A simple and rapid shaking-based assay to genotype live, early developmental stage zebrafish embryos.

The use of zebrafish as an animal model for biomedical and toxicological research has increased dramatically over the past decade, alongside a growing need to adopt the 3Rs principles to ensure ethically acceptable animal experimentation. Currently, one of the main challenges concerns 'surplus' animals that are unavoidably generated as part of an experimental procedure and are unsuitable for experimental analysis because they do not have the desired genotype, are too old or have the wrong sex. However, justifying the sacrifice of animals for these reasons is morally debatable and current ethics legislation in some countries insists they should nevertheless be maintained and left to die of natural causes. It is therefore imperative to develop strategies which can identify unwanted animals at a sufficiently early, non-sentient developmental stage so that they can then be sacrificed in an ethically more acceptable manner. In this manuscript we present a reliable medium-throughput method for non-invasive genotyping of zebrafish at developmental stages when sacrifice is considered ethically acceptable. This method is based on the use of low frequency shaking to induce the detachment of a limited number of cells from the embryos. These cells are then analysed by polymerase chain reaction-based genotyping approaches.