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

Abstract

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.

Introduction

Pain is one of the most important considerations for animal health and welfare. Since Huntingford et al. described the “Five Freedoms” for fish welfare in 2006, specifically, freedom from hunger and thirst, discomfort, pain, injury, disease, fear, and distress, discussions on animal rights for fish have intensified, bringing even more attention to the importance of improving the standards of fish welfare.¹ While our understanding of pain perception in fish remains incomplete, assessments of their welfare should thus be more conservative, rather than less.²

Examples of such conservative decision making are observed in legislation on cephalopods and decapods in European countries. Notably, the European Union is considering cephalopods as being subject to regulation in scientific procedures based on the Directive 2010/63/EU,^3,4 while Switzerland and Norway are including decapods in regulation as well.^5,6 Such legislative actions are based on the knowledge of pain response-related neuroanatomical, behavioral, and pharmacological criteria in the animals,⁷ which are common to fish in so far that their absence of a neocortex makes direct judgment impossible.

One main use of animals by humans is for scientific research. The use of fish as a laboratory animal for research continues to increase⁸ and, at the same time, zebrafish is the most widely used experimental fish species ever since it was first presented as a model organism for genetic analysis in the 1980s.⁹ Zebrafish have various advantages as an animal model, including its capacity to produce large litter sizes and its optically transparent embryos that enable high-resolution organism-level imaging, as well as well-established protocols that guide researchers to generate transgenic organisms.

These characteristics and more altogether make zebrafish an excellent experimental animal species for investigating cellular dynamics and to decipher the underlying molecular mechanisms for tissue homeostasis, disease, and aging.^10–12 In addition, given the worldwide practice of 3Rs (replacement, reduction, and refinement) in conducting research on animals, the use of zebrafish represents a viable, alternative animal model to reduce and replace the use of mammals.¹³ For these reasons and more, the use of zebrafish in research continues to rise.¹⁴

The increase in the usage of zebrafish must be met with improved care and welfare considerations for fish in animal research, particularly in the ethical considerations for pain, pain relief, and euthanasia. Indeed, the ethical and humane practice of euthanasia as an approach to preventing continuous harm to fish, or to prevent unnecessary or excessive pain in experimental situations, or both, is a top consideration in zebrafish research.¹⁵

Particularly, the “how” and “when” to implement euthanasia are important considerations, together with decision making on euthanasia methods and humane endpoint settings. Accordingly, the need for establishing guidelines for zebrafish euthanasia and humane endpoint is growing. Most of the global guidelines existing are from individual institutions and only a few cases are from the national level. In case of the Republic of Korea, which is the country of focus for this study, no national-level guideline exists and, even at the institutional level, there is only one set of guidelines for zebrafish euthanasia.

To establish effective guidelines, it is important to derive implications for the necessity of guidelines for field experts based on the precise objectification of current situations and recent knowledge in the field. When establishing a guideline at a nation-wide level, the methodology of expert elicitation could be an appropriate method to gather critical knowledge beyond what is reported in publications.

In this study, we conducted an extensive literature survey to describe the world-wide status of euthanasia and humane endpoint setting for zebrafish. Also, we carried out expert elicitation to understand current practices in euthanasia and humane endpoint treatment of zebrafish in the Republic of Korea, and to evaluate the necessity and feasibility of establishing guidelines. Through this, we drew attention to the importance of zebrafish welfare and have piqued interest from field experts about zebrafish welfare. Finally, through our expert elicitation and literature search, we have developed a set of national guidelines for the Republic of Korea, for the use of zebrafish in research.

Materials and Methods

Literature survey: Global status of zebrafish studies

Annual numbers of publications from either Google Scholar or Web of Science with the following keywords were counted from 1980 to 2022: “zebrafish”; “zebrafish” and “euthanasia”; “zebrafish” and “humane endpoint”; and “zebrafish” and “IACUC.”

National trend of research animal use in the Republic of Korea

Data on the number of animals used for research purposes in the Republic of Korea from 2016 to 2021 were collected from press releases issued by the Ministry of Agriculture, Food and Rural Affairs. The proportional changes in animal clades used were analyzed.

Literature survey: Global status of euthanasia and humane endpoint application for zebrafish studies

Four hundred sixty-eight articles were selected from PubMed, SCOPUS, and CAB Direct for trend analysis on euthanasia and humane endpoint applications in zebrafish studies, using keywords [“zebrafish[Mesh]” and (“endpoint determination” or “animal use alternatives” or “ending point” or “humane endpoint” or “humane end point” or “humane endpoints” or “pain scale” or “pain level” or “refinement” or “welfare” or “animal welfare” or “euthanasia”)]. Among the 468 articles, 223 full articles with descriptions on the zebrafish life stages and methods of euthanasia were selected for the trend analysis.

Global guidelines for euthanasia and humane endpoint for zebrafish experimental use

Both institutional and national guidelines for zebrafish euthanasia and humane endpoint were collected and summarized.

Nation-wide survey for zebrafish facilities: National status of zebrafish euthanasia in the Republic of Korea

Major zebrafish facilities in the Republic of Korea were subjected to the survey, with personnel at those facilities asked to respond to questions relating to the following topics: Life stages of the zebrafish in the facility; euthanasia methods used; number of cages; and location of the facility.

Nation-wide subject matter expert elicitation: Current knowledge of experts on zebrafish euthanasia and humane endpoint setting

Subject matter experts were selected carefully based on both the depth of their experience in zebrafish research as well as in their understanding of field situations in the Republic of Korea. Selected experts included the following: professors from the Lab of Laboratory Animal Medicine, professors from the Lab of Aquatic Biomedicine, professors well experienced with zebrafish research recommended by the Korea ZebraFish Society (KZFS); and professors with zebrafish research experience of over 20 years. A formal survey was carried out for 2 weeks in which questionnaires of three major categories were provided: (1) “Give us suggestion about zebrafish euthanasia method and explain the reason why”; (2) “Give us suggestions about zebrafish humane endpoint and explain the reason why”; and (3) “Give us additional comments related to nation-wide euthanasia and humane endpoint guideline setting.” Survey questionnaires were sent through email and the results treated as confidential.

Results

The number of research publications featuring zebrafish as an animal model is increasing

We began by analyzing the number of published documents that featured “zebrafish,” with data collected from a search through the internet resources of Google Scholar as well as Web of Science. As shown, from the 1980s when this animal began to gain prominence as a research animal model, research publications that featured zebrafish grew exponentially (Fig. 1).

FIG. 1.

The number of zebrafish-related publications indexed in Google Scholar and the Web of Science from 1980 to 2022. The number of publications is represented in 4-year periods, and items were retrieved by a search with the keyword “zebrafish” from each search engine.

Next, we focused on research from the Republic of Korea. We investigated datasets on animals used for research purpose in the Republic of Korea to find that, from the period between 2016 and 2022, the use of fish has increased in absolute number (Fig. 2A), consistent with the international trend observed in Figure 1. Next, we compared the proportions of fish used with that of rodents, which are widely used in research. As shown, the proportion of fish used has increased by 14.86% (from 4.07% in 2016 to 18.93% in 2021), while the proportion of rodents used has decreased by 18.97% (from 91.46% in 2016 to 72.49% in 2021; Fig. 2B) among the annually used research animals. In terms of proportional change in particular, the trend is opposite to that of rodents as is increasing, in general, since 2016.

FIG. 2.

Annual trends of research animal use in the Republic of Korea. (A) The number of animals used for research purposes in the Republic of Korea. (B) Annual change of rodent and fish usage for research purposes in the Republic of Korea. Data based on the animal experiment survey press release from the Ministry of Agriculture, Food and Rural Affairs of the Republic of Korea.

Global status of zebrafish euthanasia and humane endpoint for research

Next, we wanted to study the publication landscape for zebrafish research that described euthanasia as their humane endpoint. For this, we performed a search for articles using dual keywords “zebrafish” and “euthanasia” (Fig. 3A), “zebrafish” and “IACUC” (Fig. 3B), or “zebrafish” and “humane endpoint” (Fig. 3C), and related these to the number of articles retrieved from searches with the keyword “zebrafish” alone. As shown, we found that the proportion of publications with our dual search terms relative to publications with the term “zebrafish” alone was very low, at <0.2%, across the time points examined.

FIG. 3.

Ratio of euthanasia, humane endpoint, and IACUC-related articles among zebrafish articles from Google Scholar and Web of Science. The proportion of the number of articles found from dual keywords (A) [“zebrafish” and “euthanasia”] or (B) [“zebrafish” and “IACUC”] or (C) [“zebrafish” and “humane endpoint”] to the number of articles found from the sole keyword “zebrafish” is plotted to the graphs. IACUC, Institutional Animal Care and Use Committee.

Among the categories for dual search terms, the proportion of articles retrieved from searching “zebrafish” and “euthanasia” was highest, yet were no more than 0.16% of publications featuring “zebrafish” alone (Fig. 3A). Articles with the dual search terms “zebrafish” and “IACUC” (Fig. 3B), or from searching “zebrafish” and “humane endpoint” (Fig. 3C), were all proportionally lower still. These data suggest that, at least for published research items available online, the terms “euthanasia,” “humane endpoint,” and “IACUC” are not frequently mentioned in studies featuring zebrafish as a search term.

Guided by these results, we next performed a literature search on the online resources of from PubMed, SCOPUS, and CAB Direct to select 468 documents with defined search keywords as described (please see Materials and Methods for details). Of these, we found 223 documents that met our selection criteria to investigate the global status of euthanasia methods used in zebrafish research. Of the methods of euthanasia described in these documents, which were counted, assessed in accordance with procedures relative to the life stages of zebrafish, we found that “tricaine methanesulfonate (MS-222) overdose” was the most frequently used method from all life stages, including embryo, larva, juvenile, and adult (Fig. 4), with the next most frequently mentioned methods being “hypothermic shock” and “other anesthetics overdose” (Fig. 4). Notably, for a significant majority of articles, the method of euthanasia across all life stages was not mentioned.

FIG. 4.

Proportions of zebrafish euthanasia methods described from published articles. Two hundred twenty-three selected articles were further classified according to the life stages of used zebrafish and the proportion of euthanasia methods used in each life stage group was analyzed. A major portion of articles did not even mention the euthanasia methods used.

Subject matter expert elicitation about the status and practice of euthanasia in zebrafish research in the Republic of Korea

We conducted a broad study across ten major zebrafish facilities in the Republic of Korea to better understand the status and practice of zebrafish euthanasia methods within the Republic of Korea. From this study, we found that the average number of fish tanks from the facilities was 413 and 80% of the facilities were accommodating zebrafish of all life stages from embryo to adult. From our study, we found that “hypothermic shock” represented the most frequently used euthanasia method from across all facilities surveyed (40.0%), followed by “MS-222 overdose and successive hypothermic shock” (33.3%) and “MS-222 overdose” (20.0%; Fig. 5).

FIG. 5.

Euthanasia methods that are currently being used in the zebrafish facilities of the Republic of Korea. All euthanasia methods being used were based on responses from our study of facilities that permitted single or combined methods of euthanasia of zebrafish. All methods represented in this graph are represented regardless of life stages of zebrafish that were euthanized.

Subject matter expert elicitation for zebrafish euthanasia and recommendations for improvements in humane endpoints for zebrafish research

Eleven experts were carefully selected as the representative subject matter experts in the Republic of Korea. All such experts strictly meet the following conditions to (1) possess significant experience (>20 years' experience) in zebrafish research, (2) have an appreciation of the current status of the field of zebrafish research in Korea, and (3) to continue to be involved in zebrafish research. The response rate of our survey was 100%, and experts were asked to offer suggestions on euthanasia and humane endpoint methods for each life stage for zebrafish based on their expert knowledge, regardless of the currently used methods in practice.

As per our results, we found that “MS-222 overdose” was the most frequently suggested euthanasia method for zebrafish across all life stages, from embryo to adult (Fig. 6A). This was followed by “hypothermic shock” (16 suggestions), then by the combined method of “MS-222 overdose followed by hypothermic shock” (8 suggestions). For embryo and larva stages of zebrafish, suggestions for “hypothermic shock with sodium hypochlorite” (six suggestions) and “MS-222 with sodium hypochlorite” (four suggestions) were suggested for embryo and larva. Sodium hypochlorite usage was suggested specifically for the embryo and larval stages, with emphasis on secondary method choosing different methods according to the life stages. Few suggestions for “anesthesia and decapitation” and “hypothermic shock and decapitation” were offered for adult staged zebrafish.

FIG. 6.

Data collected from zebrafish research experts in the Republic of Korea on the practice of euthanasia and humane endpoint monitoring for zebrafish. (A) Eleven experts were surveyed to understand current euthanasia methods for zebrafish research, relative to the life stages of zebrafish. (B) Results from our survey seeking suggestions on human endpoint setting for juvenile and adult zebrafish used in research. Multiple responses were permitted across both survey items.

Further to our investigation of the humane endpoint for zebrafish research animals, suggestions were presented separately from “embryo and larva,” and “juvenile and adult.” At the same time, the experts mentioned the need for monitoring zebrafish to guide decision making on humane endpoints for zebrafish research, depending on the types of experiments undertaken. For example, for embryo and larval zebrafish research, we received recommendations to set a humane endpoint with reference to the toxicological reactions criteria described from OECD,¹⁶ as this can provide guidance for abnormal development of an embryo. For juvenile and adult, “loss of balance,” “deep wound,” “lying down,” “dropsy and ascites,” and “abnormal swimming” were recommended by multiple experts as humane endpoints, followed by “hernia,” “scoliosis,” and other criteria featured in our graph (Fig. 6B).

National and institutional guidelines of euthanasia and humane endpoint for experimental use of zebrafish

National-level guidelines for zebrafish euthanasia have been established in several countries (Table 1). The guidelines are published by government departments of each respective country. Different national guidelines recommend different methods of euthanasia for zebrafish in research, depending on life stage, as shown in the Table 1 with four countries as examples, including United States, Canada, India, and Qatar. In addition, guidelines published by the American Veterinary Medical Association (AVMA; Table 2), recognized as among the most highly cited, also recommended the use of secondary methods for euthanasia of zebrafish to assure the loss of consciousness and death through their recommended procedures. Taken altogether, our results show that, despite the increasing use of zebrafish in research globally and in the Republic of Korea, there is significant need in developing best practice for the implementation of zebrafish euthanasia for researchers in the Republic of Korea and the rest of the world.

Table 1.

Global Guidelines for Zebrafish Euthanasia

Country	Department	Life stage	Euthanasia method	Dose	Others
National guideline
United States	NIH²⁹	≥15 dpf	Immobilization by submersion in ice water	5 parts ice/1 part water, 0–4°C	For 10 min after cessation of opercular movement
			MS-222	200–300 mg/L	Same above
			MS-222	168 mg/L	Followed by rapid freezing in LN
		8–15 dpf	Same above		Should be followed by a secondary method (bleaching)
		≤7 dpf	Bleach solution (Sodium hypochlorite)	6.15% 1 part/water 5 part	Single use without anesthesia
Canada	The Canadian Council on Animal Care (CCAC)³⁰	Adult	Chemical agent	Agents listed by AVMA	Concerns on the use of MS-222
			Hypothermal shock	Rapid chilling followed by adjunctive method	At least 40 min, more humane that using MS-222 Direct contact between the fish and ice should be avoided
			Physical methods	Maceration Decapitation	Decapitation should be followed by pithing
		Fry	Less effective
		Embryo	Hypothermal shock or immersion in MS-222 + sodium or calcium hypochlorite solution (500 mg/mL)		<3 dpf
India	Ministry of Fisheries³¹	Adult	MS-222	>200 mg/L	For 10 min after cessation of opercular movement
			Clove oil (Eugenol)	>100 mg/L	Same above
			Lidocaine hydrochloride	>400 mg/L	Same above
			2-Phenoxyethanol	>800 mg/L	For 5 min
			Hypothermia	0–4°C	For 5 min
		Larvae	MS-222	>1800 mg/L	3–8 dpf for over 1hfollowed by adjunctive method
			Clove oil	>1500 mg/L	14 dpf for 20 min after cessation of heartbeat
			Hypothermia	0–4°C	Followed by bleach, decapitation
Qatar	Ministry of Public Health³²	≥15 dpf	Immobilization by submersion in Ice water	5 parts ice/1 part water, 0–4°C	For 10 min after cessation of peculiar movement
			MS-222	200–300 mg/L	Same above
			MS-222	168 mg/L	Followed by rapid freezing in LN
		8–15 dpf	Same above		Should be followed by a secondary method (same below)
		≤7 dpf	Bleach solution (Sodium hypochlorite)	6.15%1 part/water 5 part	For 5 min
Institutional guideline
United States	University of California San Francisco³³	Adult (≥7 dpf)	MS-222 (Finquel or Tricane-S)	Stock: 4 g/L (2:1 bicarb to MS-222)Working 300 μg/mL or 7.5 mL stock sol. to a total of 100 mL	Buffered with sodium bicarbonate (pH 7.0–7.5)For 10 min after cessation of opercular movement
		Adult (≥7 dpf)	Rapid chilling	Submerge in 5 parts ice/1 part water, 2–4°C	Confirm with a thermometer that the water is ≤4°C before placing the fishFor 10 min after cessation of opercular movement
		Fry (4–7 dpf)	MS-222	Same above	For 20 min after cessation of opercular movement
		Fry (4–7 dpf)	Rapid chilling	Same above	For 20 min after cessation of opercular movement
		Embryo (0–3 dpf)	MS-222	Same above	Should be followed with an adjunctive method. Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
		Embryo (0–3 dpf)	Rapid chilling	Same above
United States	University of Berkeley³⁴	≥8 dpf	MS-222 (Finquel or Tricane-S)	—	Buffered with sodium bicarbonate (pH 7.0–7.5).For 10 min after cessation of opercular movement
		≥8 dpf	Rapid chilling	Submerge in 5 parts ice/1 part water, 2–4°C	For 10 min after cessation of opercular movement
		Fry (4–7 dpf)	Rapid chilling	Submerge in 5 parts ice/1 part water, 2–4°C	For 20 min after cessation of opercular movement
		Embryo (0–3 dpf)	MS-222 (Finquel or Tricane-S)	—	Buffered with sodium bicarbonate (pH 7.0–7.5). Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
United States	Saint Mary's College of California	Same with University of California San Francisco
United States	Texas Tech University³⁵	≥8 dpf	MS-222 (CAS no.886-86-2)	200–300 mg/L	For 10 min after cessation of opercular movement
			MS-222 (CAS no.886-86-2)	Anesthesia at 168 mg/L	Followed by rapid freezing in LN
			Rapid chilling	Submerge in 5 parts ice/1 part water, 0–4°C	For 10 min after cessation of opercular movement
		4–7 dpf	Rapid chilling	Same above	For 20 min after cessation of opercular movement
		4–7 dpf	Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
		0–3 dpf	Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
Canada	McGill University³⁶	≥15 dpf	MS-222	250–500 mg/L	Should be buffered (pH7.0–7.5)For 10 min after cessation of opercular movementFollowed by an adjuntive method
		≥15 dpf	Rapid chilling	Insulated tank (5 parts ice to 1 part water, 2–4°C), 10-min submersion first	For 20 min after cessation of opercular movementFollowed by an adjuntive method
		8–15 dpf	MS-222	250–500 mg/L	For 20 min after cessation of opercular movementAdd dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
		8–15 dpf	Rapid chilling	Insulated tank (5 parts ice to 1 part water, 2–4°C), 20 min submersion first	Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min
		≤7 dpf	Add dilute bleach solution (1 part sodium hypochlorite 6.15% to 5 parts DW) to the water for 5 min

dpf, days postfertilization; DW, distilled water; LN, liquid nitrogen.

Table 2.

Fish Euthanasia Method Suggestion from American Veterinary Medical Association Guideline¹⁵

	Method	Detailed descriptions
Noninhaled agentsImmersion	Benzocaine; Benzocaine hydrochloride	≥250 mg/LSolutions for immersion should be buffered
	Carbon dioxide	CO₂ saturated water
	Ethanol	10–30 mL of 95% ethanol/L
	Eugenol; Isoeugenol; Clove oil	<170 mg/LIsoeugenol is a potential carcinogen, so human safety in the application is of concern
	Isoflurane; Sevoflurane	>5–30 mL/LBoth anesthetics are highly volatile and human safety is of concern and use in a well-ventilated area is imperative
	Quinaldine sulfate	≥100 mg/LSolution for immersion should be buffered
	MS-222	250–500 mg/LSolutions must be buffered, and concentrations required for euthanasia may vary depending upon the species, life stage, and water chemistry parameters
	2-phenoxyethanol	≥0.5–0.6 mL/L
	Lidocaine	400 mg/LResponse to lidocaine by immersion varies considerably across species
Noninhaled agentsInjection	Pentobarbital	Sodium pentobarbital (60–100 mg/kg) IV, IC, IP injection
	Ketamine followed by a lethal dose of pentobarbital	66–88 mg/kg IM injection
	Ketamine-medetomidine followed by lethal dose of pentobarbital	Ketamine (1–2 mg/kg) with medetomidine (0.05–0.1 mg/kg) IM injection
	Propofol followed by lethal dose of pentobarbital	1.5–2.5 mg/kg IV injection
Physical methods	Decapitation followed by pithing	Rapid severance of the head and brain from the spinal cord, followed by pithing of the brain, will cause rapid death and unconsciousnessDecapitation alone is not considered a humane approach to euthanasia, especially for species tolerant to low O₂ concentrations
	Cervical transection followed by pithing	Destruction of the connection between brain and spinal cord using a knife or other sharp instrument followed by pithing
	Cranial concussion followed by pithing or exsanguination	A rapid, accurately placed blow of sufficient energy to the cranium with an appropriate-sized club can cause immediate unconsciousness and potentially death, but should be followed by pithing or exsanguination to ensure death
	Penetrating captive bolt or NPCB	Can be applied to large fish species
	Maceration	When applied correctly, death is nearly instantaneous
	Rapid chilling (hypothermic shock)	Rapid chilling (2–4°C) until loss of orientation and operculum movementsAdult zebrafish should be exposed for a minimum of 10 min and fry 4–7 dpf for at least 20 min following loss of operculum movement to ensure deathTo ensure embryonic lethality, rapid chilling or MS-222 overdose methods should be followed with an adjunctive method such as use of dilute sodium or calcium hypochlorite solution at 500 mg/LRapid chilling is a suitable killing method for small tropical and subtropical fish species (<3.8 cm in length) having lower lethal temperatures above 4°C

IC, intracardiac; IM, intramuscular; IP, intraperitoneal; IV, intravenous; NPCB, nonpenetrating captive bolt.

Discussion

The number of research studies that employs zebrafish is increasing; however, the issue of euthanasia and the considerations for humane endpoints for zebrafish experiments seem to be reported infrequently. Given the expansion in the number of publications in the field of zebrafish research, suitable guidelines for euthanasia and the considerations for humane endpoints for zebrafish do not appear to follow an upward trend in their description in research publications.

From our findings, we found a few nations worldwide with guidelines for zebrafish euthanasia, with the majority of institutional guidelines sourced from North America. With regard to guidelines for humane endpoint for zebrafish research practices, no national guide was found globally, and such guidelines were identified in only a few institutions. Thus, more can be done to improve the monitoring of zebrafish welfare in research with regard to pain relief, and their appropriate methods for euthanasia when required, as well as the awareness among researchers to carry out good research practice for zebrafish research worldwide.

Currently, euthanasia methods undertaken in academic research for many countries worldwide require established guidelines that do not have such guidelines in place. Data from our literature study showed that the “MS-222 overdose” was the most used euthanasia method, regardless of life stages of the animal, the trend of which is consistent with the previous international survey study by Mocho and Krogh.¹⁷ However, guidelines from different countries recommend using different methods of euthanasia for zebrafish, according to the life stage.

We found that, for countries where guidelines are in place, the use of MS-222 overdose alone for embryo and larval zebrafish was not indicated, likely owing to the different sensitivities and resistance to MS-222.¹⁸ Such discrepancy in the choice of euthanasia between published studies and guidelines is noted as a consideration for the establishment of a future consensus. Nevertheless, such an inconsistency across reported instances of euthanasia in zebrafish publications could be attributable to the lack of guidelines for the location of origin for a study, or possibly the time gap between enactment of guidelines and the increase in the use of zebrafish in academia. Based on our data from researchers in the Republic of Korea, experts agree on the need for animal welfare for lower vertebrates, including fish.

In our study, we find that the practice of using optimal methods for euthanasia and their reporting in academic research publications fall behind global trends and improvements in animal welfare. In the Republic of Korea, our survey data indicate that the most frequently reported method of euthanasia for zebrafish facilities is hypothermic shock, regardless of the fish's life stage.

However, the fact that applying hypothermic shock alone to embryo and larva zebrafish is not effective enough for euthanasia has been known through previous studies.^19,20 Not even the world trends and guidelines are consistent with the Korean trend, as the MS-222 overdose was found to be the most widely used method worldwide from both this study and a previous reference.¹⁷ Indeed, the development of national guidelines for the Republic of Korea will bring practice standards closer to that observed in countries with established guidelines, including the recommendations to use specific methods of euthanasia according to the life stage of zebrafish under experimentation. In our survey, respondents cited the lack of regulations or guidelines, as well as the accessibility to the practice of hypothermic shock method as reasons for its widespread application.

We found only one accessible set of euthanasia guidelines in the Republic of Korea, which was established by a private institution. Nevertheless, all 11 respondents in our survey agreed with the need to establish guidelines to formalize the welfare considerations for zebrafish in research. Given the growing awareness that the differences in susceptibility to a certain method of euthanasia are related to the life stages of zebrafish, particularly with regard to the time point for the development of an animal,¹⁸ we conclude from the results of our survey that guidelines will have to be established, which align with the most up-to-date knowledge of existing guidelines, and our understanding of zebrafish pain perception. Also important is the development of training guidelines to ensure that zebrafish researchers are aware of the scientific basis and implications of appropriate versus inappropriate use of specific methods of euthanasia on zebrafish in research.

From our survey, the top-ranked euthanasia method suggested in the Republic of Korea is to expose zebrafish to an overdose of MS-222, regardless of life stage. Given that the sensitivity to MS-222 is different for zebrafish larva and embryo zebrafish, compared to juveniles and adults,¹⁸ our results indicate that even experts require greater awareness regarding their choice of euthanasia. In addition, euthanasia methods of choice were limited mostly to the MS-222 overdose and hypothermic shock, not opening the door for other potential and effective methods, that is, lidocaine hydrochloride or electric stunning.^20–22

In the case of humane endpoint, the frequency of descriptions documented in research publications is much less than in descriptions of euthanasia. We found that guidelines for the humane endpoint for zebrafish experiments were scant, both nationally and internationally. Our expert elicitation found repeated mentions of lack of an agreed mechanism for setting humane endpoints for zebrafish with respect to their life stage, for guidelines on the use of specific methods of euthanasia, and the need for establishing guidelines to guide decision making for zebrafish welfare in experiments. As an example, for embryo and larva stage zebrafish, abnormal development can be used as the criterion for determination and thus the use of developmental toxicity reference, as suggested by multiple respondents.

As a result of the intensive literature survey and expert elicitation reflecting current biological knowledge and the trend of world guidelines, we suggest that an agreement could be reached on suggestions for zebrafish euthanasia method and humane endpoint setting. For euthanasia, an agreement was reached on leaving both hypothermic shock and MS-222 overdose as possible choices for juvenile and adult zebrafish.²³

In the case of larvae, even though MS-222 overdose was the most recommended by experts, a practically remarkable point was that the second and third most recommended euthanasia methods were all using dual methods, that is, MS-222 overdose followed by hypothermic shock, MS-222 overdose followed by sodium hypochlorite, and hypothermic shock followed by sodium hypochlorite. The need for dual methods of euthanasia in a single procedure was recommended based on studies of the difference in sensitivities to MS-222 or hypothermic shock between life stages of zebrafish,^18,19 a consideration already reflected within guidelines worldwide. Based on such discussions, dual euthanasia methods could be given greater consideration, despite their lower preference as second and the third most recommended choices, as reflected in responses from our survey.

Although MS-222 is currently the most suggested anesthetic method for adult zebrafish in diverse guidelines, it is well known that this chemical could induce aversive reactions from diverse fish species.^21,24 Such a fact reflects the need for excavating better option for fish euthanasia based on thoroughly reviewed scientific studies, opening the door for possible and better effective euthanasia methods. Lidocaine hydrochloride and electrical stunning both could be one of the other applicable euthanasia methods for fish based on the previous study.^20,25

Lidocaine hydrochloride (1 g/L) mixed with EtOH (50 mL/L) buffered with NaHCO₃ (2 g/L) shows least aversive effect compared with MS-222 usage in zebrafish. Although lidocaine is not frequently used for zebrafish in laboratories currently, this could be suggested as successive and even better euthanasia method with an efficient and stress-free manner for zebrafish. Electrical stunning can be another choice of euthanasia, a method that has been mainly applied for farm fish culling for the purpose of food, fish oil, or fertilizer production, as is a nonchemical euthanasia method applicable for massive sacrifice.²² As such, electrical stunning can also be used for zebrafish according to the experimental needs, including nonchemical euthanasia and/or massive sacrifice when proper equipment is set.

In the case of humane endpoint, diverse criteria, including loss of balance, deep wound, lying down behavior, and ascites, were suggested for adult and juvenile, based on recommendations from many experts, as described by Sabrautzki et al.²⁶ Such criteria are widely consistent with the previously proposed concept of humane endpoint by Ellis and Katsiadaki,²⁷ based on moribundity defined with a lack of response to external stimuli, loss of equilibrium (i.e., loss of the righting reflex), and a slow opercular ventilation rate.

For embryo and larva stage zebrafish, embryo coagulation, lack of somite formation, nondetachment of the tail, and lack of heartbeat were suggested as criteria for determining the humane endpoint based on the abnormalities that can be seen through fish development until their maturation.¹⁶ However, since the criteria for the humane endpoint can be set differently depending on the type of experiment, it is important to first evaluate the effectiveness of such physiological traits through an examination of existing reported studies, or to conduct a small-scale pilot study^.28 It was also mentioned through the elicitation that accumulating references of the humane endpoint setting in diverse zebrafish experiments is another reason for the needs of such efforts, for later use of extrapolation.

We conclude that expert elicitation can be a methodology to understand and report on the direction of nationwide decision making on this area of zebrafish research. By reflecting the situation in front line, beyond reported research in publications, and by drawing consensus based on the knowledge of field experts, we could evaluate zebrafish welfare in the Republic of Korea alongside world trends based on our field data. Our study was also valuable to reveal that knowledge standards for the use of methods of zebrafish euthanasia and humane endpoint setting could be raised. Also, our expert elicitation makes the call for the establishment of national guidelines and has attracted the attention of field experts. Thus, expert elicitation may be useful to other institutions and governments that may be interested in establishing their guidelines.

Conclusion

This study highlights the growing use of zebrafish in research field and finds that both the reporting of methods of euthanasia and humane endpoint practices, as well as the availability of guidelines for such practices, could altogether be significantly improved. The disparity between the increasing number of zebrafish studies and the limited availability of national and institutional guidelines is observed worldwide.

We conducted expert elicitation in the Republic of Korea to find that guidelines for and the practice of zebrafish research methods for euthanasia and humane endpoint application could be improved. Based on our survey results, we conclude that experts agree to the establishment of national guidelines so as to improve zebrafish welfare and ensure adherence to best practices. We find that expert elicitation was proved effective as an approach to assess the current state of zebrafish welfare and advocate the establishment of national guidelines, and to invite participation from field experts. We offer the recommendation that countries and institutions in need of such guidelines may consider employing expert elicitation as an effective method to address such concerns.

Footnotes

Acknowledgment

Article editors Julian Heng and Xin Du (Remotely Consulting, Australia) provided professional English-language editing of this article (Article Certificate No. 7Gh9Ne4F).

Authors' Contributions

S.W.K.: Methodology, investigation, data curation, and article writing; W.J.J., S.H.H., and S.K.K.: Investigation and data curation; H.R.: Methodology and investigation; H.J.K.: Methodology, investigation, and funding; Y.R.N. and S.H.S.: Conceptualization, methodology, investigation, validation, article review, supervision, and funding.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT; Grant No. 2022R1C1C2010114). This work was also supported by a fund (Grant No. F-1543071-2022-23-01) from Animal and Plant Quarantine Agency, South Korea.

References

Huntingford

, Adams

, Braithwaite

, et al. Current issues in fish welfare. J Fish Biol, 2006; 68:332–372; doi: 10.1111/j.0022-1112.2006.001046.x

Browman

, Cooke

, Cowx

, et al. Welfare of aquatic animals: Where things are, where they are going and what it means for research, aquaculture, recreational angling and commercial fishing. ICES J Mar Sci, 2019; 76:82–92; doi: 10.1093/icesjms/fsy067

European Directive on the Protection of Animals Used for Scientific Purposes (Directive 2010/63/EU, European Parliament and Council of the European Union, 2010).

Smith

, Andrews

PLR

, Hawkins

, et al. Cephalopod research and EU Directive 2010/63/EU: Requirements, impacts and ethical review. J Exp Mar Biol Ecol, 2013; 447:31–45; doi: 10.1016/j.jembe.2013.02.009

Swiss Federal Veterinary Office. ‘Legislation’ link to German & French versions of Swiss Animal Welfare Act (2008); 2011. Available from: http://www.bvet.admin.ch/themen/tierschutz/index.html?lang=en [Last accessed: September 6, 2023].

Norwegian Government. Norwegian Animal Welfare Act (2009). English translation; 2011. Available from: http://www.regjeringen.no/en/doc/laws/Acts/animal-welfare-act.html?id=571188 [Last accessed: September 6, 2023].

Rowe

. Should scientific research involving decapod crustaceans require ethical review?. J Agric Environ Ethics, 2018; 31:625–634; doi: 10.1007/s10806-018-9750-7

Utne-Palm

, Smith

. Fish as Laboratory Animals. In: The Welfare of Fish. Animal Welfare, vol 20. (Kristiansen T, Fernö A, Pavlidis M, et al. eds.) Springer: Cham;, 2020; doi: 10.1007/978-3-030-41675-1_16

Streisinger

, Walker

, Dower

, et al. Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature, 1981; 291:293–296; doi: 10.1038/291293a0

10.

Shin

, Fishman

. From Zebrafish to human: Modular medical models. Annu Rev Genomics Hum Genet, 2002; 3:311–340; doi: 10.1146/annurev.genom.3.031402.131506

11.

Lieschke

, Currie

. Animal models of human disease: Zebrafish swim into view. Nat Rev Genet, 2007; 8:353–367; doi: 10.1038/nrg2091

12.

Choi

, Choi

, Lee

, et al., Zebrafish as an animal model for biomedical research. Exp Mol Med, 2021; 53:310–317; doi: 10.1038/s12276-021-00571-5

13.

National Research Council (NRC). Guide for the Care and Use of Laboratory Animals, 8th ed. National Academies Press: Washington, DC; 2010.

14.

Bailone

, Fukushima

HCS

, Fernandes

BHV

, et al. Zebrafish as an alternative animal model in human and animal vaccination research. Lab Anim Res, 2020; 36:13; doi: 10.1186/s42826-020-00042-4

15.

AVMA Guidelines for the Euthanasia of Animals; 2020. Available from: https://www.avma.org/sites/default/files/2020-02/Guidelines-on-Euthanasia-2020.pdf [Last accessed: September 6, 2023].

16.

OECD. Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Guidelines for the Testing of Chemicals, Section 2. OECD Publishing, 2013; doi: 10.1787/20745761

17.

Mocho

, Krogh

. A FELASA Working Group Survey on Fish Species Used for Research, Methods of Euthanasia, Health Monitoring, and Biosecurity in Europe, North America, and Oceania. Biology (Basel), 2022; 11(9):1259.

18.

Strykowski

, Schech

. Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio). J Am Assoc Lab Anim Sci, 2015; 54(1):76–79.

19.

Wallace

, Bright

, Marx

, et al. Effectiveness of rapid cooling as a method of euthanasia for young zebrafish (Danio rerio). J Am Assoc Lab Anim Sci, 2018; 57(1):58–63.

20.

Mocho

, Lang

, Valentin

, et al. A multi-site assessment of anesthetic overdose, hypothermic shock, and electrical stunning as methods of euthanasia for zebrafish (Danio rerio) embryos and larvae. Biology (Basel), 2022; 11(4):546.

21.

Collymore

, Banks

, Turner

. Lidocaine hydrochloride compared with MS222 for the euthanasia of zebrafish (Danio rerio). J Am Assoc Lab Anim Sci, 2016; 55:816–820.

22.

Bouwsema

, Ellis

, Lines

, et al. In-water electric stunning as a humane commercial method for culling juvenile salmonids. Aquac Eng, 2022; 99:102286.

23.

Wilson

, Bunte

, Carty

. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio). J Am Assoc Lab Anim Sci, 2009; 48:785–789.

24.

Readman

, Owen

, Murrell

, et al. Do fish perceive anaesthetics as aversive?. PLoS ONE, 2013; 8:e73773.

25.

Von Krogh

, Higgins

, Saavedra Torres

, et al. Screening of anaesthetics in adult zebrafish (Danio rerio) for the induction of euthanasia by overdose. Biology (Basel), 2021; 10(11):1133.

26.

Sabrautzki

, Miller

, Kague

, et al. Welfare assessment of adult laboratory zebrafish: A practical guide. Zebrafish, 2021; 18(4):235–296; doi: 10.1089/zeb.2021.0021

27.

Ellis

, Katsiadaki

. Clarification of early end-points for refinement of animal experiments, with specific reference to fish. Lab Anim, 2021; 55(3):244–253.

28.

Animal Research Advisory

Committee

, 2023. Guidelines for Endpoints in Animal Study Proposals. Available from: http://oacu.od.nih.gov/ARAC/documents/ASP_Endpoints.pdf [Last accessed: September 6, 2023].

29.

National Institutes of Health (NIH). Animal Research Advisory Committee (ARAC) Guidelines, Animal Study Proposals Using Zebrafish in the NIH Intramural Research Program (2023). Available from: https://oacu.oir.nih.gov/animal-research-advisory-committee-arac-guidelines [Last accessed: September 6, 2023].

30.

Batt

, Bennett-Steward

, Couturier

, et al. CCAC Guidelines On: The Care and Use of Fish in Research, Teaching, and Testing. Ottawa, ON: Canadian Council on Animal Care; 2005.

31.

Department of Animal Husbandry and Dairying Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of

Fisheries

, Government of

India

. Guidelines of CPCSEA for Experimentation on Fishes; 2021. Available from: https://ccsea.gov.in/Content/54_1_ACTS,RULESANDGUIDELINES.aspx [Last accessed: November 1, 2023].

32.

Ministry of Public

Health

, Department of

Research

. Policy on Zebrafish Research; 2020. Available from: https://www.qu.edu.qa/static_file/qu/research/documents/qu_iacuc/Policy%20on%20Zebrafish%20Research.pdf [Last accessed: November 1, 2023].

33.

UCSF Office of Research Institutional Animal Care and Use Program. Euthanasia IACUP Policy; 2022. Available from: https://iacuc.ucsf.edu/guidelines [Last accessed: November 1, 2023].

34.

University of

California

, Berkeley. Euthanasia Guidelines - Animal Care and Use Program; 2021. Available from: https://acuc.berkeley.edu/guidelines/euthanasia.pdf [Last accessed: November 1, 2023].

35.

Texas Tech University Institutional Animal Care and Use Committee, Health Sciences Center, Office of Research. IACUC Policy 23: Use of Zebrafish for Research and Teaching (2021). Available at: https://www.ttuhsc.edu/research/divisions/integrity-office/animal-care/documents/P23_Zebrafish_031221.pdf [Last accessed: November 1, 2023].

36.

McGill University Standard Operating Procedure #303.03. Fish and Aquatic Amphibian Euthanasia (2022). Available from: https://www.mcgill.ca/research/files/research/303-fish_and_aquatic_amphibian_euthanasia_-_march_2022_v2.pdf [Last accessed: November 1, 2023].