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NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research
Guidance

Skin swabbing for DNA sampling of zebrafish

Introduction

Small bony fishes, such as zebrafish and stickleback, are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals for genetic identification (genotyping). The current standard procedure to sample DNA is fin clipping, which involves placing the fish under nonterminal anaesthesia and removing a portion of the caudal fin with a scalpel. While fin clipping reliably generates good quality DNA samples for identifying animals by PCR, there is evidence that it affects fish health and welfare, leading to pain, stress and changes in behaviour [1-7]. This in turn can result in greater variation in physiological and behavioural data [8].

An alternative to fin clipping is skin swabbing, where a rayon-tipped swab or a cotton bud is used to collect mucus from the flank of the fish, which can then be processed to extract DNA [9-11]. Behavioural and physiological evidence suggests that skin swabbing has a lesser impact on fish welfare [8,11], providing an opportunity to refine DNA sampling procedures for laboratory fishes. In terms of practicality, the swabbing method is comparable to fin clipping and has some potential advantages (see table below).

Dr Will Norton and colleagues, at the University of Leicester and Nottingham Trent University, have shown that skin swabbing can be used to successfully collect DNA from zebrafish and stickleback. Interest in skin swabbing as a refinement is growing, and researchers and technicians have an increasing number of questions about whether this technique is applicable to their work, and how to perform skin swabbing correctly. This resource contains detailed information to help research groups learn more about skin swabbing as a welfare refinement, and how to establish a protocol for skin swabbing in their laboratory.

A stickleback being swabbed
Skin swabbing has also been validated as a refined method for collecting DNA from three-spined stickleback (Gasterosteus aculeatus).

Fin clipping and skin swabbing comparison table

 

 

Traditional method: fin clipping

Refined method: skin swabbing

Animal size

Larvae > 3.5 mm

Currently validated for juvenile fish ≥ 20 mm

Sample collection time

~60 seconds to anaesthetise and fin clip (not including time for recovery)

~30 seconds to restrain, swab and return to holding tank

Analgesia

Required before and after procedure

Not required 

Anaesthesia

Surgical anaesthesia required, MS-222 (tricaine) commonly used

Not required

Extraction method

HotShot method, isopropanol extraction and commercial kits

HotShot method or isopropanol extraction; commercial kits are less effective

Quantity

Medium (ng/µl), suitable for PCR

Low (ng/µl), suitable for PCR

Cost

Cost per sample is comparable to skin swabbing (e.g. using HotShot method). Reusable scalpels or scissors may be used for multiple fish.

Cost per sample is comparable to fin clipping (e.g. using HotShot method). Consumable swabs are considerably cheaper than single-use scalpels; purchase of anaesthetic is not required.

Skill level

Requires training and individual authorisation; chemical restraint of the fish is necessary (anaesthesia)

Requires training; physical restraint of the fish is necessary

 

Ethical approval

Licensed procedure requiring Home Office approval in the UK

Local ethical approval needed, e.g. from the AWERB, IACUC or equivalent

Frequently asked questions

We spoke to Dr Will Norton (WN), Associate Professor of Animal Biology at the University of Leicester, Dr Gregory Paull (GP), Aquatic Facilities Manager and NACWO at the University of Exeter and Karin Finger-Baier (KFB), Staff Scientist at Max Planck Institute for Biological Intelligence to answer your questions and address common concerns on the skin swabbing technique.

Protocol

See the Detailed skin swabbing protocol for information on how to carry out the swabbing technique to collect DNA from small-bodied fish species such as zebrafish. The protocol includes information on the equipment required and how to set it up, the preparation of reagents and DNA extraction. 

Video

Below is a video clip of the swabbing technique, which is also available to download from Vimeo for use in staff training. You can also download a close-up demonstration of swabbing.

 

Research papers on skin swabbing of laboratory fishes

Tilley C et al. (2021). Skin swabbing protocol to collect DNA samples from small-bodied fish species [version 1; peer review: awaiting peer review]. F1000Research 10:1064 doi: 10.12688/f1000research.73115.1

Tilley CA et al. (2020). Skin swabbing is a refined technique to collect DNA from model fish species. Scientific Reports 10(1): 1-17. doi: 10.1038/s41598-020-75304-1

Venta PJ et al. (2020) A 13-plex of tetra-and penta-STRs to identify zebrafish. Scientific Reports 10(1): 1-7. doi: 10.1038/s41598-020-60842-5

Breacker C et al. (2017). A low-cost method of skin swabbing for the collection of DNA samples from small laboratory fish. Zebrafish 14(1): 35-41. doi: 10.1089/zeb.2016.1348

Le Vin AL et al. (2011). Validation of swabs as a non‐destructive and relatively non‐invasive DNA sampling method in fish. Molecular Ecology Resources 11(1): 107-109. doi: 10.1111/j.1755-0998.2010.02909.x

Acknowledgements

We gratefully acknowledge the contributions of Dr Will Norton (University of Leicester), Dr Ceinwin Tilley (University of Leicester), Dr Gregory Paull (University of Exeter), Karin Finger-Baier (Max Planck Institute for Biological Intelligence), Dr Chrissy Hammond (University of Bristol), Mollie Millington (The Francis Crick Institute), Dr Stewart Owen (AstraZeneca), and Dr Anke Lange (University of Exeter).

References

  1. De Lombaert et al. (2017). Behavioral characteristics of adult zebrafish (Danio rerio) after MS222 anesthesia for fin excision. Journal of the American Association for Laboratory Animal Science 56: 377–381. PMID: 28724486
  2. Schroeder PG and Sneddon LU (2017). Exploring the efficacy of immersion analgesics in zebrafish using an integrative approach. Applied Animal Behaviour 187: 93–102. doi: 10.1016/j.applanim.2016.12.003
  3. White LJ et al. (2017). The impact of social context on behaviour and the recovery from welfare challenges in zebrafish, Danio rerio. Animal Behaviour 132: 189–199. doi: 10.1016/j.anbehav.2017.08.017
  4. Deakin AG et al. (2019a). Automated monitoring of behaviour in zebrafish after invasive procedures. Scientific Reports 9(1):9042. doi: 10.1038/s41598-019-45464-w
  5. Deakin AG et al. (2019b). Welfare challenges influence the complexity of movement: fractal analysis of behaviour in zebrafish. Fishes 4(1): 8. doi: 10.3390/fishes4010008
  6. Thomson JS et al. (2019). Assessment of behaviour in groups of zebrafish (Danio rerio) using an intelligent software monitoring tool, the chromatic fish analyser. Journal of neuroscience methods 328: 108433. doi: 10.1016/j.jneumeth.2019.108433
  7. Thomson JS et al. (2020). Acute and chronic stress prevents responses to pain in zebrafish: evidence for stress-induced analgesia. Journal of Experimental Biology 223(14). doi: 10.1242/jeb.224527
  8. Tilley CA et al. (2020). Skin swabbing is a refined technique to collect DNA from model fish species. Scientific Reports 10(1): 1-17. doi: 10.1038/s41598-020-75304-1
  9. Breacker C et al. (2017). A low-cost method of skin swabbing for the collection of DNA samples from small laboratory fish. Zebrafish 14(1): 35-41. doi: 10.1089/zeb.2016.1348
  10. Venta PJ et al. (2020). A 13-plex of tetra-and penta-STRs to identify zebrafish. Scientific Reports 10(1): 1-7. doi: 10.1038/s41598-020-60842-5
  11. Tilley C et al. (2021). Skin swabbing protocol to collect DNA samples from small-bodied fish species [version 1; peer review: awaiting peer review]. F1000Research 10:1064 doi: 10.12688/f1000research.73115.1
  12. Le Vin AL et al. (2011). Validation of swabs as a non‐destructive and relatively non‐invasive DNA sampling method in fish. Molecular Ecology Resources 11(1): 107-109. doi: 10.1111/j.1755-0998.2010.02909.x
  13. Díaz C et al. (2019). Fast Multiplex real time PCR method for sex-identification of medaka (Oryzias latipes) by non-invasive sampling. MethodsX 6: 587-593. doi: 10.1016/j.mex.2019.03.011
  14. Lange A et al. (2020). A newly developed genetic sex marker and its application to understanding chemically induced feminisation in roach (Rutilus rutilus). Molecular ecology resources 20(4): 1007-1022. doi: 10.1111/1755-0998.13166
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  16. Okada M et al. (2017). An efficient, simple, and noninvasive procedure for genotyping aquatic and nonaquatic laboratory animals. Journal of the American Association for Laboratory Animal Science 56(5): 570-573. PMID: 28903830
  17. McMurtrie J et al. (2021). Relationships between pond water and tilapia skin microbiomes in aquaculture ponds in Malawi. bioRxiv. doi: 10.1101/2021.12.06.470702
  18. Readman GD et al. (2013). Do fish perceive anaesthetics as aversive? PLoS One. doi: 10.1371/journal.pone.0073773
  19. von Krogh K et al. (2021). Screening of anaesthetics in adult zebrafish (Danio rerio) for the induction of euthanasia by overdose. Biology 10(11): 1133. doi: 10.3390/biology10111133
  20. Collymore C (2020). Anesthesia, analgesia, and euthanasia of the laboratory zebrafish. In: The Zebrafish in Biomedical Research (Eds. Cartner S, Eisen J, Farmer S, Guillemin K, Kent M, Sanders G). 1st edition. Academic Press.
  21. Köhler A and Valentim AM (2022). Analgesia, anesthesia, and euthanasia in zebrafish. In: Laboratory Fish in Biomedical Research (Eds. D'Angelo L, de Girolamo P). 1st edition. Academic Press.
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