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