Ethical considerations regarding animal experimentation


Animal experimentation
Animal model
4Rs principle
Animal welfare


Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees’ adoption of the ‘four Rs’ principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued.


[1] Fernandes MR, Pedroso AR. Animal experimentation: a look into ethics, welfare and alternative methods. Rev Assoc Med Bras 2017;63:923-8.
[2] LaFollette H. Ethics in practice: an anthology, 4th ed. New York: Wiley & Sons 2020.
[3] Franco NH. Animal experiments in biomedical research: a historical perspective. Animals 2013;3:238-73.
[4] Animal Research at Stanford.. Avalable at: https://med.stanford. edu/animalresearch/why-animal-research.html. Accessed on: 30/05/2021.
[5] Simmons D. The use of animal models in studying genetic disease: transgenesis and induced mutation. Available at: Accessed on: 30/05/2021.
[6] National Research Council (US) Committee on Developmental Toxicology. Using Model Animals to Assess and Understand Developmental Toxicity In: scientific frontiers in developmental toxicology and risk assessment. Washington (DC): National Academies Press (US) 2000.
[7] Regenberg A, Mathews DJ, Blass DM, Bok H, Coyle JT, Duggan P, Faden R, Finkel J, Gearhart JD, Hillis A, Hoke A, Johnson R, Johnston M, Kahn J, Kerr D, King P, Kurtzberg J, Liao SM, McDonald JW, McKhann G, Nelson KB, Rao M, Siegel AW, Smith K, Solter D, Song H, Sugarman J, Vescovi A, Young W, Greely HT, Traystman RJ. The role of animal models in evaluating reasonable safety and efficacy for human trials of cell-based interventions for neurologic conditions. J Cereb Blood Flow Metab 2009;29:1-9.
[8] Williams ED. Federal protection for human research subjects: an analysis of the common rule and its interactions with FDA regulations and the HIPAA Privacy Rule. Congressional Research Service 2005. Available at: pdf. Accessed on: 30/05/2021.
[9] Fotuhi M, Mian A, Meysami S, Raji CA. Neurobiology of COVID-19. J Alzheimers Dis 2020;76:3-19.
[10] Iadecola C, Anrather J, Kamel H. Effects of COVID-19 on the nervous system. Cell 2020;183:16-27. cell.2020.08.028
[11] Marshall M. How COVID-19 can damage the brain. Nature 2020;585:342-3.
[12] Marshall M. COVID’s toll on smell and taste: what scientists do and don’t know. Nature 2021;589:342-3.
[13] Paolo G. Does COVID-19 cause permanent damage to olfactory and gustatory function? Med Hypotheses 2020;143:110086.
[14] Reichard RR, Kashani KB, Boire NA, Constantopoulos E, Guo Y, Lucchinetti CF. Neuropathology of COVID-19: a spectrum of vascular and acute disseminated encephalomyelitis (ADEM)-like pathology. Acta Neuropathol 2020;140:1-6.
[15] Taquet M, Geddes JR, Husain M, Luciano S, Harrison PJ. 6-month neurological and psychiatric outcomes in 236379 survivors of COVID-19: a retrospective cohort study using electronic health records. Lancet Psychiatry 2021;8:416-27.
[16] Lakdawala SS, Menachery VD. The search for a COVID-19 animal model. Science 2020;368:942-3.
[17] Neff EP. Meeting the need for COVID-19 models. Lab Anim 2021;50:111-2.
[18] Dinnon KH 3rd, Leist SR, Schäfer A, Edwards CE, Martinez DR, Montgomery SA, West A, Yount BL Jr, Hou YJ, Adams LE, Gully KL, Brown AJ, Huang E, Bryant MD, Choong IC, Glenn JS, Gralinski LE, Sheahan TP, Baric RS. A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures. Nature 2020;586 560-6.
[19] Gurumurthy CB, Quadros RM, Richardson GP, Poluektova LY, Mansour SL, Ohtsuka M. Genetically modified mouse models to help fight COVID-19. Nat Protoc 2020;15:3777-3787.
[20] Nainu F, Rahmatika D, Emran TB, Harapan H. Potential application of Drosophila melanogaster as a model organism in COVID-19-related research. Front Pharmacol 2020;11:588561.
[21] Galindo-Villegas J. The zebrafish disease and drug screening model: a strong ally against Covid-19. Front Pharmacol 2020;11:680.
[22] Adhikary PP, Ul Ain Q, Hocke AC, Hedtrich S. COVID-19 highlights the model dilemma in biomedical research. Nat Rev Mater 2021;17:1-3.
[23] Hein WR, Griebel PJ. Road less travelled: large animal models in immunological research. Nat Rev Immunol 2003;3:79-84.
[24] Hild SA, Chang MC, Murphy SJ, Grieder FB. Nonhuman primate models for SARS-CoV-2 research: infrastructure needs for pandemic preparedness. Lab Anim 2021;50:140-1.
[25] Kumar S, Yadav PK, Srinivasan R, Perumal N. Selection of animal models for COVID-19 research. Virus disease 2020;31:1-6.
[26] Muñoz-Fontela C, Dowling WE, Funnell SGP, Gsell PS, Riveros-Balta AX, Albrecht RA, Andersen H, Baric RS, Carroll MW, Cavaleri M, Qin C, Crozier I, Dallmeier K, de Waal L, de Wit E, Delang L, Dohm E, Duprex WP, Falzarano D, Finch CL, Frieman MB, Graham BS, Gralinski LE, Guilfoyle K, Haagmans BL, Hamilton GA, Hartman AL, Herfst S, Kaptein SJF, Klimstra WB, Knezevic I, Krause PR, Kuhn JH, Le Grand R, Lewis MG, Liu WC, Maisonnasse P, McElroy AK, Munster V, Oreshkova N, Rasmussen AL, Rocha-Pereira J, Rockx B, Rodríguez E, Rogers TF, Salguero FJ, Schotsaert M, Stittelaar KJ, Thibaut HJ, Tseng CT, Vergara-Alert J, Beer M, Brasel T, Chan JFW, García-Sastre A, Neyts J, Perlman S, Reed DS, Richt JA, Roy CJ, Segalés J, Vasan SS, Henao-Restrepo AM, Barouch DH. Animal models for COVID-19. Nature 2020;586:509-15.
[27] Pandey K, Acharya A, Mohan M, Ng CL, Reid SP, Byrareddy SN. Animal models for SARS-CoV-2 research: a comprehensive literature review. Transbound Emerg Dis 2020;68:1868-85.
[28] Pechanova O. Why we still need animal models. Pathophysiology 2020;27:44-5.
[29] Natoli S, Oliveira V, Calabresi P, Maia LF, Pisani A. Does SARS-Cov-2 invade the brain? Translational lessons from animal models. Eur J Neurol 2020;27:1764-73.
[30] Mahajan A, Mason GF. A sobering addition to the literature on COVID-19 and the brain. J Clin Invest 2021;131:e148376.
[31] Renn M, Bartok E, Zillinger T, Hartmann G, Behrendt R. Animal models of SARS-CoV-2 and COVID-19 for the development of prophylactic and therapeutic interventions. Pharmacol Ther 2021;228:107931. pharmthera.2021.107931
[32] Shi J, Wen Z, Zhong G, Yang H, Wang C, Huang B, Liu R, He X, Shuai L, Sun Z, Zhao Y, Liu P, Liang L, Cui P, Wang J, Zhang X, Guan Y, Tan W, Wu G, Chen H, Bu Z. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science 2020;368:1016-20.
[33] Genzel L, Adan R, Berns A, van den Beucken JJJP, Blokland A, Boddeke EHWGM, Bogers WM, Bontrop R, Bulthuis R, Bousema T, et al. How the COVID-19 pandemic highlights the necessity of animal research. Curr Biol 2020;30:R1014-R1018.
[34] Deb B, Shah H, Goel S. Current global vaccine and drug efforts against COVID-19: Pros and cons of bypassing animal trials. J Biosci 2020;45:82.
[35] Delahaye P. Animal studies, animal ethics. In: a semiotic methodology for animal studies. 1st ed. Cham: Springer 2019.
[36] Singer P. All Animals are Equal. Available at: https://spot.,Spr07/singer.pdf. Accessed on: 30/05/2021.
[37] Machan TR. Why human beings may use animals. J Value Inq 2002;36:9-14.
[38] Gruen L. The Moral Status of Animals In: Zalta EN, ed. Stanford Encyclopedia of Philosophy. Metaphysics Research Lab, Stanford University 2021. Available at: https://plato. Accessed on: 30/05/2021.
[39] Cohen C. The case for the use of animals in biomedical research. N Engl J Med 1986;315:865-70.
[40] Cohen AI, Wellman CH. Contemporary debates in applied ethics. Malden, 1st ed. MA: Blackwell Pub 2005.
[41] Liou S. The Ethics of Animal Experimentation. 2010. Available at: Accessed on: 30/05/2021.
[42] Bateson P. When to experiment on animals. New Sci 1986;109:30-2.
[43] Bateson’s cube. Available at: Accessed on: 30/05/2021.
[44] Monsó S, Benz-Schwarzburg J, Bremhorst A. Animal morality: what it means and why it matters. J Ethics 2018;22:283-310.
[45] Proctor H. Animal sentience: where are we and where are we heading? Animals 2012;2:628-39.
[46] Langford DJ, Bailey AL, Chanda ML, Clarke SE, Drummond TE, Echols S, Glick S, Ingrao J, Klassen-Ross T, Lacroix-Fralish ML, Matsumiya L, Sorge RE, Sotocinal SG, Tabaka JM, Wong D, van den Maagdenberg AM, Ferrari MD, Craig KD, Mogil JS. Coding of facial expressions of pain in the laboratory mouse. Nat Methods 2010;7:447-9.
[47] National Research Council (US) Committee on Recognition and Alleviation of Pain in Laboratory Animals. Recognition and alleviation of pain in laboratory animals. Washington (DC): National Academies Press (US) 2009.
[48] Carbone L. Pain in laboratory animals: the ethical and regulatory imperatives. PLoS One 2011;6:e21578.
[49] Gregory G. Physiology and behavior of animal suffering. United Kingdom: Blackwell Publishing Company 2004, pp. 25-50.
[50] Panksepp J. Affective neuroscience: the foundations of human and animal emotions. USA: University Press 2004.
[51] Bradshaw GA, Capaldo T, Lindner L, Grow G. Building an inner sanctuary: complex PTSD in chimpanzees. J Trauma Dissociation 2008;9:9-34.
[52] Balcombe J. Animal pleasure and its moral significance. Appl Anim Behav Sci 2009;118:208-16. applanim.2009.02.012
[53] Graham KL, Burghardt GM. Current perspectives on the biological study of play: signs of progress. Q Rev Biol 2010;85:393-418.
[54] Hecht EE, Mahovetz LM, Preuss TM, Hopkins WD. A neuroanatomical predictor of mirror self-recognition in chimpanzees. Soc Cogn Affect Neurosci 2017;12:37-48.
[55] Veit L, Pidpruzhnykova G, Nieder A. Associative learning rapidly establishes neuronal representations of upcoming behavioral choices in crows. Proc Natl Acad Sci USA 2015;112:15208-13.
[56] Emery NJ, Clayton NS. The mentality of crows: convergent evolution of intelligence in corvids and apes. Science 2004;306:1903-7.
[57] Gamble JR, Cristol DA. Drop-catch behaviour is play in herring gulls, Larus argentatus. Anim Behav 2002;63:339-45.
[58] McCance D. Critical Animal Studies: An Introduction. 1st ed. USA: SUNY Press 2012.
[59] Ferdowsian HR, Beck N. Ethical and scientific considerations regarding animal testing and research. PLoS One 2011;6:e24059.
[60] Hansen LA. Institution animal care and use committees need greater ethical diversity. J Med Ethics 2013;39:188-90.
[61] Tannenbaum J, Bennett BT. Russell and Burch’s 3Rs then and now: the need for clarity in definition and purpose. J Am Assoc Lab Anim Sci 2015;54:120-32.
[62] Lee KH, Lee DW, Kang BC. The ‘R’ principles in laboratory animal experiments. Lab Anim Res 2020;36:1-3.
[63] Di Salvo L. The Ethical and Moral Status of Invasive Animal Research: The Dilemma and Alternative Approaches. Rome: LUISS Thesis Guido Carli 2017. Available at: https://tesi.luiss. it/19067/. Accessed on: 30/05/2021.
[64] Dondossola D, Santini A, Lonati C, Zanella A, Merighi R, Vivona L, Battistin M, Galli A, Biancolilli O, Maggioni M, Villa S, Gatti S. Human red blood cells as oxygen carriers to improve ex-situ liver perfusion in a rat model. J Clin Med 2019;8:1918.
[65] Dondossola D, De Falco S, Kersik A, Maggioni M, Di Girolamo L, Biancolilli O, Busana M, Lonati C, Carù F, Zanella A, Gatti S. Procurement and ex-situ perfusion of isolated slaughterhouse-derived livers as a model of donors after circulatory death. ALTEX 2019.
[66] Furka I, Brath E, Nemeth N, Miko I. Learning microsurgical suturing and knotting techniques: comparative data. Microsurgery 2006;26:4-7.
[67] Tolba RH, Czigány Z, Osorio Lujan S, Oltean M, Axelsson M, Akelina Y, Di Cataldo A, Miko I, Furka I, Dahmen U, Kobayashi E, Ionac M, Nemeth N. Defining standards in experimental microsurgical training: recommendations of the European Society for Surgical Research (ESSR) and the International Society for Experimental Microsurgery (ISEM). Eur Surg Res 2017;58:246-62.
[68] Banks RE. The 4th R of research. Contemp Top Lab Anim Sci 1995;34:50-1.
[69] Neal JM, Bernards CM, Butterworth JF 4th, Di Gregorio G, Drasner K, Hejtmanek MR, Mulroy MF, Rosenquist RW, Weinberg GL. ASRA practice advisory on local anesthetic systemic toxicity. Reg Anesth Pain Med 2010;35:152-61.
[70] Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. NC3Rs Reporting Guidelines Working Group. Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 2010;160:1577-9.
[71] The Capacity of Animals to Experience Pain, Distress and Suffering In: The Ethics of Research Involving Animals. Available at: Accessed on: 30/05/2021.
[72] Peterson NC, Nunamaker EA, Turner PV. To treat or not to treat: the effects of pain on experimental parameters. Comp Med 2017;67:469-82.
[73] Canadian Council on Animal Care. Guide to the Care and Use of Experimental Animals. Available at: https:// Standards/ Guidelines/Experimental_Animals_ Vol1.pdf. Accessed on: 30/05/2021.
[74] Singh VP, Pratap K, Sinha J, Desiraju K, Bahal D, Kukreti R. Critical evaluation of challenges and future use of animals in experimentation for biomedical research. Int J Immunopathol Pharmacol 2016;29:551-61.
[75] Buchanan K, Burt de Perera T, Carere C, Carter T, Hailey A, Hubrecht R, Jennings D, Metcalfe N, Pitcher T, Péron F, Sneddon L, Sherwin C, Talling J, Thomas R, Thompson M. Guidelines for the use of animals: guidelines for the treatment of animals in behavioural research and teaching. Anim Behav 2013;85:285-97.
[76] Rai J, Kaushik K. Reduction of animal sacrifice in biomedical science & research through alternative design of animal experiments. Saudi Pharm J 2018;26:896-902.
[77] Marafante E, Smyrniotis T, Balls M. ECVAM: the European centre for the validation of alternative methods. Toxicol in Vitro 1994;8:803-5.
[78] Stokes WS, Schechtman LM, Hill RN. The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM): a review of the ICCVAM test method evaluation process and current international collaborations with the European Centre for the Validation of Alternative Methods (ECVAM). Altern Lab Anim 2002;30:23-32.
[79] Animal and plant health inspection service. Available at: Accessed on: 30/05/2021.
[80] Rollin BE. The regulation of animal research and the emergence of animal ethics: a conceptual history. Theor Med Bioeth 2006;27:285-304.
[81] Kehinde EO. They see a rat, we seek a cure for diseases: the current status of animal experimentation in medical practice. Med Princ Pract 2013;22:52-61.
[82] Clovis Y.Worms. Flies or fish? A Comparison of common model organisms-part 1: models for biomedical research. Available at: Accessed on: 30/05/2021.
[83] Prüßing K, Voigt A, Schulz JB. Drosophila melanogaster as a model organism for Alzheimer’s disease. Mol Neurodegener 2013;8:1-12.
[84] Milan DJ, Peterson TA; Ruskin JN, Peterson RT, MacRae CA. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 2003;107:1355-8.
[85] Petroianu A. Aspectos éticos na pesquisa em animais. Acta Cir Bras 1996;11:157-64.
[86] Iki Y, Ito T, Kudo K, Noda M, Kanehira M, Sueta T, Miyoshi I, Kagaya Y, Okada Y, Unno M. Animal ethics and welfare education in wet-lab training can foster residents’ ethical values toward life. Exp Anim 2017;66:313-20.
[87] Neves SM. Manual de Cuidados e Procedimentos com Animais de Laboratório do Biotério de Produção e Experimentação. São Paulo: Universidade de São Paulo 2013. Available at: Accessed on: 30/05/2021.
[88] Muller CA, Ramos S, Saisse AO, Almosny NRP. Videocâmeras em biotérios de experimentação: importante ferramenta no controle da contaminação ambiental na microbiota de camundongos. Arq Bras Med Vet Zootec 2015;67:689-97.
[89] Guia brasileiro de boas práticas para eutanásia de animais. Conselho Federal de Medicina Veterinária do Brasil Brasilia 2013. Available at: Accessed on: 30/05/2021.
[90] O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW. 1,026 experimental treatments in acute stroke. Ann Neurol 2006;59:467-77.
[91] Schardein J. Drugs as Teratogens. 1st ed. Cleveland, Ohio: CRC Press. Inc. 1976.
[92] Akhtar A. The flaws and human harms of animal experimentation. Camb Q Healthc Ethics 2015;24:407-19.
[93] Bailey J. An assessment of the role of chimpanzees in AIDS vaccine research. Altern Lab Anim 2008;36:381-428.
[94] Akhtar AZ, Pippin JJ, Sandusky CB. Animal studies in spinal cord injury: a systematic review of methylprednisolone. Altern Lab Anim 2009;37:43-62.
[95] Lane E, Dunnett S. Animal models of Parkinson’s disease and L-dopa induced dyskinesia: how close are we to the clinic? Psychopharmacology 2008;199:303-12.
[96] Mole DR, Tomson CR, Mortensen N, Winearls CG. Renal complications of jejuno-ileal bypass for obesity. QJM 2001;94:69-77.
[97] Balls M, Combes R. Animal experimentation and alternatives: revealed preferences. London: SAGE Publications Sage 2017.
[98] Hartung T. Toxicology for the twenty-first century. Nature 2009;460:208-12.
[99] Langley G, Evans T, Holgate ST, Jones A. Replacing animal experiments: choices, chances and challenges. Bioessays 2007;29:918-26.
[100] Washio T, Okada J, Takahashi A, Yoneda K, Kadooka Y, Sugiura S, Hisada T. Multiscale heart simulation with cooperative stochastic cross-bridge dynamics and cellular structures. Multiscale Model Simul 2013;11:965-99.
[101] Azer SA, Azer S. 3D anatomy models and impact on learning: a review of the quality of the literature. Health Prof Educ 2016;2:80-98.
[102] Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E, Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB, Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P, Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, van Loveren H, Vinken M, Worth A, Zaldivar JM. Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010. Arch Toxicol 2011;85:367-485.
[103] Huh D, Torisawa YS, Hamilton GA, Kim HJ, Ingber DE. Microengineered physiological biomimicry: organs-on-chips. Lab Chip 2012;12:2156-64.
[104] Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 2014;12:207-18.
[105] Wright AK, Ferreira DM, Gritzfeld JF, Wright AD, Armitage K, Jambo KC, Bate E, El Batrawy S, Collins A, Gordon SB. Human nasal challenge with Streptococcus pneumoniae is immunising in the absence of carriage. PLoS Pathog 2012;8:e1002622.
[106] Gray AC, Sidhu SS, Chandrasekera PC, Hendriksen CFM, Borrebaeck CAK. Animal-friendly affinity reagents: replacing the needless in the haystack. Trends Biotechnol 2016;34:960-9.
[107] Dübel S, Stoevesandt O, Taussig MJ, Hust M. Generating recombinant antibodies to the complete human proteome. Trends Biotechnol 2010;28:333-9.