cost to the each substance used in the respective processes.
Then our main objectives will be to investigate the best methodology to be used in euthanasia procedure, that is, contemplate the respective requirements: a) Fast death dynamics; b) Minimal effects on the animal biological systems; c) Low acquisition cost.
We hope that our results can add effectively to the scientific and technological innovation knowledge laboratory animal science area, specifically, on this very controversial topic, which is euthanasia, but to which everyone is in search of the same purpose, the animal welfare.

Euthanasia Chamber Elaboration and Calculation of the Flow of Inhalation Agents
Prototype of the Euthanasia Chamber: Made of transparent acrylic with a thickness of 0.5mm, a euthanasia chamber with dimensions of 20 x 20 x 15 cm, making a total area of 7,4 cm 3 , with a retractable cover and with two specific valves for gas with 3/8"x 1,5 mm silicone hose coupling. The insufflation valves are located on the side and the outlet on the chamber cover ( Figure 1).

Figure 1:
Study of the flow of insufflation by carbon dioxide in the euthanasia chamber. The validation of the methodologies used came from the design of an euthanasia chamber in which it was possible to carry out all the evaluation parameters. At first we kept the commercial valve for the insufflation of CO 2 V (A). Our perception was not satisfactory when using CO 2 V, so we developed a system composed of activated carbon, paper filter and metallic protection to minimize odor and noise (B), allowing the use of the euthanasia chamber with CO 2 insufflation with the valve modified -CO 2 M (C).

Insufflation valves
We initially used the standard insufflation valve for carbon dioxide (CO 2 V) (Fig. 1A) and performed the valve modification by applying a flow diffusion and odor retention system composed of: Aluminum cube perforated (used in microisolators racks) associated with a filter paper Ø 18.5 cm and an amount of activated carbon (ac) calculated using the formula: Weight ac = Chamber Volume x Insufflation Speed / Insufflation Pressure ( Figure 1B) being coupled directly in front valve (CO 2 M) ( Figure 1C). In the first stage, we conducted the study of the insufflation flow (in the absence of animals) through the ethereal dissipation of "dry ice stones" and the recording and comparison between the methods.
We performed the evaluation of all the parameters proposed in the study comparing the two types of valve.

CO 2 flow
Regardless of the valve used, we follow the guidelines of

Isoflurane saturation
We compared the efficacy of euthanasia induction in mice by the inhalation agent Isoflurane 100 ml (2L/min) in respective

Injectable Anesthetics
Barbiturates and dissociative anesthetics Instruments ® ) and the software capturing signals in the frequency of 0.1 to 100 Hz. We determined the death at the moment of absence of respiratory movements, heart rate (heart rate per minute) and the presence of sinus and ventricular arrhythmias and/or systoles absencethrought the Scope software for Windows V3.6.10 program (PanLab Instruments ® ) each 15 to 30 seconds until the moment of death.

Open Field Test
This parameter was used to enable the behavioral assessment of distress and suffering that each euthanasia method could promote to the animal. Our chamber prototype was designed to have dimensions of 20 x 20 x 15 cm. Thus, we divided its area of 40 cm 2 into 12 quadrants of 3,5 cm 2 to evaluate the animal's displacement and the height of 15 cm to determine the exploratory activity. We determined from the moment the mouse was placed inside until the moment of death confirmation, the number of horizontal quadrants crossed (motor activity), the number of vertical surveys (Rearing) and the presence/absence of urination and defecation.

Animal Welfare and Influence on Biological Systems: Invasive Parameters
Hematology, Biochemistry and Immunophenotypic Analysis: After death confirmation, we performed the mouse cardiac puncture, obtaining 0,8 to 1,0 ml of blood for processing using EDTA K2 (Vacuplast ® ) for the hematological evaluation a from the following parameters: i) RBC: Red blood cell count (millions per mm 3 ); ii) Hemoglobin measurement (gram/deciliter); iii) Hematocrit assessment (percentage) and iv) WBC: Leukocyte count (millions per mm 3 ) and the mean leukocyte value between the methodologies (± VM). Biochemical assessment was performed using serum using the following parameters: i) Liver

Detection of the release of reactive oxygen species (ROS)
The ROS detection by the cerebral cortex region was performed

Financial and Comparative Planning of Euthanasia Methodologies
Each euthanasia methodology was compared by financial cost

Statisticalanalysis
Statistical analyzes were performed using the Graph Pad Prism program, version 5.0 (Graph-Pad Software Inc.), calculating the group's mean value and ± SD. In addition, we applied the One Way ANOVA test (between different groups of animals). The statistical significance was confirmed by the post-test Turkey, considering a significant difference when p ≤ 0.05.

Results
Our experimental design was based on the use of male and adult Swiss Webster mouse. We performed the non-invasive parameters before and after the start of the euthanasia procedure. Only after animal death we collect biological material to evaluated invasive parameters. Our longest period of evolution to death was 6 minutes and 30 seconds, however we evaluated the animal every 15 to 30 seconds ( Figure 2).

Figure 2:
Experimental design: Our scheme was based on the assessment of the presence of pain and suffering during euthanasia induction, the time of death confirmation and the interference in the biological systems of each methodology used through the collection of blood and organs. We used adult Swiss Webster (male) mice to evaluate injectable and inhaled agents. Immediately after the induction of euthanasia, inside the euthanasia chamber, we performed the evaluation by the non-invasive parameters of the ethogram and electrocardiography (ECG) for the presence of distress in the animal and after confirming the death, we collected biological material for carrying out the hemogram, biochemical markers of tissue injury, flow cytometry and brain tissue immunofluorescence (IF).  Figure 3A. However, when the valve is modified, according to our prototype, there is a significant change in the CO 2 insufflation flow. The CO 2 M, prevents the direct flow force, promoting an initial resistance, thus the gas is blown into the chamber smoothly and tends to descend evenly, we do not observe swirling of the insufflation flow. We suggest that the association between the Copy@ Gabriel Melo de Oliveira paper filter and activated carbon also reduce CO 2 odor ( Figure 3B).
The placement of the gauze soaked in Isoflurane inside the chamber does not alter the flow inside the chamber, however there is a uniform saturation of the environment ( Figure 3C). The association between the saturation of the environment with isoflurane and subsequently the insufflation of 20% CO 2 M until the total saturation time (100%) of the chamber by CO 2 maintains the same pattern described for both products and methodologies, or in other words, total saturation of the chamber with Isoflurane and CO 2 insufflation in a smooth way and without swirling the environment ( Figure 3D). Figure 3: Evaluation of the saturation flow and insufflation of inhalation agents. In our euthanasia chamber, we insert dry ice stones, which, at room temperature, begins to give off a smoke which during continuous filming we could observe that during the insufflation of CO 2 V (A) the gas flow enters the chamber in the opposite direction. Straight and crashes against the wall of the chamber forming a whirlwind of air. However, when we modify the valve (B) the insufflation of CO 2 M demonstrates that the resistance promoted by the valve prevents the swirling, forcing the flow to the floor of the chamber. In the case of the insertion of Isoflurane, there is no insufflation, there is only saturation of the environment which does not modify the air flow (C), so the ideal was the association between Isoflurane saturation and CO 2 insufflation with the modified valve (D).
After the insufflation and saturation mechanical tests, we performed the non-invasive parameters comparing the animal welfare and the effectiveness of the different euthanasia methodologies. Etological parameters, it was possible to evaluate the animal behavior death dynamics of each euthanasia procedure.
During CO 2 V insufflation, the animal shows irritability and tries to "clean its nose" as if there is a strong odor bothering. Despite the relatively rapid dynamics of death, the mouse shows a marked sensation of discomfort and distress. CO 2 M use demonstrated the same characteristics as the CO 2 V, but with significantly lower intensity and faster evolution to death. ISO saturation demonstrates that its action takes a little longer than CO 2 flow, but this period is not marked by the presence of behavioral signs of discomfort or suffering. Only the sensation of odor is very striking in this situation. in 67% of the animals used and sinus tachycardia characterized by 961±104 heart beats per minute (bpm). Within 45 seconds, it was not possible to calculate the heart rate by severe sinus arrhythmia in 100% of the animals. After 60 seconds, there is a dizzying drop in heart rate and asystole and death within 120 seconds ( Figure 4A).
Through the modification of the valve (CO 2 M) we observed that the death of the animal occurs in about 120 seconds, however, the euthanasia induction begins with 30 seconds (553±25 bpm) with a smooth and progressive sinus bradycardia , with only 33% of the animals showing mild sinus arrhythmia ( Figure 4B).

Figure 4:
Cardiac monitoring for the presence of suffering during euthanasia induction and the time to confirm death. Initially, we performed through cardiac monitoring using the ECG the influence on the cardiac electrical conduction system of pain or suffering, to the percentage of the arrhythmias (ART%) during the induction of euthanasia and also the confirmation of death. The ECG tracings demonstrated that the use of CO 2 V between 30 and 45 seconds promotes intense discomfort in the animal due to the presence of cardiac arrhythmias (A) and increased of the Heart Rate (bpm), which only in 33% of the mice was perceived by the animals within 30 seconds when using the CO 2 M (B). The use of the Isoflurane saturated euthanasia chamber (ISO) did not demonstrate the presence of discomfort / arrhythmias, only after 30 seconds of exposure to the presence of cardiac fibrillation and the confirmation of death with 240 seconds (C). However, the association between Isoflurane and CO 2 M did not, at any time, show changes in the electrocardiographic tracing compatible with discomfort, and confirmation of death can be indicated within 90 seconds (D).

Copy@ Gabriel Melo de Oliveira
Still in relation to inhalation anesthetics, we performed the ECG evaluation of euthanasia induction by saturation of the chamber with ISO. The results demonstrate that the death confirmation occurs in 240 seconds, however the induction process starts from 15 seconds, with an evident sinus bradycardia (547 ± 144 bpm), however 33% of the animals show cardiac fibrillation in the period between 30 to 180 seconds ( Figure 4C). The association between ISO and CO 2 M, interestingly, demonstrates that euthanasia induction occurs quickly, progressively and smoothly. After 30 seconds of exposure to the respective association, the animals demonstrate a significant decrease in heart rate (512 ± 100 bpm) that will confirm the death with asystole in 90 seconds. We emphasize that during this period, as shown in Figure 4D, the presence of arrhythmias or any other alteration in the cardiac electrical conduction system was not observed in any animal.
The evaluation of injectable anesthetics, we did not observe at any time, after the administration of the drugs, the presence of cardiac arrhythmias or any other alteration in the cardiac electrical system that was not characterized by the progressive decrease in heart rate (sinus bradycardia). Confirmation of death occurred around 420 seconds for both Thiopental and for the association between Ketamine and Xylazine. However, after a period of 180 seconds, we can observe the presence of severe respiratory depression, transitory apnea, which only characterized death with the complete absence of respiratory movements after only 360 seconds (Table 1).Thus, we chose, for the welfare of the animals, to continue using CO 2 M and Thiopental excluded in the rest of the study.   Figure 7A) and CD19+ ( Figure 7B) cells, respectively by the use of the combination of Ketamine and Xylazine. This association also promoted, after death, a 20% decrease in CD4+ cells ( Figure   7C) and a 2% increase in CD8 cells positively marked in the liver tissue ( Figure 7D).  Copy@ Gabriel Melo de Oliveira

Am J Biomed Sci & Res
Copy@ Gabriel Melo de Oliveira  Regarding the costs of acquisition and use of the respective inhaled and injectable anesthetics used in this study, we performed a calculation basis for the euthanasia induction an event with a number of 10 animals, starting from the animal's weight and estimating for younger ages, that is, lighter weights. Initially, we describe the main injectable and used agents such as Thiopental, Ketamine and Xylazine (Table 3). Euthanasia due to overdose of Thiopental (150 mg/kg) in mice weighing 45 grams determines a cost for each event R$ 21,00 and the association in ketamine and xylazine overdose (300 + 50 mg/kg) defines a cost of R $ 7,80 for each event. When we estimated the cost of inhalation agents ( Table   4), we determined that for mice weighing 45g, the costs were R$ 0,10 for CO 2 M (2,0 L/min ); R$ 0,55 for ISO (10%) at a saturation of 2 L/min and R$ 0,35 for a saturation of 2,0 L/min for ISO (5%) and CO 2 M (1 L/min).

Discussion
The objective of our study was in accordance with national The cost of the higher doses of agents required to cause death may substantially exceed that of an approved euthanasia methodology. On the other hand, the use of injectable anesthetics, both Thiopental and the combination of Ketamine and Xylazine, Regarding the mechanisms of action of injectable anesthetics (Brabiturics and association of dissociative anesthetics and alpha-2 adrenoreceptor agonists), as well as inhalation agents (Isoflurane and CO 2 ) are well described. However, the innovative character in our work was the association between combination between induction with ISO and CO 2 with modified valve. Thus, we are convinced that the Good Death proposal is closely related to the concept of Speed Death [20].

Copy@ Gabriel Melo de Oliveira
The Speed Death suggest arehighly released ROS. This reactive molecules produced in the cell endoplasmic reticulum, peroxisosomes, cytosol, plasma membrane, extracellular space and mainly in the energy metabolism process, that is, in the mitochondria [13,7,26]. This organelle acts in the redox/oxidative balance, regulating cell death and survival [24,30]. Thus, it assists in the proper functioning of brain cells and other essential organs for life [11,25]. So that we have not yet been able to elucidate the association between ISO and CO 2 M, it quickly demonstrates altering mitochondrial functioning leading to redox/oxidative imbalance, and/or change in ATP production, resulting in damage to the mitochondria, causing greater production of ROS, leading the cell to an irreversible oxidative stress state, causing cell death.
Thus, the dynamics of death by this respective association suggests promoting a fast death (by means of necrosis or apoptosis ways) in a rapid and extensive manner, preventing the possibility of disconfort and suffering of the animal due to the irreversible involvement of the cells of the Central Nervous System [23].

Conclusions
Initially, we would like to emphasize that the value of our study was its ethical aspect of developing and investigating new techniques, procedures and knowledge that will directly raise the quality of life of animals used for didactic and scientific purposes, in our case the animal model, the lab mouse.
Our results demonstrate that the best methodology to induce euthanasia in the lab mouse during its use for didactic and scientific purposes is through the euthanasia chamber with an environment saturated with 5% Isoflurane (through a hospital gauze soaked in anesthetic liquid and protected from contact with and the insufflation of Carbon Dioxide 1 L/min, initially in 20% to 100% of the total area of the chamber, with the restriction of the use of a flow meter and modification of the insufflation valve [32,33]. However, other methodologies also have their applicability. We suggest that for neonates or animals from breeding colonies the ideal protocol consists of using a chamber with an environment saturated with 10% Isoflurane, also soaked in hospital gauze and protected from contact with animals. Regarding injectable anesthetics, we suggest not using Thiopental (150 mg/kg) in mice and using the combination of ketamine (300 mg/kg) and xylazine (50 mg/kg) overdose by i.p. only in cases of Humanitarian Finally, we believe that this study can add of the laboratory animal science, demonstrating through relevant scientific methodologies, that the induction of euthanasia must use materials, methodologies and procedures that allow the absence of pain, discomfort and distress to the animal, a speed death dynamics and a relatively low financial cost.