Bioethical and practical aspects of experimentation with zebrafish embryos and adults

ABSTRACT

Zebrafish, also known as ‘peixe-zebra’ and ‘Paulistinha’ in Brazil, have been increasingly used for studies in various areas of Medicine and Biology. This fish has several characteristics that make it more suitable for scientific experimentation than other animal models, such as a large number of embryos per mating (about 100 to 200 eggs), a short life cycle (about 2 years), development fast (60 to 90 days to have all organs and systems fully mature), sequenced genome, easy adult breeding, low maintenance cost, transparent eggs and embryos, making it possible to monitor their development.

In this qualitative review we discuss the advantages and disadvantages of using the zebrafish model (Danio rerio) in the embryo and adult life stages, considering the principle of the 3 R’s (reduction, replacement and refinement) which are the basis of Bioethics applied to Animal Science Laboratory.

Keywords
ethical aspects regarding the use of zebrafish; Danio rerio ; experimentation with zebrafish; Laboratory Animal Science

Bioethics is a branch of science that allows us to carry out high-quality research while respecting the animal’s limits and identifying which is the best to be used and at which stage of life an effective and rapid result must be achieved. Using animal science with bioethics encompasses applying the ethical principles of the 3 R’s, namely reduction, replacement, and refinement.

The theme of this article is that the zebrafish is one of the models that is highly used in more than 15 research areas due to its numerous advantages in its life stages, not just in the adult phase. This fish, from the cyprinid family Zebrafish or Zebra Fish (Danio rerio, Hamilton-Buchanan, 1822), as it is also called, has been increasingly used as an excellent experimental model in scientific research (SANTIAGO et al., 2023) in areas such as genetics, toxicology, oncology, and ecology.

Despite all the millions of scientific articles about this fish, it is difficult to find concrete comparisons between carrying out experiments with embryos, larvae, and adults of Danio rerio, which is difficult to obtain from the information we found about the research object itself, as in articles on effective euthanasia the comparison is made by many. A type of euthanasia is more discussed in larvae, and not in adults, there is nothing very in-depth on what the best types of research in different larval developments are. Therefore, in this work, we will present our considerations.

Even an adult zebrafish is small in size, making it easier to care for the specimens, making them low in cost and breeding. As it has a sequenced genome similar to that of humans, it is easy to manipulate and becomes attractive for research in areas such as immunology and hematology. As for its life cycle, it is as fast as its development, with high reproduction and fertilization, which is external (WESTERFIELD, 2007; CANEDO et al., 2022; MOCHO et al., 2022). GOLDSMITH (2004) provides more complete information regarding reproduction, considering that this is a factor of great importance when we think about model animals, saying that a pair of adults can produce at least once a week from 100 to 200 descendants. Compared to other models used for much longer, such as the rat, the cost of breeding is low.

The same author states that there are certain areas of an adult D. rerio made for a disease model. An example given by him is ophthalmology, an area little mentioned regarding possible research with this fish, but which GOLDSMITH (2004) brings to light. In ophthalmology, even with the growing population of age-related macular degeneration, there are few drugs in the testing or even production phase, most likely, he explains, that the models previously used and cited, rats. They developed night vision, and their retina is predominantly made of rods, making the adult D. rerio a great competitor for the role of a model animal.

Furthermore, the object of study of this article in its adult phase is used in the studies of many other recurrent and important diseases for society, such as tuberculosis and cancer. MYLLYMÄKI et al. (2017) say in their study that “Mycobacterium marinum infection in adult zebrafish resembles human tuberculosis,” which is why it is a model for pre-clinical screening of vaccines against this disease. Regarding cancer, thousands of studies in this field are treated with adults. FEITSMA; CUPPEN (2008) state “some or more sensitivity to cancer in heterozygous adults, confirming the strong link between genomic instability and cancer,” regarding the connection of this serious disease that affects and degenerates people around the world. However, larvae are “also widely used in cancer research, becoming a tiny model of cancer.”

Using the genomic relationship cited by previous authors, CANEDO et al. (2022) state in their work that the adult species has a complex behavior, transgenic, and knockdown mutant lines available (FERNANDES; PEDROSO, 2017; AKSOY et al., 2019). MARUYAMA et al. (2015) and other authors later (SONG et al., 2016; TANG et al., 2017; AKSOY et al., 2019) explain that “despite the detailed and human-like genome, the widespread genome editing in the zebrafish embryo has been limited, indicating that such research must occur, for the most part, in fully formed fish.”

Even though this limitation arose in the embryos, these immature fish have one of the most important points of this species, transparency, enabling the visualization of changes in this stage of life, used as an alternative in toxicity tests together with the larvae (NUSSLEIN-VOLHARD; DAHM, 2002; BERMAN et al., 2003; LIESHKE; CURRIE, 2007; WESTERFIELD, 2007; MEEKER; TREDE, 2008; HARPER; LAWRENCE, 2011; LOPES; TAVARES, 2013; WALLACE et al., 2018; CANEDO et al., 2022; MOCHO et al., 2022). FUKUSHIMA et al. (2020) show that, according to international ethical regulations, D. rerio “larvae until five days post-fertilization are considered in vitro models” (Directive 2010/63/EU) and are accepted as an alternative to animal testing, as, differently from other models, the zebrafish embryo has a complex and functional organism even at this stage of life (CORNET et al., 2017), allowing access to developing pathologies in real-time through the previously mentioned transparency, emerging refinement procedures, such as software capable of measuring physiology and behavior per animal, following the line of the 3 R’s, also already brought to light. However, even with major organs developed in these five days, “pain, sedation, tumor metastasis, vascular tone, and intestinal motility are disease-relevant phenotypic examples that are observable in zebrafish but completely inaccessible in vitro approaches” (MACRAE; PETERSON, 2015), while this possibility already exists in adults.

By separating the species by their life stages, a lot of research can be done individually, even for the same diseases or areas of study, because, as already mentioned, even immature fish have their organs and metabolic systems developed and complex. Therefore, the third vision brought in this work is to use the complete life cycle of the zebrafish for experiments in general, since it is not a high-cost fish, it reproduces quickly and its lifespan is short, with elaborate work aimed at all stages of D. rerio and, as euthanasia in this fish is also simple to do, as soon as they are useless, they can be euthanized. As an example of what works, FEITSMA; CUPPEN (2008) cite in their article LEE et al. (2005), who used “from the zebrafish blastula stage, applying a melanoma cell, to the adult stage, noting that this cell survived and remained present until the individuals matured,” making their work more complete and effective.

Particularly, in the field of toxicology, the use of adult D. rerio as standardized by the Brazilian Association of Technical Standards (ABNT), in contrast to the Organization for Economic Cooperation and Development recommendation (OECD, 2013) for the use of zebrafish embryos, is a subject of importance in scientific research and environmental monitoring.

Zebrafish (D. rerio) is widely used as a sensitive alternative toxicity model, due to embryonic transparency, rapid reproduction, and development, sequenced genome (ROZMÁNKOVÁ et al., 2020), following the 3R’s principle, which deals with the use of animals in research with ethics and principles aimed at reducing, replacing animals and refining research as already mentioned (RUSSELL; BURCH, 1959; ROZMÁNKOVÁ et al., 2020; SANTIAGO et al., 2023), ease of breeding and manipulation, high number of offspring per mating (around 200 eggs), allowing significant sampling in a single experiment (SANCHES et al., 2017; MOURA et al., 2018; LECHINOVSKI et al., 2022), small size of these fish when adults (4–5 cm), easy to buy and more than 70% similarity with the human genome, even being recommended by the ABNT (2022) for ecotoxicological tests.

Here we address the advantages and disadvantages of each stage of development, adult, and embryo, for use in toxicological and ecotoxicological assays, bearing in mind that the option between them depends on the research objectives, ethical considerations, and laboratory conditions.

Regarding the research objectives, in studies to evaluate the chronic and long-term effects of a given phytosanitary product, it is generally more appropriate to use adult zebrafish, because they are larger than their counterpart’s embryos, facilitating the collection of accurate data, and have already reached organ maturity. In this case, the use of adult fish is a toxicological assessment closer to reality, as fish in rivers and lakes probably suffer exposure to pollutants for long periods in their lives.

Furthermore, adults can be used to analyze specific effects, such as toxicity to the reproductive system or the central nervous system. They can be used to evaluate the toxicity of compounds, for example, that cause infertility or birth defects, that cause damage to the brain or spinal cord, as well as other organs such as the liver, kidneys, and heart. However, zebrafish breeding is more complex than the development and study of embryos, which can imply more costs and difficulties in carrying out ecotoxicological tests.

On the other hand, zebrafish embryos are more used in acute toxicity studies because they are more sensitive, as they are in the early stages of development. This is useful when you want to quickly identify potential risks from chemical exposure or for initial screening of compounds. Furthermore, embryos have not yet developed the capacity to suffer and have less complexity compared to adult organisms. These can be important factors from an ethical point of view in research involving toxicity tests.

Considering the costs and ease of breeding, raising adult zebrafish is more expensive and time-consuming than maintaining embryos. Long-term studies may be also affected because it is more difficult to raise zebrafish to adulthood than to the embryonic stage, affecting the viability of long-term studies, as creating embryos is simpler and requires less space and resources, which means it can be beneficial in terms of costs and practicality.

The choice between adult zebrafish and embryos may depend on the degree of proximity to reality in the study of environmental or human health impacts.

It should be noted that local and international regulations when carrying out toxicology studies have the greatest weight in the decision between using adults or D. rerio embryos.

Each approach has its advantages and limitations, and the choice should be based on the relevance of the results to the research problem and the ethical principles involved. Furthermore, adjustment between different guidelines, such as those from ABNT and OECD, is desirable to ensure consistency in research and regulation.

ACKNOWLEDGEMENTS

Not applicable.

• FUNDING
  This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
• ETHICAL APPROVAL
  Not applicable.

AVAILABILITY OF DATA AND MATERIAL

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

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Edited by

• Associate Editor: Silvia Galleti https://orcid.org/0000-0002-0745-5716

Publication Dates

• Publication in this collection
  13 Dec 2024
• Date of issue
  2024

History

• Received
  05 Jan 2024
• Accepted
  15 Oct 2024