Quantifying the potential of skin swabbing as a refinement for DNA sampling of laboratory fish

Sampling DNA to identify small lab fish is typically achieved by fin clipping, an invasive, regulated procedure carried out under non-terminal anaesthesia. A recent global survey indicated that 85% of zebrafish (Danio rerio) labs use the technique. With an estimated 3,250 zebrafish facilities worldwide, each genotyping 100s-1000s of animals per year, this equates to millions of procedures annually, and this figure is likely to increase dramatically with the utility the zebrafish model for CRISPR/Cas9 genome editing. Yet, despite its routine use as an SOP, fin clipping has the potential to impact fish welfare by causing them pain, altering their physiology, swimming ability or other behaviour, and increasing the subsequent risk of pathogen infections. Furthermore, the biological effects of anaesthesia potentially impact the quality of behavioural, physiological and other data derived from fin-clipped fish. The identification of refinements to standard DNA sampling SOPs to improve animal welfare is therefore warranted, both ethically and scientifically.

An alternative approach for sampling the DNA of aquatic vertebrates involves swabbing the skin surface. We recently developed a reliable swabbing technique that provides sufficient DNA for PCR reactions from different zebrafish strains and sticklebacks (Gasterosteus aculeatus) >20mm. However, while swabbing appears to offer a less invasive alternative, it still requires fish to be netted, held in air and handled; procedures that themselves are potentially stressful. Therefore, despite its putative advantages, the potential of swabbing to provide a refinement to standard DNA collection protocols remain untested. In this project we will provide evidence on the nature and magnitude of any welfare benefits of swabbing over fin clipping. Such information is essential to validate the technique as a refinement, and assure its wider adoption by a growing community of researchers using fish as laboratory models.

Fin clipping under non-terminal anaesthesia is the SOP for sampling DNA from fish in lab studies. However, fin clipping causes pain and induces stress in fish, and the use of anaesthesia potentially impacts data quality. Globally, we estimate ca. 1M fish are fin-clipped each year. The recent development of skin swabbing techniques - which produce sufficient DNA for PCR amplification - potentially offers a refined procedure with enormous impact for fish welfare. Our group has recently developed such a technique for zebrafish that has been well received by the community. However, as swabbing still requires fish to be netted, air-exposed and restrained, the magnitude of any actual welfare benefits over fin clipping remain unquantified, and the value of swabbing as a refinement untested.

Our main aim is to test the hypothesis that swabbing has a significantly lower impact on stress-related behaviour and physiology than fin clipping, and so represents a valid refinement to current regulated procedures. We propose a series of experimental studies comparing the effects of swabbing and fin clipping on a range of indicators of stress and wellbeing. To demonstrate the potential for cross-species benefits, we will study both zebrafish and sticklebacks. We will expose fish to a range of control, swabbing or fin clipping treatments, designed to separate the effects of various components involved in each procedure, and quantify behavioural and physiological correlates of stress over appropriate timescales. We will additionally compare the effects of analgesia on the stress responses of fish subjected to swabbing and fin clipping, to investigate the relative pain associated with each approach. Finally, we will examine the potential of proprietary water treatments to provide further refinement to swabbing procedures. We will publicise our results to a wide network of end users to ensure that our recommendations are taken up by the community

Background
Fish are increasingly used in regulated scientific research, accounting for 14% of procedures in the UK in 2015 (294K; up by 35K from 2013 [1]). Numbers are dominated by the zebrafish Danio rerio - the primary model for biomedical science - with sticklebacks Gasterosteus aculeatus being used for environmental / ecological studies. The frequent use of fish in genetic studies means in vivo DNA samples are often required for genotyping; however, the standard procedure - fin clipping under non-terminal anaesthesia - is regulated, and requires refinement [2]. We recently developed a reliable skin swabbing procedure for sampling DNA from zebrafish without anaesthesia [2], and in this project we propose to validate its potential to provide a refined fish DNA sampling protocol to benefit fish welfare.

Opportunity for impact
We used data from our lab and those of associates to estimate impact. In our zebrafish lab (ca. 1500 brood stock), prior to developing the swabbing procedure, we fin-clipped ca. 200 fish annually for genotyping. In a recent survey of 98 zebrafish facilities, brood stock numbers were spread evenly from <500 to >10,000 individuals, and most labs carried out fin-clip genotyping [3]. We invited a number of labs to share fin-clip genotype counts with us. Of the 7 responding labs, most genotyped 500-1250 fish per year, but one UK facility carried out 5700 such procedures in 2014, and 7300 in 2015. There are 53 registered zebrafish labs in the UK, and 3,250 globally [4]. Taking 500 as a conservative estimate of annual fin-clip procedures per lab, we estimate that over 20K DNA sampling procedures in the UK, and over 1M worldwide, could be refined each year if a validated swabbing technique was adopted. The popularity of CRISPR/Cas9 genome editing, and suitability of zebrafish for this approach [5], predicts even greater future impact of a refined sampling procedure.

Ensuring end user support
The beneficiaries of our proposed project are researchers in industry and academia. In developing the swabbing procedure, we engaged widely with the zebrafish community, sharing the technique with other researchers, technical staff, HO Inspectors and facility managers, through our NC3Rs representative and through presentations given by our senior aquarium technician Carl Breacker and NVS. Our approach has support from end users in industry and advisory bodies. The President of the Zebrafish Husbandry Association has agreed to support us by publicizing the project. In a letter of support, Dr Stewart Owen - Senior Environmental Scientist and 3Rs Lead at AstraZeneca's Council for Science and Animal Welfare - remarked that our project "represents an approach that could impact very large numbers of animals in thousands of laboratories around the world". We are also collaborating with Cefas (Weymouth) who have committed financial and technical support to our project (see attached letter). We are therefore extremely well positioned to ensure the findings of our project, and refinements to current procedures, are disseminated widely across the community. To facilitate this, we will present our results at meetings such as LASA, FELASA, AALAS, IAT Congress or the European Zebrafish Meeting, and hold a half-day workshop aimed at end-users from UK academia, industry and regulatory authorities. We will also commission a professionally-produced online training video tutorial providing detailed instruction on performing the skin swabbing technique to minimize adverse effects, for the NC3Rs website.

[1] Home Office (2016) Annual Statistics of Scientific Procedures on Living Animals Great Britain 2015 Office for National Statistics: London, UK. 63p. [2] Lidster et al. (2017) J. Fish Biol., in press. [3] Breacker et al. (2016) Zebrafish 13(6) in press. [4] Kinth et al. (2013) Zebrafish 10 510-17. [5] Hwang et al. (2013). Nat. Biotech. 31, 227-29.