Zebrafish (Danio rerio) meets bioethics: the 10Rs ethical principles in research

Aryelle Canedo Patrícia Saiki Andressa Liberal Santos Karla da Silva Carneiro Andreza Martins de Souza Gabriel Qualhato Rafaella da Silva Brito Francyelli Mello-Andrade Thiago Lopes Rocha About the authors

Abstract

Zebrafish (Danio rerio) is a tropical fish species widely used in research, worldwide. The development of genetically modified animals and the increasing number of zebrafish breeding facilities due to their emerging use in several research fields, opened room for new ethical challenges for research carried out with this species. It is necessary to raise the scientific community’s awareness of the ethical standards and laws in force, on animal research. Thus, the aim of the current study is to describe 10 Rs ethical principles by using zebrafish as model system in research. The classical 3 Rs concerning animal welfare, namely replacement, reduction and refinement; and the added 7 Rs related to scientific (registration, reporting, robustness, reproducibility and relevance) and conduct principles (responsibility, and respect) in zebrafish research are herein presented and critically discussed. The use of these 10 Rs by researchers, institutions and the Animal Ethics Committee is recommended to support regulations, decision-making about and the promotion of zebrafish health and welfare in research.

Keywords:
animal health; animal welfare; animal ethics committees; fish; laboratory animals

Resumo

O peixe-zebra (Danio rerio) é um peixe tropical amplamente usado em pesquisas em todo o mundo. Devido ao seu uso emergente em várias áreas de pesquisa, o desenvolvimento de animais geneticamente modificados e o aumento das instalações de peixe-zebra, novos desafos éticos surgem na pesquisa com o peixe-zebra. Além disso, é necessária a conscientização da comunidade científica sobre as normas éticas e leis vigentes na pesquisa com animais. Assim, o presente estudo teve como objetivo descrever os princípios éticos de 10 Rs usando o peixe-zebra como sistema modelo em pesquisa. Os 3 Rs clássicos relativos ao bem-estar animal (substituição, redução e refinamento) e 7 Rs adicionais relacionados aos princípios científicos (registro, relatório, robustez, reprodutibilidade e relevância) e de conduta (responsabilidade e respeito) na pesquisa do peixe-zebra são apresentados e discutido criticamente. Recomendamos o uso desses 10 Rs pelos pesquisadores, instituições e Comitê de Ética Animal na regulamentação, decisão e promoção da saúde e bem-estar do peixe-zebra em pesquisas.

Palavras-chave:
saúde animal; bem-estar animal; comitês de ética animal; peixe; animais de laboratório

Introduction

Zebrafish, Danio rerio (Hamilton 1822), is a small tropical fish species popular in aquariums. In the late 1960s, zebrafish was introduced as vertebrate model system by George Streisinger. Thenceforth, zebrafish has been used as well-established model system in several research fields, such as developmental biology(11 Selderslaghs IW, Blust R, Witters HE. Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reproductive toxicology. 2012;33(2):142-154. doi:10.1016/j.reprotox.2011.08.003
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), pharmacology and toxicology(33 Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?. Comparative biochemistry and physiology. Toxicology & pharmacology: CBP,2009;149(2):196-209. doi:10.1016/j.cbpc.2008.11.006
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, 44 CanedoA, Rocha TL. Zebrafish (Danio rerio) using as model for genotoxicity and DNA repair assessments: historical review, current status and trends. The Science of the total environment. 2021;762:144084. doi:10.1016/j.scitotenv.2020.144084
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, 55 Canedo A, de Jesus LWO, Bailão EFLC, Rocha TL. Micronucleus test and Nuclear Abnormality assay in zebrafish (Danio rerio): past, present and future trends. Environment Pollution. 2021:290:118019. doi:10.1016/j.envpol.2021.118019
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), ecotoxicology(66 Cristiano W, Lacchetti I, Mancini L, Corti M, Di Domenico K, Di Paolo C, Hollert H, Carere M. Promoting zebrafish embryo tool to identify the effects of chemicals in the context of Water Framework Directive monitoring and assessment. Microchemical Journal. 2019:149:104035. doi:10.1016/j.microc.2019.104035
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, 77 Ribeiro RX, da Silva Brito R, Pereira AC, Monteiro KBES, Gonçalves BB, Rocha TL. Ecotoxicological assessment of efuents from Brazilian wastewater treatment plants using zebrafish embryotoxicity test: A multi-biomarker approach. The Science of the total environment. 2020;735:139036. doi:10.1016/j.scitotenv.2020.139036
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), veterinary sciences(8)8 Nowik N, Podlasz P, Jakimiuk A, Kasica N, Sienkiewicz W, Kaleczyc J. Zebrafish: an animal model for research in veterinary medicine. Polish journal of veterinary sciences. 2015;18(3):663-674. doi:10.1515/pjvs-2015-0086
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, evolution biology(9)9 Parichy DM. The Natural History of Model Organisms: Advancing biology through a deeper understanding of zebrafish ecology and evolution. eLife 2015;4:e05635. doi: 10.7554/eLife.05635.
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, nanotechnology and nanomedicine(1010 Sieber S, Grossen P, Bussmann J, et al. Zebrafish as a preclinical in vivo screening model for nanomedicines. Advanced drug delivery reviews. 2019;151-152:152-168. doi:10.1016/j.addr.2019.01.001
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, 1111 Pereira AC, Gomes T, Ferreira Machado MR, Rocha TL. The zebrafish embryotoxicity test (ZET) for nanotoxicity assessment: from morphological to molecular approach. Environmental pollution. 2019;252(Pt B):1841-1853. doi:10.1016/j.envpol.2019.06.100
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), human disease(12)12 Dooley K, Zon LI. Zebrafish: a model system for the study of human disease. Current opinion in genetics & development. 2000;10(3):252-256. doi:10.1016/s0959-437x(00)00074-5
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, vaccination(13)13 Bailone RL, R, Fukushima HCS, Fernandes BHV, DeAguiar LK, Corrêa T, Janke H, Setti PG, Roça RO, Borra RC. Zebrafish as an alternative animal model in human and animal vaccination research. Laboratory Animal Research. 2020;36(13):1-10. doi:10.1186/s42826-020-00042-4
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, food safety(14)14 Bailone RL, Aguiar LKD, Roca RDO, Borra RC, Corrêa T, Janke H, Fukushima HCS. Zebrafish as an animal model for food safety research: trends in the animal research. Food Biotechnology. 2019;33(4): 283-302. http://dx.doi.org/10.1080/08905436.2019.1673173
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, among others (Figure 1). Zebrafish is also a preclinical screening model used in vivo for personalized medicine(15)15 Baxendale S, van Eeden F, Wilkinson R. “The Power of Zebrafish in Personalised Medicine.” Advances in experimental medicine and biology. 2017;1007:179-197. doi:10.1007/978-3-319-60733-7_10
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and nanomedicine(10)10 Sieber S, Grossen P, Bussmann J, et al. Zebrafish as a preclinical in vivo screening model for nanomedicines. Advanced drug delivery reviews. 2019;151-152:152-168. doi:10.1016/j.addr.2019.01.001
https://doi.org/10.1016/j.addr.2019.01.0...
. Interestingly, it is an emerging suitable vertebrate model system in the Global South(16)16 Trigueiro, N., Canedo, A., Braga, D., Luchiari, A. C., Rocha, T. L., 2020. Zebrafish as an Emerging Model System in the Global South: Two Decades of Research in Brazil. Zebrafish. 10.1089/zeb.2020.1930. Advance online publication. doi:10.1089/zeb.2020.1930
https://doi.org/10.1089/zeb.2020.1930...
, since it accounts for decentralizing scientific publications.

Figure 1
Zebrafish use in several scientific research fields.

There are several advantages in using zebrafish as model system, such as transparent embryos, high reproduction and fertilization rate, rapid development, short life cycle, small size, low-cost and easy maintenance under laboratory conditions, sequenced genome, genetic similarity to humans, complex behaviour(1717 Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Developmental dynamics: an official publication of the American Association of Anatomists. 1995;203(3):253-310. doi:10.1002/aja.1002030302
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, 1818 Aksoy YA, Nguyen DT, Chow S, et al. Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos. Communications biology. 2019;2:198. doi:10.1038/s42003-019-0444-0
https://doi.org/10.1038/s42003-019-0444-...
), as well as mutant strains, transgenic reporters and knockdown(19)19 Fernandes MR, Pedroso AR. Animal experimentation: A look into ethics, welfare and alternative methods. Revista da Associação Medica Brasileira (1992). 2017;63(11):923-928. doi:10.1590/1806-9282.63.11.923
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. Furthermore, zebrafish embryos and larvae are an alternative to using adults in toxicity tests(3)3 Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?. Comparative biochemistry and physiology. Toxicology & pharmacology: CBP,2009;149(2):196-209. doi:10.1016/j.cbpc.2008.11.006
https://doi.org/10.1016/j.cbpc.2008.11.0...
. According to international ethical regulations, zebrafish larvae (up to 120 h post-fertilization (hpf)) are considered models in vitro (Directive 2010/63/EU)(20)20 Cornet C, Calzolari S, Miñana-Prieto R, et al. ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish. International journal of molecular sciences. 2017;18(4):864. doi:10.3390/ijms18040864
https://doi.org/10.3390/ijms18040864...
. The Fish Embryo Acute

Toxicity (FET) test can determine the toxic effects of different pollutants (e.g., nanomaterials, pesticides, pharmaceuticals, industrial chemicals, metals, among others)(21)21 OECD, 2013. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 236: Applicability of the Fish Embryo Acute Toxicity (FET) Test. Organization for Economic Cooperation and Development, Paris, France.. Using animals in research involves ethical challenges; in 1959, Russell and Burch published the book titled “The principles of humane experimental technique”, which became a reference in animal management based on the “3 Rs” (replacement, reduction and refinement) principle(22)22 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biology. 2010;8(6):e1000412. doi:10.1371/journal.pbio.1000412
https://doi.org/10.1371/journal.pbio.100...
. This principle recommends replacing vertebrates by animals who have lower potential for pain perception, based on techniques in vitro or computational methods, to reduce the number of animals per experiment and to use techniques that minimize animals’ pain and distress(23)23 Council of Europe, 1986, European convention for the protection of vertebrate animals used for experimental and other scientific purposes, European Treaty Series 123, Council of Europe, Strasbourg.. Guidelines were created to determine the ethical procedures for handling animals in research; they incorporated the “3 Rs” principle. Among them, one finds the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes(24)24 European Union, 2010, ‘Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes’, Official Journal of the European Union 276, 33–79., Directive 2010/63/EU on the protection of animals used for scientific purposes (25)25 Basel Declaration Society, 2010, Basel Declaration: A call for more trust, transparency and communication on animal research, Basel Declaration Committee, Basel., Basel Declaration(26)26 National Research Council, 2011, Guide for the care and use of laboratory animals: Eighth edition, National Academic Press, Washington, DC., Guide for the care and use of laboratory animals(27)27 Society of Environmental Toxicology and Chemistry, n.d., SETAC code of ethics, viewed 30 August 2021, from https://www.setac.org/?page=SETACEthics
https://www.setac.org/?page=SETACEthics...
, and Code of Ethics(28)28 American Fisheries Society, 2014, ‘Guidelines for the use of fishes in research’, American Fisheries Society, Bethesda, MD.. Accordingly, researchers account for the ethical and scientific validity of their studies, whereas institutions and government agencies make researchers follow the ethics codes, to apply appropriate strategies, techniques and facilities to use animals in research(2424 European Union, 2010, ‘Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes’, Official Journal of the European Union 276, 33–79., 2626 National Research Council, 2011, Guide for the care and use of laboratory animals: Eighth edition, National Academic Press, Washington, DC., 2727 Society of Environmental Toxicology and Chemistry, n.d., SETAC code of ethics, viewed 30 August 2021, from https://www.setac.org/?page=SETACEthics
https://www.setac.org/?page=SETACEthics...
, 2929 Sloman KA, Bouyoucos IA, Brooks EJ, Sneddon LU. Ethical considerations in fish research. Journal of Fish Biology. 2019;94(4):556-577. https://doi.org/10.1111/jfb.13946
https://doi.org/10.1111/jfb.13946...
).

Despite the several scientific fields focused on using fish as model system in research, the application of welfare concepts to fish has received less attention than that paid to mammals (30)30 Canadian Council on Animal Care, 2005, Guidelines on: The care and use of fish in research, teaching and testing, Canadian Council on Animal Care, Ottawa.. However, procedural guidelines, mainly the ones regarding fish, have created Guidelines on the care and use of fish in research, teaching and testing (31)31 Osborne N, Paull G, Grierson A, et al. Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care. Zebrafish. 2016;13(6):584-589. doi:10.1089/zeb.2016.1324
https://doi.org/10.1089/zeb.2016.1324...
, and guidelines for fish use in research (29)29 Sloman KA, Bouyoucos IA, Brooks EJ, Sneddon LU. Ethical considerations in fish research. Journal of Fish Biology. 2019;94(4):556-577. https://doi.org/10.1111/jfb.13946
https://doi.org/10.1111/jfb.13946...
. These guidelines aim at ensuring fish well-being in fish breeding facilities and under laboratory conditions; they point out that it is necessary (i) maintaining adequate water and food levels, and avoiding malnutrition; (ii) animals must be free from disease or injury; (iii) behavioural-interactive restriction must not be allowed; (iv) animals cannot suffer with stress or physical conditions (2929 Sloman KA, Bouyoucos IA, Brooks EJ, Sneddon LU. Ethical considerations in fish research. Journal of Fish Biology. 2019;94(4):556-577. https://doi.org/10.1111/jfb.13946
https://doi.org/10.1111/jfb.13946...
, 3131 Osborne N, Paull G, Grierson A, et al. Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care. Zebrafish. 2016;13(6):584-589. doi:10.1089/zeb.2016.1324
https://doi.org/10.1089/zeb.2016.1324...
).

A meeting on “Contemporary Topics in Zebrafish Management and Care” was held in the UK in 2014 to bring together researchers, animal technicians and veterinarians who have discussed topics such as (i) larvae rearing protocol; (ii) 3Rs implementation; (iii) management practices, cryopreservation, and environmental enrichment (31)31 Osborne N, Paull G, Grierson A, et al. Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care. Zebrafish. 2016;13(6):584-589. doi:10.1089/zeb.2016.1324
https://doi.org/10.1089/zeb.2016.1324...
. All these measures helped improving zebrafish’s welfare condition in laboratory environment. Furthermore, it is essential standardizing specific lines and protocols to bring benefits to research (32)32 Lidster K, Readman GD, Prescott MJ, Owen SF. International survey on the use and welfare of zebrafish Danio rerio in research. Journal of Fish Biology. 2017;90(5):1891-1905. https://doi.org/10.1111/jfb.13278
https://doi.org/10.1111/jfb.13278...
.

The aim of the current study was to describe 10 Rs ethical principles by using zebrafish as model system in research given the growing use of this species as model system and the need of ensuring these animals’ welfare and health. Implementing the 7 Rs is an additional initiative to contribute to Animal Ethics Committees at the time to regulate, guide studies, report research carried out with zebrafish, increase the availability of information about this animal model and to minimize unnecessary studies, as well as to contribute to ensure zebrafish welfare and health both in fish breeding facilities and research centres.

The 10 Rs for research carried out with zebrafish

The classical 3 Rs for animal welfare (replacement, reduction, and refinement) were added with 7 Rs related to scientific (registration, reporting, robustness, reproducibility and relevance) and conduct principles (responsibility and respect) in order to help scientists improving zebrafish welfare and health in research carried out with it, as well as experiments’ integrity and relevance. These principles encompass different developmental stages of zebrafish used in research, namely, at the embryo, larvae, juveniles and adult stages (Figure 2). The 10 Rs for research carried out with zebrafish have no scale of importance, but they must be recommended for zebrafish use in research.

Figure 2
The 10 Rs ethical principles for zebrafish using as model system in research. The principles were classified into three categories, namely: welfare (replacement, reduction and refinement), scientific (registration, reporting, robustness, reproducibility and relevance) and conduct principles (responsibility and respect).

Animal welfare

1R – Replacement

The discussion on ethical principles and welfare in animals’ use for educational and scientific purposes lights up the need of replacing model systems in order to eliminate or reduce pain, stress and suffering(25)25 Basel Declaration Society, 2010, Basel Declaration: A call for more trust, transparency and communication on animal research, Basel Declaration Committee, Basel.. Such a replacement can be total or partial; total replacement of animals - without compromising research results - can be accomplished through the adoption of emerging and well-established techniques(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
. Cell lines, tissue and organoid cultures, organ chips, and computational and mathematical modeling are among the commonly applied methodologies(3434 Ford KA. Refinement, Reduction, and Replacement of Animal Toxicity Tests by Computational Methods. ILAR J. 2016;57(2):226-233. doi:10.1093/ilar/ilw031
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, 3535 Schaeck M, Van den Broeck W, Hermans K, Decostere A. Fish as research tools: alternatives to in vivo experiments. Alternatives to laboratory animals: ATLA. 2013;41(3):219-229. doi:10.1177/026119291304100305
https://doi.org/10.1177/0261192913041003...
).

For many years, research was carried out with mammals, because of their common ancestry with human beings; therefore, it allowed extrapolating the results and applying them to human health conditions(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
. However, several regulatory authorities recommended reducing, or eliminating the use of mammal models in research(2525 Basel Declaration Society, 2010, Basel Declaration: A call for more trust, transparency and communication on animal research, Basel Declaration Committee, Basel., 3636 USEPA, 2020. New approach methods work plan: Reducing use of animals in chemical testing. U.S. Environmental Protection Agency, Washington, DC. EPA 615B2001. https://www.epa.gov/sites/default/fles/2020-06/documents/epa_nam_work_plan.pdf
https://www.epa.gov/sites/default/fles/2...
). The United States Environmental Protection Agency (USEPA) will stop conducting, or funding, studies involving mammal models by 2035(36)36 USEPA, 2020. New approach methods work plan: Reducing use of animals in chemical testing. U.S. Environmental Protection Agency, Washington, DC. EPA 615B2001. https://www.epa.gov/sites/default/fles/2020-06/documents/epa_nam_work_plan.pdf
https://www.epa.gov/sites/default/fles/2...
.

Many guidelines to assess chemicals’ toxicity have been published; they provide on the use of different animal groups, other than mammals, such as diptera, crustaceans, earthworm species, molluscs and fish(3737 OECD, 2010. OECD Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 233: Sediment-water Chironomid life-cycle toxicity test using spiked water or spiked sediment. Organization for Economic Cooperation and Development, Paris, France., 3838 OECD, 2012. OECD Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 211: Daphnia magna Reproduction Test. Organization for Economic Cooperation and Development, Paris, France., 3939 OECD, 2016a. OECD Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei). Organization for Economic Cooperation and Development, Paris, France., 4040 OECD, 2016b. OECD Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 243: Lymnaea stagnalis Reproduction Test. Organization for Economic Cooperation and Development, Paris, France., 4141 OECD, 2019. OECD Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 203: Fish, acute toxicity testing. Organization for Economic Cooperation and Development, Paris, France., 4242 Streisinger G, Walker C, Dower N, Knauber D, Singer F. Production of clones of homozygous diploid zebra fish (Brachydanio rerio). Nature. 1981;291(5813):293-296. doi:10.1038/291293a0
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). Thus, zebrafish emerged as experimental animal model back in the 1980s, and remained so onwards(43)43 Eisen JS. History of zebrafish research. The Zebrafish in Biomedical Research Biology Husbandry, Diseases and Research Applications. 2020: 3-14. doi: 10.1016/B978-0-12-812431-4.00001-4
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- one can observe the significant increase in the number of publications based on this model since the 1990s(44)44 Khan FR, Alhewairini SS. Zebrafish (Danio rerio) as a model organismo. In: Streba L, Gheonea DI, Schenker M. Current Trends in cancer mangement. IntechOpen. 2018. doi: 10.5772/intechopen.81517
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. This small teleost fish presents a whole variety of features that turns it into a gold standard in scientific research, mainly to replace mammals(45)45 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Mufato M, Collins JE, Humphray S, Mclaren K, Mathews L, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013; 496:498-503. https://doi.org/10.1038/nature12111
https://doi.org/10.1038/nature12111...
. Zebrafish genome, among other features, was fully sequenced and it shares 70% genetic similarity with humans(46)46 ZFIN, 2021. Zebrafish strains. University of Oregon. EPA 615B2001. https://zfin.org/zf_info/zfbook/zfstrn.html. Access in 14 sep 2021.
https://zfin.org/zf_info/zfbook/zfstrn.h...
; moreover, its homology is very similar to that of mouse models (approximately 75%)(47)47 Lee KY, Jang GH, Byun CH, Jeun M, Searson PC, Lee KH. Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications. Bioscience reports. 2017;37(3):BSR20170199. doi:10.1042/BSR20170199
https://doi.org/10.1042/BSR20170199...
. In addition, there are thousands of mutant and transgenic zebrafish strains(48)48 Belanger SE, Rawlings JM, Carr GJ. Use of fish embryo toxicity tests for the prediction of acute fish toxicity to chemicals. Environmental toxicology and chemistry. 2013;201332, 1768-1783. doi:10.1002/etc.2244
https://doi.org/10.1002/etc.2244...
.

Several studies have shown that zebrafish embryos and larvae have similar responses to those of adult individuals, and it optimizes model system replacement from the welfare viewpoint, since it saves resources and minimizes waste generation(33 Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T. Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test?. Comparative biochemistry and physiology. Toxicology & pharmacology: CBP,2009;149(2):196-209. doi:10.1016/j.cbpc.2008.11.006
https://doi.org/10.1016/j.cbpc.2008.11.0...
, 4949 Braunbeck T, Kais B, Lammer E, Otte J, Schneider K, Stengel D, Strecker R. The fish embryo test (FET): origin, applications, and future. Environmental science and pollution research international. 2014; 22, 16247-16261. doi:10.1007/s11356-014-3814-7
https://doi.org/10.1007/s11356-014-3814-...
, 5050 Sneddon LU, Halsey LG, Bury NR. Considering aspects of the 3Rs principles within experimental animal biology. The Journal of experimental biology. 2017;220(Pt 17):3007-3016. doi:10.1242/jeb.147058
https://doi.org/10.1242/jeb.147058...
). Furthermore, tests with zebrafish embryos and larvae do not suffer with regulation, since there is no extra-chorionic feeding at these developmental stages(2525 Basel Declaration Society, 2010, Basel Declaration: A call for more trust, transparency and communication on animal research, Basel Declaration Committee, Basel., 5151 Bayne K, Ramachandra GS, Rivera EA, Wang J. The Evolution of Animal Welfare and the 3Rs in Brazil, China, and India. Journal of the American Association for Laboratory Animal Science. 2015;54(2):181-191.). Zebrafish embryos stand out for animal replacement - even at early developmental stages – since numerous biochemical reactions, organ diferentiation and morphological formations take place at this stage, and it opens room for evaluating pollutants and drugs in a whole living organism(18)18 Aksoy YA, Nguyen DT, Chow S, et al. Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos. Communications biology. 2019;2:198. doi:10.1038/s42003-019-0444-0
https://doi.org/10.1038/s42003-019-0444-...
, at real-time and at researcher's naked eye, due to their transparency.

2R – Reduction

Reduction is an important principle to be taken into consideration when animals’ replacement during scientific research is not possible(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
. Reduction means decreasing the number of animals used in protocols and experimental designs(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
; therefore, the minimum number of zebrafish must be used to achieve scientific or educational aims when no alternative methods such as mathematical models, computer simulation, biological systems in vitro, or other non-animal (adjuvant) approaches are available to replace the use of living animals(52)52 Rácz A, Allan B, Dwyer T, Thambithurai D, Crespel A, Killen SS. Identification of Individual Zebrafish (Danio rerio): A Refined Protocol for VIE Tagging Whilst Considering Animal Welfare and the Principles of the 3Rs. Animals. 2021;11(3):616. https://doi.org/10.3390/ani11030616
https://doi.org/10.3390/ani11030616...
.

Researchers must initially choose an adequate model system for the analyses carried out in their experiments in order to avoid the unnecessary use of animals. Furthermore, the number of animals used in experiments must be consistent and well-justifed. This principle should be applied in cases where reducing the number of nimals does not impair the scientific rigor of the analysis(30)30 Canadian Council on Animal Care, 2005, Guidelines on: The care and use of fish in research, teaching and testing, Canadian Council on Animal Care, Ottawa.. Some resources can be used to estimate the fish-sample size needed to start an experiment, among them there are software and power analysis, which aim at determining the smallest sample size necessary to detect a given effect at a specific significance level(30)30 Canadian Council on Animal Care, 2005, Guidelines on: The care and use of fish in research, teaching and testing, Canadian Council on Animal Care, Ottawa..

In addition, Directive 2010/63/EU suggests that researchers must share organs and tissue of euthanized animals to easily establish new methods in vitro(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
or to use the same individuals in different analyses, whenever possible(53)53 Workman P, Aboagye E, Balkwill F. et al. Guidelines for the welfare and use of animals in cancer research. British Journal of Cancer, 2010;102(11):1555–1577. doi:10.1038/sj.bjc.6605642
https://doi.org/10.1038/sj.bjc.6605642...
. These are respectful ways to minimize the use of animals in research, rather than to just discard unused biological structures(33)33 Días L, Zambrano E, Flores ME, Contreras M, Crispin JC, Alemán G, Bravo C, Armenta A, Valdés VJ, Tovar A, gamba G, Barrios-Payán J, Bobadilla NA. Ethical considerations in animal research: The principle of 3R's. Revista de investigacion clinica; organo del Hospital de Enfermedades de la Nutricion. 2021;73(4):199-209. doi:10.24875/ric.20000380
https://doi.org/10.24875/ric.20000380...
.

Zebrafish models reduce the time and resource demands, as well as space demands and costs, in comparison to more popular animal models, such as rodents. Zebrafish also provides greater informational and predictive potential that results from experiments in vitro. Thus, this model can reduce the use of mammals in research and mitigate issues related to these animals’ well-being(13)13 Bailone RL, R, Fukushima HCS, Fernandes BHV, DeAguiar LK, Corrêa T, Janke H, Setti PG, Roça RO, Borra RC. Zebrafish as an alternative animal model in human and animal vaccination research. Laboratory Animal Research. 2020;36(13):1-10. doi:10.1186/s42826-020-00042-4
https://doi.org/10.1186/s42826-020-00042...
.

3R – Refinement

Although using non-human models remains necessary in scientific research, excellence standards on the care provided to animals involved in it should fully comply with research conduct(5454 Rinkwitz S, Mourrain P, Becker TS. Zebrafish: An integrative system for neurogenomics and neurosciences. Progress in Neurobiology, 2011;93(2):231-243. doi:10.1016/j.pneurobio.2010.11.003
https://doi.org/10.1016/j.pneurobio.2010...
, 5555 Garcia GR, Noyes PD, Tanguay RL. Advancements in zebrafish applications for 21st century toxicology. Pharmacology and Therapeutics, 2016;161:11-21. doi: 10.1016/j.pharmthera.2016.03.009.
https://doi.org/10.1016/j.pharmthera.201...
, 5656 Seixas MM. et al. Consciência Na Substituição Do Uso De Animais No Ensino: Aspectos Históricos, Éticos E De Legislação. Revista Brasileira de Direito Animal, 2014; 5(6):71-96. doi:10.9771/rbda.v5i6.11073
https://doi.org/10.9771/rbda.v5i6.11073...
). Refining the way experiments are carried out means ensuring that animals suffer as little as possible with them, and it includes better housing, adequate water parameters, regular feeding, adequate anaesthesia and analgesia procedures(5757 Augustsson, H, van de Weerd, HA, Kruitwagen, CL, Baumans, V. Effect of enrichment on variation and results in the light/dark test. Laboratory Animals, 2003;7(4), 328-340. doi:10.1258/002367703322389898
https://doi.org/10.1258/0023677033223898...
, 5858 Russell WMS, Burch RL. The Principles of Humane Experimental Technique. special ed ed. [s.l: s.n.]., 5959 Huntingford FA, Adams C, Braithwaite VA, Kadri S, Pottinger TG, Sandoe P, Turnbull JF. Current issues in fish welfare. Journal of Fish Biology, 2006;68(2), 332-372. doi:10.1111/j.0022-1112.2006.001046.x
https://doi.org/10.1111/j.0022-1112.2006...
, 6060 Stevens CH, Reed BT, Hawkins P. Enrichment for Laboratory Zebrafish-A Review of the Evidence and the Challenges. Animals (Basel). 2021;11(3):698. doi:10.3390/ani11030698
https://doi.org/10.3390/ani11030698...
, 6161 Hawkins P, Dennison N, Goodman G, et al. Guidance on the severity classification of scientific procedures involving fish: Report of a Working Group appointed by the Norwegian Consensus-Platform for the Replacement, Reduction and Refinement of animal experiments (Norecopa). Laboratory Animals, 2011;45(4):219-224. doi:10.1258/la.2011.010181
https://doi.org/10.1258/la.2011.010181...
).

Just as all other vertebrates, fish are driven to environmental challenges due to a whole series of adaptive neuroendocrine adjustments known as stress response; moreover, the prolonged activation of this response is detrimental and leads to immunosuppression, reduced growth and reproductive dysfunction. Indicators associated with response to chronic stress (physiological outcomes, disease status and behaviour) are a source of information about fish’s welfare state(6262 Collymore C, Tolwani RJ, Rasmussen S. The behavioral effects of single housing and environmental enrichment on adult zebrafish (Danio rerio). Journal of the American Association for Laboratory Animal Science, 2015;54(3):280-285., 3030 Canadian Council on Animal Care, 2005, Guidelines on: The care and use of fish in research, teaching and testing, Canadian Council on Animal Care, Ottawa.). Developing alternatives to increase welfare not only favours fish health and welfare, but also improves scientific data reliability and repeatability, a fact that has the potential to reduce the number of animals used in research(63)63 Kistler C, Hegglin D, Wurbel H, Konig B, Preference for structured environment in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis). Applied Animal Behaviour Science, 2011, 135(4);318–327. doi: 10.1016/j.applanim.2011.10.014
https://doi.org/10.1016/j.applanim.2011....
.

Zebrafish is often kept in water recirculation systems, in commercial polycarbonate tanks with bluish coloration or glass aquarium, at density of up to 5 animals per litter of water. This practical measure enables the formation of social groups typical of zebrafish’s behaviour(6464 White LJ, Thomson JS, Pounder KC, Coleman RC, Sneddon LU. The impact of social context on behaviour and the recovery from welfare challenges in zebrafish, Danio rerio. Animal Behaviour, 2017;132:189-199. doi:10.1016/j.anbehav.2017.08.017
https://doi.org/10.1016/j.anbehav.2017.0...
, 6565 Engeszer RE, Patterson LB, Rao AA, Parichy DM. Zebrafish in the wild: A review of natural history and new notes from the field. Zebrafish, 2007;4(1):21-40. doi: 10.1089/zeb.2006.9997.
https://doi.org/10.1089/zeb.2006.9997...
, 6666 Best J, Adatto I, Cockington J, James A, Lawrence C. A novel method for rearing frst-feeding larval zebrafish: Polyculture with type L saltwater rotifers (Brachionus plicatilis). Zebrafish, 2010;7(3):289-295. doi:10.1089/zeb.2010.0667
https://doi.org/10.1089/zeb.2010.0667...
). In addition to using aquarium coloration, it is also recommended to mimic its natural environment, namely: controlled photoperiod and availability of living food in order to stimulate the natural prey capture behaviour and to improve nutritional quality(6767 CONSTITUIÇÃO FEDERAL. LEI Nº 11.794 DE 8 DE OUTUBRO DE 2008.Brasil, 2008. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/111794.htm
http://www.planalto.gov.br/ccivil_03/_at...
, 6868 Neifer DL, Stamper MA. Fish sedation, anesthesia, analgesia, and euthanasia: Considerations, methods, and types of drugs. ILAR Journal, 2009;50(4):343-360. doi:10.1093/ilar.50.4.343
https://doi.org/10.1093/ilar.50.4.343...
, 3232 Lidster K, Readman GD, Prescott MJ, Owen SF. International survey on the use and welfare of zebrafish Danio rerio in research. Journal of Fish Biology. 2017;90(5):1891-1905. https://doi.org/10.1111/jfb.13278
https://doi.org/10.1111/jfb.13278...
). Zebrafish are kept in tanks at temperature ranging from 26 °C to 28°C, water pH ranging from 7 to 8, alkalinity ranging from 50 to 150 mg of CaCO3/L, water hardness at levels higher than 75 mg/L, salinity close to 0.5-2 g/L and dissolved oxygen levels of at least 4 mg/L(69)69 Dammski AP, Muller BR, Gaya C, Regonato D. Zebrafish: Manual de Criação em Biotério. Curitiba: Universidade Federal do Paraná. 2011;1-107.. Strains used in laboratory come from strains stabilized with genetic background, since it leads to the loss of natural adaptive capacity and reduces the tolerance to environmental changes, mainly to those related to water physiochemical conditions(70)70 Trevarrow B, Robison B. Genetic backgrounds, standard lines, and husbandry of zebrafish. Methods in cell biology. 2004;77:599-616. doi:10.1016/s0091-679x(04)77032-6
https://doi.org/10.1016/s0091-679x(04)77...
. Thus, it is necessary to always keep the physicochemical parameters within the reference values recommended in the species' breeding guides and manuals in order to avoid stressful situations(69)69 Dammski AP, Muller BR, Gaya C, Regonato D. Zebrafish: Manual de Criação em Biotério. Curitiba: Universidade Federal do Paraná. 2011;1-107..

Experimental manipulations that can cause pain or anguish must be carried out under sedation, analgesia or anesthesia, since fish have robust neuroendocrine and physiological responses to stress caused by harmful stimuli(7171 Martins S, Monteiro JF, Vito M, Weintraub D, Almeida J, Certal AC. Toward an Integrated Zebrafish Health Management Program Supporting Cancer and Neuroscience Research. Zebrafish, 2016a; 13(00):S47–S55. doi: 10.1089/zeb.2015.1198
https://doi.org/10.1089/zeb.2015.1198...
, 7272 Martins T, Valentim AM, Pereira N, Antunes LM. Anaesthesia and analgesia in laboratory adult zebrafish: A question of refinement. Laboratory Animals, 2016b; 50(6), 476-488. doi:10.1177/0023677216670686
https://doi.org/10.1177/0023677216670686...
). It is necessary using anaesthetic, sedative or analgesic drugs when fish have to be immobilized in order to help handling them, collecting them beforehand, or subjecting them to euthanasia. These are fundamental procedures to achieve fish welfare. It is important highlighting that these vertebrates can show signs of stress and/or pain during handling, transport, marking, sampling and invasive procedures(72)72 Martins T, Valentim AM, Pereira N, Antunes LM. Anaesthesia and analgesia in laboratory adult zebrafish: A question of refinement. Laboratory Animals, 2016b; 50(6), 476-488. doi:10.1177/0023677216670686
https://doi.org/10.1177/0023677216670686...
.

Using inadequate anaesthetic protocol can compromise not only fish welfare, but also research results’ reliability(73)73 Readman GD, Owen SF, Murrell JC, Knowles TG. Do fish perceive anaesthetics as aversive?. PLoS One. 2013;8(9):e73773. doi:10.1371/journal.pone.0073773
https://doi.org/10.1371/journal.pone.007...
. Studies have been carried out to investigate the most suitable drugs for zebrafish by taking into account the welfare principles as way to refine techniques applied to this species’ analgesia and anaesthesia procedures(7373 Readman GD, Owen SF, Murrell JC, Knowles TG. Do fish perceive anaesthetics as aversive?. PLoS One. 2013;8(9):e73773. doi:10.1371/journal.pone.0073773
https://doi.org/10.1371/journal.pone.007...
, 7474 Valentim AM, Félix LM, Carvalho L, Diniz E, Antunes LM. A new anaesthetic protocol for adult zebrafish (Danio rerio): Propofol combined with lidocaine. PLoS ONE, 2016;11(1):e0147747. doi:10.1371/journal.pone.0147747
https://doi.org/10.1371/journal.pone.014...
, 7575 Martins T, Valentim A, Pereira N, Antunes LM. Anaesthetics and analgesics used in adult fish for research: A review. Laboratory Animals, 2019; 53(4): 325-341. doi: 10.1177/0023677218815199
https://doi.org/10.1177/0023677218815199...
, 7676 Xing L, Quist TS, Stevenson TJ, Dahlem TJ, Bonkowsky JL. Rapid and efficient zebrafish genotyping using PCR with high-resolution melt analysis. Journal of Visualized Experiments, 2014;(84):e51138. doi:10.3791/51138
https://doi.org/10.3791/51138...
). Tricaine methanesulfonate (60-300 mg/L), clove oil (25-100 mg/L) and 2-phenoxyethanol (2-PE) (0.1-0.5 mg/L) are the most used anaesthetic agents in cyprinid fish. In addition, several factors can interfere with anaesthesia, such as developmental stages and environmental factors(77)77 Neifer DL, Stamper MA. Fish sedation, analgesia, anesthesia, and euthanasia: considerations, methods, and types of drugs. ILAR journal. 2009;50(4):343-360. doi:10.1093/ilar.50.4.343
https://doi.org/10.1093/ilar.50.4.343...
.

Studies focused on obtaining DNA usually have to cut the fin of adult fish under non-terminal anaesthesia(78)78 Schroeder PG, Sneddon LU. Exploring the eficacy of immersion analgesics in zebrafish using an integrative approach. Applied Animal Behaviour Science, 2017;187:93-102. doi:10.1016/j.applanim.2016.12.003
https://doi.org/10.1016/j.applanim.2016....
. The fin clipping effect can have negative influence on fish and lead to infection and change its survival, growth and behaviour, as well as decrease feeding and induce cortisol release(6565 Engeszer RE, Patterson LB, Rao AA, Parichy DM. Zebrafish in the wild: A review of natural history and new notes from the field. Zebrafish, 2007;4(1):21-40. doi: 10.1089/zeb.2006.9997.
https://doi.org/10.1089/zeb.2006.9997...
, 7979 De Lombaert MC, Rick EL, Krugner-Higby LA, Wolman MA. Behavioral characteristics of adult zebrafish (Danio rerio) after MS222 anesthesia for fin excision. Journal of the American Association for Laboratory Animal Science, 2017;56(4):377-381., 8080 Le Vin AL, Adam A, Tedder A, Arnold KE, Mable BK. Validation of swabs as a non-destructive and relatively non-invasive DNA sampling method in fish. Molecular Ecology Resources, 2011;11(1):107-9. doi: 10.1111/j.1755-0998.2010.02909.x.
https://doi.org/10.1111/j.1755-0998.2010...
), a fact that affects fish welfare. Eforts to improve and refine DNA collection techniques have increased, since such changes can drastically interfere with the quality of experimental data(8181 Breacker C, Barber I, Norton WH, McDearmid JR, Tilley CA.ALow-Cost Method of Skin Swabbing for the Collection of DNA Samples from Small Laboratory Fish. Zebrafish. 2017;14(1):35-41. doi: 10.1089/zeb.2016.1348.
https://doi.org/10.1089/zeb.2016.1348...
, 8282 Campanella JJ, Smalley JV. A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples. BMC Genetics, 2006,7, 5-8. doi:10.1186/1471-2156-7-32
https://doi.org/10.1186/1471-2156-7-32...
). Alternative DNA collection methods, such as scaling, mucus and sperm sampling, have been successfully used and they are gaining popularity. Some of these techniques are lesser invasive than fin clipping, in addition to have the potential to improve animal welfare(83)83 Tilley, C.A., Carreño Gutierrez, H., Sebire, M. et al. Skin swabbing is a refined technique to collect DNA from model fish species. Scientific Reports. 2020;10(1):1-17. doi:10.1038/s41598-020-75304-1
https://doi.org/10.1038/s41598-020-75304...
. A new collection technique was developed from skin swab smearing; it is lesser invasive and stressful for fish than fin clipping - such a finding confirms that ‘swab’ is a refined method for fish DNA collection(84)84 Mason, G. Stereotypic behaviour in captive animals: Fundamentals and implications for welfare and beyond. Stereotypic Animal Behaviour: Fundamentals and Applications to Welfare: Second Edition, 2006:326-356..

Many refinement techniques can be applied during scientific experiments, as well as throughout the animals’ lives and they can improve fish living conditions in breeding facilities(85)85 Gouveia K, Hurt JL. Optimising reliability of mouse performance in behavioural testing: The major role of non-aversive handling. Scientific Reports, 2017;7:1-12. doi.org/10.1038/srep44999
https://doi.org/10.1038/srep44999...
. Refinement not only favours animals, but it can also improve the quality of research results. Evidence suggests that pain and suffering can alter animals' behaviour and physiology during experimental periods. These changes can lead to variations in experimental results, and it can affect both studies’ reliability and repeatability(8686 Mulder A. Journal of Applied Animal Welfare Effects of Environmental Enrichment for Mice: Variation in Experimental Results. Journal of Applied Animal Welfare Science, 2010;8705(776099595):7-41., 8787 Strech D, Dirnagl U. 3Rs missing: animal research without scientific value is unethical. BMJ Open Science. 2019; 3(1)e000048. doi:10.1136/BMJOS-2018-000048
https://doi.org/10.1136/BMJOS-2018-00004...
). The development of lesser invasive and more refined methods must be taken into consideration whenever possible.

Scientific principles

4R – Registration

Scientific principles add value to science and society through relevant studies using zebrafish as model system; therefore, we must consider the 4th R: Registration(87)87 Strech D, Dirnagl U. 3Rs missing: animal research without scientific value is unethical. BMJ Open Science. 2019; 3(1)e000048. doi:10.1136/BMJOS-2018-000048
https://doi.org/10.1136/BMJOS-2018-00004...
. Adherence to methods focused on registering information available in studies conducted with animals has been inadequate in some cases, as well as has been leading to biased, delayed studies or unreported results(88)88 Percie du Sert, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M. T., Baker, M., Browne, W. J., Clark, A., Cuthill, I. C., Dirnagl, U., Emerson, M., Garner, P., Holgate, S. T., Howells, D. W., Karp, N. A., Lazic, S. E., Lidster, K., MacCallum, C. J., Macleod, M., Pearl, E. J., … Würbel, H. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS biology. 2020;18(7), e3000410. https://doi.org/10.1371/journal.pbio.3000410
https://doi.org/10.1371/journal.pbio.300...
. The Declaration of Helsinki includes Registration as mandatory principle. Platforms for these Registries, such as http://www.preclinicaltrials.eu and http://www.animalstudyregistry.org, were created by academic initiatives and governmental organizations, respectively. Protocol registrations can be quickly accomplished in these platforms, and it can increase research value. Thus, the use of zebrafish in research must be followed by the prior approval by local or national ethics committees on animals’ use in research. Information that is required for experiments’ Registration, as described in the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments), are related to study design, sample size, inclusion and exclusion criteria, randomization, outcome measurements, statistical methods, experimental animals, experimental procedures, results, abstract, background, objectives, ethical statements, housing and husbandry, animal care and monitoring, interpretation/scientific implications, generalisability/translation, protocol registration, data access and declaration of interests(88)88 Percie du Sert, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M. T., Baker, M., Browne, W. J., Clark, A., Cuthill, I. C., Dirnagl, U., Emerson, M., Garner, P., Holgate, S. T., Howells, D. W., Karp, N. A., Lazic, S. E., Lidster, K., MacCallum, C. J., Macleod, M., Pearl, E. J., … Würbel, H. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS biology. 2020;18(7), e3000410. https://doi.org/10.1371/journal.pbio.3000410
https://doi.org/10.1371/journal.pbio.300...
.

Registration of studies with zebrafish should be free for public access because the registered protocols allow observations on studies presenting the best results(87)87 Strech D, Dirnagl U. 3Rs missing: animal research without scientific value is unethical. BMJ Open Science. 2019; 3(1)e000048. doi:10.1136/BMJOS-2018-000048
https://doi.org/10.1136/BMJOS-2018-00004...
. Many controversies and embarrassments can be mitigated by this fast and practical registration of studies with zebrafish(89)89 Wieschowski S, Silva DS, Strech D. Animal Study Registries: Results from a Stakeholder Analysis on Potential Strengths, Weaknesses, Facilitators, and Barriers. PLoS biology. 2016;14(11):1-12. doi:10.1371/journal.pbio.2000391
https://doi.org/10.1371/journal.pbio.200...
. Zebrafish has several strains that may have different responses; thus, it is necessary having the Registry of these features(90)90 Séguret A, Collignon B, Halloy J. Strain diferences in the collective behaviour of zebrafish (Danio rerio) in heterogeneous environment. Royal Society open science. 2016;3(10):160451. doi:10.1098/rsos.160451
https://doi.org/10.1098/rsos.160451...
. It is likely thinking about increased administrative tasks or even about stealing ideas through the Register of Studies(89)89 Wieschowski S, Silva DS, Strech D. Animal Study Registries: Results from a Stakeholder Analysis on Potential Strengths, Weaknesses, Facilitators, and Barriers. PLoS biology. 2016;14(11):1-12. doi:10.1371/journal.pbio.2000391
https://doi.org/10.1371/journal.pbio.200...
; however, Registry’s aim, and its major outcomes, focus on making research with zebrafish even more efficient.

5R – Reporting

In short, a scientific report is a document to describe research processes and/or results, as well as the status of a problem faced by a technical research. It may also contain recommendations, cautions and conclusions for the research(91)91 Lobban, C. S and Schefter, M., 2021. Write Scientific Reports – The Library : University of Waikato [WWW Document]. Cambridge Univ. Press Cambridge. URL https://www.waikato.ac.nz/library/guidance/guides/write-scientific-reports#Elementsofa ScientificReport (accessed 9.17.21).
https://www.waikato.ac.nz/library/guidan...
. The aforementioned data can be published in technical notes, film communications and scientific articles; thus, methods and techniques must be disclosed in order to make techniques’ standardization easier, and to make sure of results’ integrity and reliability (91)91 Lobban, C. S and Schefter, M., 2021. Write Scientific Reports – The Library : University of Waikato [WWW Document]. Cambridge Univ. Press Cambridge. URL https://www.waikato.ac.nz/library/guidance/guides/write-scientific-reports#Elementsofa ScientificReport (accessed 9.17.21).
https://www.waikato.ac.nz/library/guidan...
. Despite their importance, the 3Rs may be inadequate for experiments run with animals, given their limited focus on the fundamental ethical principle, due to the complexity of the "animal welfare" principle. This situation can lead to lack of practical guiding principles aimed at promoting the second fundamental ethical principle, namely: "scientific value".

Scientific reports play fundamental role in developing methods, standards and parameters that can i) reinforce the 3 Rs, and ii) formulate new thoughts that can lead to discoveries to support new bioethical pillars. Documenting and publishing methods and reports based on models are actions directly influencing ethical and bioethical aspects of animal using. In case there is no detailed record of new methods, techniques, or even studies that have already been tested, there is no possibility of optimizing animals’ use and testing; therefore, there is not even the guarantee of compliance with the already established 3Rs. Furthermore, there is intrinsic and dependent relationship among research ethics, reports and records generated from the use of each animal model employed in science.

Accordingly, a meaningful report must have reliable methods and analyses based on statistical reliability to make sure of the quality and reliability of a given experiment or report. However, many experimental scientific papers are not published (50%), or do not leave the pre-print status(87)87 Strech D, Dirnagl U. 3Rs missing: animal research without scientific value is unethical. BMJ Open Science. 2019; 3(1)e000048. doi:10.1136/BMJOS-2018-000048
https://doi.org/10.1136/BMJOS-2018-00004...
; these uncertainties only contribute to the well-known 'reproducibility crisis' affecting those seeking new methods and techniques for their experiments(92)92 Mogil J, Macleod M. No publication without confirmation. Nature. 2017; 542:409-411. doi:10.1038/542409a.
https://doi.org/10.1038/542409a...
.

One way to improve reporting lies on considering the ethical and moral responsibilities of researchers, publishers, editors and funders, as well as on dealing with them in a responsible manner: i) researchers are in charge of clear and transparent reports; ii) Journal publishers and editors help putting improvements in place through their criticism and suggestions(93)93 Needleman I, Moher D, Altman DG, Schulz KF, Moles DR, Worthington H. Improving the clarity and transparency of reporting health research: a shared obligation and responsibility. Journal of dental research. 2008;87(10):894-895. doi:10.1177/154405910808701013
https://doi.org/10.1177/1544059108087010...
.

Given the challenges described above, D. rerio may be an excellent alternative to develop methods that contribute to reduce unambiguous reports, because its well-assessed biological model provides extensive support through publications and journals. Reports on D. rerio are significant due to a model that carries unique combinations of developmental and maintenance traits; it has a well-fused collection of methods that provide a system that has the advantages of running tests in vitro and in vivo(94)94 De Abreu MS, Giacomini ACVV, Echevarria DJ, Kaluef AV. Legal aspects of zebrafish neuropharmacology and neurotoxicology research. Regulatory Toxicology and Pharmacology. 2018;101: 65-70. doi:10.1016/j.yrtph.2018.11.007
https://doi.org/10.1016/j.yrtph.2018.11....
. This process will improve testing and the ability to replicate methods and experiments that can favour researchers in their technical responsibility to publish accurate and faithful reports(94)94 De Abreu MS, Giacomini ACVV, Echevarria DJ, Kaluef AV. Legal aspects of zebrafish neuropharmacology and neurotoxicology research. Regulatory Toxicology and Pharmacology. 2018;101: 65-70. doi:10.1016/j.yrtph.2018.11.007
https://doi.org/10.1016/j.yrtph.2018.11....
. This extensive collection of publications contributes to strengthen research and technological development, as well as gives strength to the other Rs, as they precisely depend on dissemination and scientific results to develop and interact with each other.

6R – Robustness

Environmental factors can change zebrafish welfare, health and response. On the other hand, robustness ensures that similar results can derive from previous experiments, even when there are small variations in reagents or experimental designs. Results are conditional and not robust when they depend on specific experimental conditions(95)95 Steward O. A Rhumba of "R's": Replication, Reproducibility, Rigor, Robustness: What Does a Failure to Replicate Mean?. eNeuro. 2016;3(4):ENEURO.0072-16.2016. doi:10.1523/ENEURO.0072-16.2016
https://doi.org/10.1523/ENEURO.0072-16.2...
.

Robustness of research carried out with zebrafish can increase when results are confirmed by different methods and collaborative research. The following aspects can improve the robustness of such research: best scientific practices; using guidelines; zebrafish quality and welfare; experimental design; addition of negative, solvent and control groups; sample size estimates; effects of sex differences; suitable statistical analysis; adequate result analysis and interpretation; inclusion of negative and validation data; training and support.

7R – Reproducibility

Study reproducibility is a fundamental science principle. Researchers should be able to recreate experiments and to get to the same (or similar) results and conclusions(96)96 Resnik DB, Shamoo AE. Reproducibility and Research Integrity. Account Res. 2017;24(2):116-123. doi:10.1080/08989621.2016.1257387
https://doi.org/10.1080/08989621.2016.12...
. However, oftentimes, research cannot be reproduced and they lead to resource, time and scientific credibility losses(9797 Ioannidis JP. How to make more published research true. PLoS Medicine. 2014;11(10):e1001747. doi:10.1371/journal.pmed.1001747
https://doi.org/10.1371/journal.pmed.100...
, 9898 Freedman LP, Cockburn IM, Simcoe TS. The Economics of Reproducibility in Preclinical Research. PLoS Biol. 2015;13(6):e1002165. doi:10.1371/journal.pbio.1002165
https://doi.org/10.1371/journal.pbio.100...
). Research irreproducibility can highlight experimental design issues, methodology implementation, data quality or integrity, statistical analysis error or study result interpretation(96)96 Resnik DB, Shamoo AE. Reproducibility and Research Integrity. Account Res. 2017;24(2):116-123. doi:10.1080/08989621.2016.1257387
https://doi.org/10.1080/08989621.2016.12...
, as well as animal quality and welfare.

Reproducibility is particularly important for emerging model systems that have available a limited number of protocols and information(99)99 Gerlai R. Reproducibility and replicability in zebrafish behavioral neuroscience research. Pharmacology, biochemistry, and behavior. 2019;178:30-38. doi:10.1016/j.pbb.2018.02.005
https://doi.org/10.1016/j.pbb.2018.02.00...
. The Organization for Economic Cooperation and Development (OECD) currently provides guidelines for Acute Toxicity Testing in Fish(100)100 OECD, 1992. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 203: Fish, Acute Toxicity Test. Organization for Economic Cooperation and Development, Paris, France., Fish Embryo Acute Toxicity (FET)(21)21 OECD, 2013. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 236: Applicability of the Fish Embryo Acute Toxicity (FET) Test. Organization for Economic Cooperation and Development, Paris, France., Fish, Juvenile Growth test(101)101 OECD, 2000. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 215: Fish, Juvenile Growth test. Organization for Economic Cooperation and Development, Paris, France., 21-day Fish Assay(102)102 OECD, 2009. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 230: 21-day Fish Assay. Organization for Economic Cooperation and Development, Paris, France. and Fish sexual development test(103)103 OECD, 2018. OECD. OECD guidelines for the testing of chemicals. Section 2: Effects on Biotic Systems Test No. 234: Fish Sexual Development Test (FSDT). Organization for Economic Cooperation and Development, Paris, France.. However, many research fields lack specific protocols for fish, such as genotoxicity(4)4 CanedoA, Rocha TL. Zebrafish (Danio rerio) using as model for genotoxicity and DNA repair assessments: historical review, current status and trends. The Science of the total environment. 2021;762:144084. doi:10.1016/j.scitotenv.2020.144084
https://doi.org/10.1016/j.scitotenv.2020...
, mutagenicity(5)5 Canedo A, de Jesus LWO, Bailão EFLC, Rocha TL. Micronucleus test and Nuclear Abnormality assay in zebrafish (Danio rerio): past, present and future trends. Environment Pollution. 2021:290:118019. doi:10.1016/j.envpol.2021.118019
https://doi.org/10.1016/j.envpol.2021.11...
, and neuroscience and behavioural studies(99)99 Gerlai R. Reproducibility and replicability in zebrafish behavioral neuroscience research. Pharmacology, biochemistry, and behavior. 2019;178:30-38. doi:10.1016/j.pbb.2018.02.005
https://doi.org/10.1016/j.pbb.2018.02.00...
. Gerlai(99)99 Gerlai R. Reproducibility and replicability in zebrafish behavioral neuroscience research. Pharmacology, biochemistry, and behavior. 2019;178:30-38. doi:10.1016/j.pbb.2018.02.005
https://doi.org/10.1016/j.pbb.2018.02.00...
, for example, describes methodological issues and solutions to ensure the reproducibility of behavioural neuroscience studies with zebrafish. Furthermore, although the 3Rs have been incorporated to the ARRIVE guidelines, additional eforts are needed to comply with laws in force, in different countries, to make sure of research integrity(104)104 Andersen ML, Winter LMF. Animal models in biological and biomedical research - experimental and ethical concerns. Anais da Academia Brasileira de Ciencias. 2019;91(suppl 1):e20170238. doi:10.1590/0001-3765201720170238
https://doi.org/10.1590/0001-37652017201...
.

Thus, measures such as (i) ensuring access to protocols used in the studies; (ii) conducting well-designed reproducible research and minimizing experimental error and the number of animals used in experiments; (iii) being transparent and clear about results found in studies; (iv) correcting analysis of complex data; (v) avoiding cognitive bias; (vi) identifying gaps in knowledge about studies carried out with zebrafish; (vii) standardizing protocols to studies with zebrafish(9898 Freedman LP, Cockburn IM, Simcoe TS. The Economics of Reproducibility in Preclinical Research. PLoS Biol. 2015;13(6):e1002165. doi:10.1371/journal.pbio.1002165
https://doi.org/10.1371/journal.pbio.100...
, 3232 Lidster K, Readman GD, Prescott MJ, Owen SF. International survey on the use and welfare of zebrafish Danio rerio in research. Journal of Fish Biology. 2017;90(5):1891-1905. https://doi.org/10.1111/jfb.13278
https://doi.org/10.1111/jfb.13278...
, 105105 Klein J. Improving the reproducibility of findings by updating research methodology Quality & quantity. 2021;1-13. doi:10.1007/s11135-021-01196-6
https://doi.org/10.1007/s11135-021-01196...
) must be applied to ensure the reproducibility of studies with this species.

8R – Relevance

Animal species must be able to meet all study aims when animals are used. Accordingly, planning must be carried out before any experiment is implemented in order to avoid the unnecessary use of animals. Therefore, researchers must know the features of the model system they intend to use, such as its anatomy, physiology, developmental stage, specific behaviours, among others(22)22 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biology. 2010;8(6):e1000412. doi:10.1371/journal.pbio.1000412
https://doi.org/10.1371/journal.pbio.100...
. Even if the aim of the study is to investigate human diseases, to perform toxicity tests or environmental monitoring, the relevance of the herein assessed principle must concern the transferability of an animal test. Experiments should only be carried out with a given species after taking into consideration its relevance to biological knowledge advancement(106)106 Mehić B Professor. Bioethical Principles of Biomedical Research Involving Animals. Bosnian Journal of Basic Medical Sciences. 2011;11(3):145-146..

Zebrafish has many relevant features for research on animal development(107)107 Veldman M, Lin S. Zebrafish as a Developmental Model Organism for Pediatric Research. Pediatric research. 2008; 64:470-476 doi:10.1203/PDR.0b013e318186e609
https://doi.org/10.1203/PDR.0b013e318186...
, genetics(108)108 Holtzman NG, Iovine MK, Liang JO, Morris J. Learning to Fish with Genetics: A Primer on the Vertebrate Model Danio rerio. Genetics. 2016;203(3):1069-1089. doi:10.1534/genetics.116.190843
https://doi.org/10.1534/genetics.116.190...
, behaviour(109)109 Basnet RM, Zizioli D, Taweedet S, Finazzi D, Memo M. Zebrafish Larvae as a Behavioral Model in Neuropharmacology. Biomedicines. 2019;7(1):23. doi:10.3390/biomedicines7010023
https://doi.org/10.3390/biomedicines7010...
, drug discovery(110)110 Kari G, Rodeck U, Dicker AP. Zebrafish: an emerging model system for human disease and drug discovery. Clinical pharmacology and therapeutics.2007;82(1):70-80. doi:10.1038/sj.clpt.6100223
https://doi.org/10.1038/sj.clpt.6100223...
, diseases(111)111 Gut P, Reischauer S, Stainier DYR, Arnaout R. Little fish, big data: zebrafish as a model for cardiovascular and metabolic disease. Physiological reviews. 2017;97(3):889-938. doi:10.1152/physrev.00038.2016
https://doi.org/10.1152/physrev.00038.20...
, vaccination(13)13 Bailone RL, R, Fukushima HCS, Fernandes BHV, DeAguiar LK, Corrêa T, Janke H, Setti PG, Roça RO, Borra RC. Zebrafish as an alternative animal model in human and animal vaccination research. Laboratory Animal Research. 2020;36(13):1-10. doi:10.1186/s42826-020-00042-4
https://doi.org/10.1186/s42826-020-00042...
, food safety(14)14 Bailone RL, Aguiar LKD, Roca RDO, Borra RC, Corrêa T, Janke H, Fukushima HCS. Zebrafish as an animal model for food safety research: trends in the animal research. Food Biotechnology. 2019;33(4): 283-302. http://dx.doi.org/10.1080/08905436.2019.1673173
http://dx.doi.org/10.1080/08905436.2019....
, among others. Unlike other vertebrate models, its embryos have a body plan, whose beating heart, blood vessels and rudimentary intestine 48 hpf can be observed(17)17 Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Developmental dynamics: an official publication of the American Association of Anatomists. 1995;203(3):253-310. doi:10.1002/aja.1002030302
https://doi.org/10.1002/aja.1002030302...
. Despite the early formation of the cardiac and metabolic systems in zebrafish, experimental manipulations are possible as long as embryos get oxygen from water through passive diffusion and nutrients from the yolk sac(112)112 Giannaccini M, Cuschieri A, Dente L, Rafa V. Non-mammalian vertebrate embryos as models in nanomedicine. Nanomedicine: nanotechnology, biology, and medicine. 2014;10(4):703-719. doi:10.1016/j.nano.2013.09.010
https://doi.org/10.1016/j.nano.2013.09.0...
. Furthermore, fertilization and zebrafish embryo development are external, and embryos are translucent – a feature that turns them into an ideal model system for imaging during development(113113 Dai YJ, Jia YF, Chen N, et al. Zebrafish as a model system to study toxicology. Environmental toxicology and chemistry. 2014;33(1):11-17. doi:10.1002/etc.2406
https://doi.org/10.1002/etc.2406...
, 114114 Cui C, Benard EL, Kanwal Z, et al. Infectious disease modeling and innate immune function in zebrafish embryos. Methods in cell biology. 2011;105:273-308. doi:10.1016/B978-0-12-381320-6.00012-6
https://doi.org/10.1016/B978-0-12-381320...
). Another relevant feature lies on the fact that zebrafish's innate immune system develops in the first two days of embryogenesis(115)115 Lam SH, Chua HL, Gong Z, Lam TJ, Sin YM. Development and maturation of the immune system in zebrafish, Danio rerio: a gene expression profling, in situ hybridization and immunological study. Developmental and comparative immunology. 2004;28(1):9-28. doi:10.1016/s0145-305x(03)00103-4
https://doi.org/10.1016/s0145-305x(03)00...
. The development and maturation of their adaptive immune system occur between 4 and 6 weeks after fertilization(116)116 National Centre for the Replacement Refinement and Reduction of Animals in Research, NC3Rs. Responsibility in the use of animals in bioscience research: expectations of the major research councils and charitable funding bodies [Internet]. 2019. Available from: https://nc3rs.org.uk/sites/default/fles/documents/Guidelines/Responsibility in the use of animals in bioscience research 2019.pdf
https://nc3rs.org.uk/sites/default/fles/...
- such feature makes them an ideal model system for immunology studies.

The zebrafish genome was sequenced in 2013 by Cambridge's Welcome Trust Sanger Institute. Zebrafish has 26,206 protein-coding genes, and approximately 70% of them have at least one ortholog in humans(45)45 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Mufato M, Collins JE, Humphray S, Mclaren K, Mathews L, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013; 496:498-503. https://doi.org/10.1038/nature12111
https://doi.org/10.1038/nature12111...
. It should be noticed that approximately 84% of genes known to be associated with human diseases have zebrafish orthologs(45)45 Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Mufato M, Collins JE, Humphray S, Mclaren K, Mathews L, et al. The zebrafish reference genome sequence and its relationship to the human genome. Nature. 2013; 496:498-503. https://doi.org/10.1038/nature12111
https://doi.org/10.1038/nature12111...
. Currently, due to the development of tools - such as CRISPR-Cas9, TALENs, ZFNs - for gene editing, several mutant zebrafish strains, transgenic reporters and knockdown have been developed for studies on diseases, developmental biology, toxicology, among other fields(19)19 Fernandes MR, Pedroso AR. Animal experimentation: A look into ethics, welfare and alternative methods. Revista da Associação Medica Brasileira (1992). 2017;63(11):923-928. doi:10.1590/1806-9282.63.11.923
https://doi.org/10.1590/1806-9282.63.11....
. Finally, information on zebrafish’s anatomy, physiology, development and genetics was deposited in a database to make scientific communication about this species easier (http://www.zfin.org). All these features turn zebrafish into a relevant model for scientific research.

Principles of conduct

9R – Responsibility

Everyone involved in research with animals account for the care provided to, and use of, them. Institutions, animal ethics committees, investigators and animal caretakers must understand and accept their responsibility(6767 CONSTITUIÇÃO FEDERAL. LEI Nº 11.794 DE 8 DE OUTUBRO DE 2008.Brasil, 2008. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/111794.htm
http://www.planalto.gov.br/ccivil_03/_at...
, 117117 National Health and Medical Research Council, NHMRC; Australian Research Council. Australian code for the care and use of animals for scientific purposes. Vol. 2013. 2021. 86 p., 118118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
). Institutions account for supporting animal ethics committees, investigators and animal caretakers. Ensuring access to veterinary advice, education and training, policies and procedures, in the process to comply with the principles of research with animals are examples of such support(6767 CONSTITUIÇÃO FEDERAL. LEI Nº 11.794 DE 8 DE OUTUBRO DE 2008.Brasil, 2008. Disponível em: http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/111794.htm
http://www.planalto.gov.br/ccivil_03/_at...
, 117117 National Health and Medical Research Council, NHMRC; Australian Research Council. Australian code for the care and use of animals for scientific purposes. Vol. 2013. 2021. 86 p.). Animal ethics committees account for validating, approving and monitoring the care provided to, and use of, animals for scientific and teaching purposes(117117 National Health and Medical Research Council, NHMRC; Australian Research Council. Australian code for the care and use of animals for scientific purposes. Vol. 2013. 2021. 86 p., 118118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
). Investigators and animal caretakers, including associated veterinaries, are responsible for applying high care and management standards to animal use in research(117117 National Health and Medical Research Council, NHMRC; Australian Research Council. Australian code for the care and use of animals for scientific purposes. Vol. 2013. 2021. 86 p., 118118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
, 119119 BRASIL. (a) Resolução Normativa CONCEA n. 30, de 02.02.2016. Baixa a Diretriz Brasileira para o Cuidado e a Utilização de Animais em Atividades de Ensino ou de Pesquisa Científica – DBCA., 120120 BRASIL. (b) Resolução Normativa CONCEA n. 32, de 06.09.2016. Baixa as Diretrizes de Integridade e de Boas Práticas para Produção, Manutenção ou Utilização de Animais em Atividades de Ensino ou Pesquisa Científica., 121121 Ogden BD. Principles of animal research: replacement, reduction, refinement, and responsibility. Anim Law [Internet]. 1996;2:167–70. Available from: https://heinonline.org/HOL/Page?public=%0Atrue&handle=hein.journals/anim2÷=12&start_page=167&collection=%0Ajournals&set_as_cursor=0&men_tab=srchresults
https://heinonline.org/HOL/Page?public=%...
).

Experimental animal use, social issues, scientific integrity and animal welfare are related to responsibility in research with animals(122)122 Franco AL, Nogueira MNM, Sousa NGK, da Frota MF, Fernandes CMS, Serra M da C. Pesquisas em animais: uma refexão bioética. Acta Bioethica. 2014;20(2):247-53. doi:10.4067/S1726-569X2014000200012.
https://doi.org/10.4067/S1726-569X201400...
. Responsibility to the experimental use of animal concerns using them for scientific and teaching purposes, ad it must be justified and conducted based on ethical acceptability(118)118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
. Responsibility to social issues includes sharing with the public the reasons why research with animals is required in order to improve health issues of animals, humans and the environment(123)123 McLeod C, Hartley S. Responsibility and Laboratory Animal Research Governance. Science Technology Human Values. 2018;43(4):723-41. doi:10.1177/0162243917727866
https://doi.org/10.1177/0162243917727866...
. The aim is to show the impact of scientific purposes on stances that go beyond developing scientific knowledge, such as on societal consequences, as well as to share the practices and procedures adopted in research with animals(124)124 Reed B; Jennings M. Guidance on the housing and care of Zebrafish (Danio rerio). Available from: https://www.aaalac.org/pub/?id=E9019693-90EC-FC4A-526E-E8236CC13B28
https://www.aaalac.org/pub/?id=E9019693-...
. Responsibility to scientific integrity comprises the design and conduct of research with animals, including legal accountabilities. It is recommended to apply the Planning Research and Experimental Procedures on Animals (PREPARE) to ensure the adoption of nowadays best practices concerning the care and management procedures for animals used in research proposals(121)121 Ogden BD. Principles of animal research: replacement, reduction, refinement, and responsibility. Anim Law [Internet]. 1996;2:167–70. Available from: https://heinonline.org/HOL/Page?public=%0Atrue&handle=hein.journals/anim2÷=12&start_page=167&collection=%0Ajournals&set_as_cursor=0&men_tab=srchresults
https://heinonline.org/HOL/Page?public=%...
. Finally, responsibility for animals’ welfare requires procedures about the proper care and management provided to the selected species throughout its lifetime(118)118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
, and it includes number of animals, animals’ source, biological status, transport, prevention of and relief from pain, suffering, distress and disease, appropriate housing and husbandry, as well as final disposal(22)22 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biology. 2010;8(6):e1000412. doi:10.1371/journal.pbio.1000412
https://doi.org/10.1371/journal.pbio.100...
.

The principles of research with animals remain a challenge; however, well-established guidelines and clear legislations allow “R” responsibility enforcement(22)22 Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biology. 2010;8(6):e1000412. doi:10.1371/journal.pbio.1000412
https://doi.org/10.1371/journal.pbio.100...
. Knowledge on zebrafish biology, physiology and behaviour is crucial at the time to apply the best practice for its care and use for scientific purposes, since it ensures zebrafish welfare over its life cycle(125125 Zagorac I. Fritz Jahr’s Bioethical Imperative. Synth Philos. 2011;51:141-50., 3131 Osborne N, Paull G, Grierson A, et al. Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care. Zebrafish. 2016;13(6):584-589. doi:10.1089/zeb.2016.1324
https://doi.org/10.1089/zeb.2016.1324...
). Therefore, responsibility in research with animals is associated with the duty to respect animals.

10R – Respect

According to the bioethical imperative defined by Fritz Jahr, "every living being [must be respected] on principle as an end in itself and treated, if possible, as such"(126)126 MacRae CA, Peterson RT. Zebrafish as tools for drug discovery. Nature reviews. Drug discovery. 2015;14(10):721-731. doi:10.1038/nrd4627
https://doi.org/10.1038/nrd4627...
. Respect can be demonstrated when everyone who uses animals considers both the quality and dignity of their lives(121)121 Ogden BD. Principles of animal research: replacement, reduction, refinement, and responsibility. Anim Law [Internet]. 1996;2:167–70. Available from: https://heinonline.org/HOL/Page?public=%0Atrue&handle=hein.journals/anim2÷=12&start_page=167&collection=%0Ajournals&set_as_cursor=0&men_tab=srchresults
https://heinonline.org/HOL/Page?public=%...
. Scientific and social impact through research with animals should be cautiously evaluated based on the necessity and justification of animals’ use for scientific proposals, and on animals’ welfare(118)118 BRASIL. Resolução Normativa CONCEA n. 51, de 19 de maio de 2021. Dispõe sobre a instalação e o funcionamento das Comissões de Ética no Uso de Animais - CEUAs e dos biotérios ou instalações animais. https://www.in.gov.br/web/dou/-/resolucao-normativa-concea-n-51-de-19-de-maio-de-2021-321534226
https://www.in.gov.br/web/dou/-/resoluca...
. Accordingly, "R" (Respect) in research with animals also consists in minimizing harms to animals; applying the 10Rs at all animals’ care and management stages; applying the best practice for animals’ care and management; and understanding and accepting one’s responsibilities, as well as following recommendations of national laws and regulatory guidelines(121)121 Ogden BD. Principles of animal research: replacement, reduction, refinement, and responsibility. Anim Law [Internet]. 1996;2:167–70. Available from: https://heinonline.org/HOL/Page?public=%0Atrue&handle=hein.journals/anim2÷=12&start_page=167&collection=%0Ajournals&set_as_cursor=0&men_tab=srchresults
https://heinonline.org/HOL/Page?public=%...
.

For research using zebrafish, "Respect" can be applied to knowledge on zebrafish life-history traits; to choose the appropriate life cycle stage for the scientific proposal and to adopt recommendations for animals’ acquisition; maintenance, such as water quality parameters; feeding; environmental enrichment; as well as all aspects involved in animal welfare(31)31 Osborne N, Paull G, Grierson A, et al. Report of a Meeting on Contemporary Topics in Zebrafish Husbandry and Care. Zebrafish. 2016;13(6):584-589. doi:10.1089/zeb.2016.1324
https://doi.org/10.1089/zeb.2016.1324...
in order to use these spaces in research experiments, besides the use of the 10 Rs presented in the current study.

The growing use of these animals for scientific and educational purposes reinforces the need of regulating scientific research based on using fish as model, including zebrafish(127)127 Message R, Greenhough B. "But It's Just a Fish": Understanding the Challenges of Applying the 3Rs in Laboratory Aquariums in the UK. Animals (Basel). 2019;9(12):1075. Published 2019 Dec 3. doi:10.3390/ani9121075
https://doi.org/10.3390/ani9121075...
. Furthermore, some previous studies described zebrafish as “tool” in several research fields(128128 Bambino K, Chu J. Zebrafish in Toxicology and Environmental Health. Current topics in developmental biology. 2017;124:331-367. doi:10.1016/bs.ctdb.2016.10.007
https://doi.org/10.1016/bs.ctdb.2016.10....
, 129129 Bertoncello KT, Bonan CD. Zebrafish as a tool for the discovery of anticonvulsant compounds from botanical constituents. European journal of pharmacology. 2021;908:174342. doi:10.1016/j.ejphar.2021.174342
https://doi.org/10.1016/j.ejphar.2021.17...
, 130130 Vierstraete J, Fieuws C, Willaert A, Vral A, Claes KBM. Zebrafish as an in vivo screening tool to establish PARP inhibitor eficacy. DNA Repair. 2021:97:103023. doi:10.1016/j.dnarep.2020.103023
https://doi.org/10.1016/j.dnarep.2020.10...
), as well as educational “tool”. However, since ethical concerns should guide animals’use in scientific and educational proposals, the herein presented initiatives focused on the 10 Rs must be considered and implemented in order to ensure zebrafish’s use in research, as a well-established as animal model, rather than just as "tool". Thus, it is essential reinforcing the urgent need of adopting "model system" or "animal model" as standard term. It is important emphasizing that respect for animals should underpin research with animals. Therefore, guaranteeing the ethical research conduct in studies with zebrafish, as well as animal welfare and compliance with scientific principles must be the very basis of principles of conduct.

Conclusions

The emerging use of zebrafish as model system in several research fields opens room for new ethical challenges. The current study describes 10 Rs ethical principles for zebrafish using as model system for research. They were classified into three categories, namely: welfare (replacement, reduction and refinement), scientific (registration, reporting, robustness, reproducibility and relevance) and conduct principles (responsibility and respect). Implementing the 10 Rs can help researchers, institutions and Animal Ethics Committees in regulating, making-decisions and promoting zebrafish health and welfare during research protocols. Standards and laws must be followed by researchers in order to ensure research integrity, as well as its relevance for human and animal heath development.

Acknowledgments

The current study was funded by the National Council for Scientific and Technological Development - CNPq (MCTIC/CNPq n. 28/2018; n. 433553/2018-9) and by the Coordination for the Improvement of Higher Education Personnel - CAPES (funding code 001). Rocha T.L. is granted with productivity scholarship from CNPq (proc. n. 306329/2020-4).

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Publication Dates

  • Publication in this collection
    30 Mar 2022
  • Date of issue
    2022

History

  • Received
    16 Nov 2021
  • Accepted
    11 Jan 2022
  • Published
    21 Feb 2022
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