Zebrafish embryos as an alternative to animal experiments—A commentary on the definition of the onset of protected life stages in animal welfare regulations

doi:10.1016/j.reprotox.2011.06.121

Reproductive Toxicology

Volume 33, Issue 2, April 2012, Pages 128-132

https://doi.org/10.1016/j.reprotox.2011.06.121 Get rights and content

Abstract

Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.

Section snippets

The zebrafish embryo as an alternative to animal experiments

Embryonic stages of fish are increasingly being used as a model in biomedical research and (eco)toxicology. According to the 3Rs (replacement, reduction and refinement of animal experiments, [1]), fish embryos are considered as replacement or refinement methods, since these developmental stages are likely to experience less or no pain, suffering, distress or lasting harm [2]. In particular, the zebrafish (Danio rerio) embryo has achieved high popularity founded on a unique set of properties

The current regulatory situation

Fish show a high variability with respect to their embryonic development and the identification of clear markers of the end of the embryonic period is difficult [38], [39]. In contrast, from a regulatory perspective, precise definitions are needed and various attempts have been made within European regulations. In Great Britain, protection of fish, amphibians and Octopus vulgaris commences from the time at which they become capable of independent feeding. According to the home office's guidance

When does a zebrafish feed freely?

In order to address the onset of independent feeding in the zebrafish, a clear definition of developmental time is required. This is an issue over which there is considerable confusion in the literature. It is difficult to refer to time after hatching, since the time of hatching itself depends on strain, stocking density and temperature, reflecting the fact that fish are poikilothermic animals. Moreover, rather than being a time point, hatching is a process that occurs over a prolonged period.

Regulatory perspectives for the use of zebrafish embryos

The exact onset of feeding in zebrafish is of great practical interest because of its far-ranging implications for animal protection. For mammals, the European Food Safety Agency (EFSA) considers the stage of development at which there is a risk of poor welfare to begin with the last third of development within the mother. For fish, it is instead defined as the stage when it is capable of feeding independently rather than being dependent on the food supply deposited by the mother in form of the

Conclusions

The stage of “independently feeding larval forms” can be defined by assessing a couple of criteria, such as development of the digestive tract and swim bladder, incorporation of food, swimming ability and yolk consumption. By 120 hpf, when the yolk is gradually used up, swimming activity increases and the majority of the larvae are able for the first time to actively hunt for food, most of these criteria would be matched. We thus conclude that developing zebrafish should be regarded as protected

Acknowledgements

This study has been created within the scope of EuFishBiomed COST Action BM0804. U. Strähle and R. Geisler are supported by the European Commission's 7th Framework Programme (ZF-HEALTH project, EC Grant Agreement HEALTH-F4-2010-242048). Critical comments on the manuscript by colleagues from the UFZ Leipzig (R. Altenburger, W. Busch, T. Luckenbach and others) are gratefully acknowledged.

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Zebrafish embryos as an alternative to animal experiments—A commentary on the definition of the onset of protected life stages in animal welfare regulations

Abstract

Section snippets

The zebrafish embryo as an alternative to animal experiments

The current regulatory situation

When does a zebrafish feed freely?

Regulatory perspectives for the use of zebrafish embryos

Conclusions

Acknowledgements

Toxicol Lett

Aquat Toxicol

Toxicol Lett

Comp Biochem Physiol C Toxicol Pharmacol

Toxicology

Neurotoxicol Teratol

Reprod Toxicol

Reprod Toxicol

Toxicology

Comp Biochem Physiol C Toxicol Pharmacol

Toxicol In Vitro

Dev Biol

Dev Biol

Dev Biol

Aquat Toxicol

The principles of humane experimental techniques

Opinion of the Scientific Panel on Animal Health and Welfare on a request from the Commission related to the aspects of the biology and welfare of animals used for experimental and other scientific purposes (EFSA-Q-2004-105)

EFSA J

Large scale genetics in a small vertebrate, the zebrafish

Int J Dev Biol

Animal models of human disease: zebrafish swim into view

Nat Rev Genet

Current perspectives in zebrafish reverse genetics: moving forward

Dev Dynam

Zebrafish: a practical approach

From zebrafish to human: modular medical models

Annu Rev Genomics Hum Genet

In vivo drug discovery in the zebrafish

Nat Rev Drug Discov

Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis

Nature

Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Nat Biotechnol

Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation

Science

The zebrafish embryo model in environmental risk assessment – applications beyond acute toxicity testing

Environ Sci Pollut Res Int