Skin Swabbing as a Noninvasive Alternative to Fin Clipping for Commercial Automated Genotyping

Summary

The zebrafish (Danio rerio) is a widely used laboratory model with significant genetic similarity to humans and is accessible for genetic manipulation. Standard genotyping involves anesthetizing the animal to obtain a fin clip, which may cause pain. We evaluate skin swabbing as a refinement for automated genotyping of adult zebrafish.

Abstract

Fin clipping remains the most common sampling method for genotyping zebrafish, yet it requires anesthesia and tissue removal, raising animal welfare concerns and potentially impacting behavior in the short term. To address this, we have validated a non-invasive skin swabbing technique that eliminates the need for fin clipping adults while maintaining genotyping accuracy during automated genotyping.

This method involves gently swabbing the fish’s skin to collect mucus and epidermal cells for DNA extraction. This approach avoids anesthesia, reduces handling stress, and is much less invasive than traditional fin clipping with limited tissue injury. We conducted a comparative analysis of swab-derived DNA against traditional fin clip samples sent for automated genotyping. Swab and fin clips samples taken from the same animal matched genotype results, demonstrating substantial promise as a refinement for automated genotyping under the 3Rs framework. Further testing of the swabbing technique, transport, and sample processing was optimized across nine sessions. Key aspects of the collection technique include focusing on consistent swabbing pressure and sample dry time. Proper technique is critical to ensuring robust and reproducible genotyping outcomes.

Our findings indicate that skin swabbing offers a welfare-improving alternative to fin clipping for automated genotyping. Broad adoption of this technique has the potential to enhance animal welfare standards while maintaining scientific rigor, aligning with the principles of the 3Rs and advancing non-invasive research practices in zebrafish facilities worldwide. While the current success rate indicates some limitations relative to fin clip genotyping results with automated genotyping, swabbing remains a promising refinement that can substantially improve welfare in zebrafish facilities.

Introduction

Zebrafish (Danio rerio) are the second most widely used laboratory animal model worldwide, after mice, owing to their high fecundity, externally fertilized transparent embryos, and 72% of human genes have a zebrafish ortholog¹. Increasing evidence demonstrates that fish are capable of experiencing pain², prompting continued efforts within the zebrafish research community to refine experimental procedures and minimize animal pain and distress. Zebrafish fins are capable of regeneration, and fin clipping remains a commonly performed procedure for tissue collection, primarily for genotyping purposes³. Analogous to ear punching in mice, fin clipping provides a reliable source of DNA but raises welfare concerns⁴, as the procedure is considered invasive and is expected to be of mild severity.

Genotyping samples obtained via fin clipping may be processed in-house or submitted to third-party service providers for automated genotyping. At the Francis Crick Institute, most fin clip samples are outsourced for automated genotyping, allowing researchers to optimize their time by continuing other experimental work while genotyping is performed externally.

Skin swabbing is an advancement in the 3Rs for zebrafish, relying on a noninvasive sampling technique rather than an invasive one⁵. It has been proven that mucus obtained from a swab of the skin can provide enough genetic material to run genotyping assays in-house^4,6, and the collection method also works on mice and Xenopus laevis⁷. Skin swabbing of adult zebrafish avoids the need for analgesia or anesthesia⁸, sparing the animal pain and the adverse effects of MS-222⁹, while also saving the technician preparation time.

As we do not typically run genotyping samples in-house, we wanted to determine if sending skin swabs for automated genotyping would provide reliable genotype information. Swabbing zebrafish provides variable DNA collection due to the nature of the collection technique as compared to tissue collection via fin clipping.

During automated genotyping, skin swab samples are processed in a similar manner to fin clips, with a reduction in the concentration of NaOH in the lysing solution. Genotyping results for each sample are calculated via relative copy number (RCN) and other metrics using a software analysis package. We improved sample handling across several sessions to reduce the reported error rates during automated genotyping.

Protocol

This work was in compliance with the Francis Crick Institute’s animal welfare and ethical review board and Home Office licensing.

1. Set up

1. Identify adult zebrafish (>3 months of age, at least 20 mm long) that need genotyping.
2. Set up an area with a soft, wet sponge in a plastic container, a 96-well plate provided by the commercial vendor, a swab, scissors for cutting the swab, and a final isolation tank for swabbed fish.
   NOTE: One must be able to individually identify each animal to match the genotyping results.
3. Use system water to wet the sponge, rather than purified or tap water.

2. Swabbing⁴

1. Net fish from the home tank onto the top of a wet sponge and secure the head gently with the net using fingers⁴. No analgesia or anesthesia is required⁸.
2. Try to gently cover the eyes with the net and fingers to limit external stimulation⁴.
3. Once the fish is in place, rub the swab gently along one side of the fish from the pectoral fin to tail⁴.
4. Repeat four more times, then roll the swab 180 degrees and repeat five more times.
5. Never swab against the scales as this could cause injury.
6. Rest the swab in the corresponding well on the plate while the fish is placed in its individual housing.
   NOTE: Small breeding tanks may also be used to provide individual housing (1 L tank with divider).
7. Ensure each container is properly labeled to avoid mix-ups.
8. Using scissors, cut the swab tip off so it fits in the well plate.
9. Repeat for all fish that need genotyping.
10. Let the plate dry for 60 min on the open bench before sealing.
11. Seal the well plate with a cap mat.

3. Submitting samples

1. Prior to submission, contact an automated genotyping supplier to create an account, order shipping documentation, arrange collection, and submit order requests.

4. Automated genotyping

1. Follow the steps below for the automated genotyping sample processing:
   1. Lyse swab samples for DNA extraction via the addition of 50 µL of 0.5 M NaOH solution.
   2. Incubate the samples for 60 min at 90 °C.
   3. Transfer 15 µL of the lysed sample to another well-plate for DNA isolation.
      NOTE: The isolation process is a proprietary, ferrous bead-based isolation method¹⁰ that utilizes EtOH washes to purify the DNA that has adhered to the beads.
   4. Use 100 µL of DNase-free H₂O to elute the DNA from the beads.
   5. Mix the DNA with qPCR assays for the targeted mutations and an internal control assay targeted to the cJun gene.
   6. Use the following thermocycler settings: 1 cycle at 95 °C for 10 min, followed by 35 cycles of 95 °C for 15 s, and 60 °C for 1 min. Refer to Supplementary Table 1 for the housekeeper and reporter genes.
   7. Add the proprietary master mix, including Taq polymerase and buffers.
   8. The total reaction size is less than 1 µL. Run each reaction in duplicate wells.
   9. Ensure the assay and Master mix are at 1× concentration.
      NOTE: The results will be emailed when ready.

Results

Genotyping results from skin swabs

Over nine different sessions, we processed 470 skin swab samples of wild type and two different genetically altered lines (Table 1). Six different animal technicians obtained the swab samples across all sessions. The overall success rate of obtaining a definitive genotyping result was 86.3%.

During session 1, all the fish were swabbed and fin clipped to verify the correct genotype results we obtain...

Discussion

It has already been shown that skin swabbing of zebrafish is an efficient DNA collection technique^4,5,6,7,8. There is evidence that zebrafish that are fin-clipped experience pain¹¹. Skin swabbing offers a refined DNA collection step for zebrafish genotyping methods, with studies showing that fish that have been fin clipped sh...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.1 L breeding tank	Tecniplast	ZB10BTE, ZB10BTI, ZB10BTD, ZB10BTL	Four components required: outer tank, inner tank, divider and lid. This is an alternative to Gene-S stand alone
96 well plate	Transnetyx
Automated Genotyping System	Transnetyx	https://www.transnetyx.com/services/genotyping
Gene-S Active Blue stand alone	Tecniplast	SAAB	Stand alone system with drawers that can hold up to 288 individual adult zebrafish
MS-222	Merck	886-86-2	Also known as Ethyl 3-aminobenzoate methanesulfonate or Tricaine
Plastic container to hold sponge			Can be any container that is leak proof and clean
PurFlock Ultra swab	Puritan	25-3318-U
QuantStudio real-time PCR	ThermoFisher	https://www.thermofisher.com/in/en/home/life-science/pcr/real-time-pcr/real-time-pcr-instruments/quantstudio-systems.html
Scissors to cut  swab
Soft car sponge		https://amzn.to/4dQwngP
Taqman genotyping assays and reagents	ThermoFisher	https://www.thermofisher.com/in/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/snp-genotyping-taqman-assays.html	qPCR assays