DOI: 10.5061/dryad.hx3ffbgdp
title: Skin swabbing protocol to collect DNA samples from small-bodied fish species

description: Fish species are commonly used as experimental models in the laboratory.
 DNA is routinely collected from these animals to permit identification of
 their genotype. The current standard procedure to sample DNA is fin
 clipping, which involves anaesthetising individuals and removing a portion
 of the caudal fin. While fin clipping reliably generates good quality DNA
 samples for downstream applications, there is evidence that it can alter
 health and welfare, leading to infection and impacting on the fish’s
 behaviour. This in turn can result in greater variation in the data
 collected. In a recent study we adapted a skin swabbing protocol to
 collect DNA from small-bodied fish, including sticklebacks and zebrafish,
 without the use of anaesthetics or sharp instruments. A rayon-tipped swab
 was used to collect mucus from the flank of the fish which was then used
 for DNA extraction. We subsequently demonstrated that – compared to fin
 clipping – skin swabbing triggered fewer changes in stress axis activation
 and behaviour. We also found that swabbing had a smaller impact on
 subsequent data collection, potentially allowing smaller sample sizes in
 experimental groups after using this technique, so reducing animal use.
 Here we provide a detailed protocol explaining how to collect DNA samples
 using skin swabs from small laboratory fish. Skin swabbing is a refined
 technique to collect DNA from fish, with the potential to reduce the
 number of animals used in experiments as well.
description: Fin
 clipping sticklebacks and zebrafish To collect DNA
 by fin clipping, a single fish was removed from its home tank using a
 small hand net. The fish was pre-treated with an anaesthetic by placing it
 in a tank containing 168 mg/L ethyl 3-aminobenzoatemethanesulfonate
 (MS-222) buffered to pH 7.2 with sodium bicarbonate dissolved in fresh
 system water. Once the fish was no longer response to touch, it was gently
 caught in a net and placed into a Petri dish containing a small amount of
 water. Fins were clipped using a sterile razor blade, taking care to only
 remove about one third of the caudal fin. The excised fin tissue was
 placed into a sterile labelled Eppendorf tube, and the fish was moved to a
 recovery tank containing fresh system. The fish’s behaviour was monitored
 until it had recovered consciousness, so that it swam around in the tank
 freely. Fin clip DNA was extracted using DNA extraction buffer containing
 15 µl of 20 mg/ml Proteinase K. This was incubated at 57˚C for 30 min,
 followed by addition of 400 µl chilled isopropanol. The solutions were
 mixed and the DNA solution was then chilled at -80˚C for 30 min. The
 solution was centrifuged for 10 min at 10,625 g, the supernatant decanted,
 and the remaining pellet washed with 190 µl 70% EtOH. After a further
 centrifugation step (2 min at 10,625 g) the DNA pellet was air dried and
 resuspended in 30 µl ddH2O. Skin
 swabbing sticklebacks and zebrafish To collect DNA
 by skin swabbing, a single fish was removed from its home tank using a
 small hand net. The fish was pre-treated with an analgesic, by placing it
 in a tank containing 2 mg/L lidocaine dissolved in fresh system water. The
 fish was immersed in this solution for 45 min immediately before skin
 swabbing was carried out. The fish was then re-caught in the net, and
 gently restrained on top of a wetted sponge. The uppermost surface of the
 fish was exposed to the air to permit DNA collection. A sterile swab was
 gently stoked along the flank of the fish, from head to tail, four or five
 times. The swab was placed into a clean labelled Eppendorf tube, and the
 fish was placed into a holding tank until DNA extraction and
 identification was complete. The DNA was extracted from the swab by adding
 DNA extraction buffer warmed to 55˚C and letting it incubate for 2 min.
 The swab was the removed, taking care to squeeze out as much liquid as
 possible. The DNA was precipitated by addition of 400 µl chilled
 isopropanol. The solutions were mixed and the DNA solution was chilled at
 -80˚C for 30 min. The solution was centrifuged for 10 min at maximum speed
 (10,625 g), the supernatant decanted, and the remaining pellet washed with
 190 µl 70% EtOH. After a further centrifugation step (2 min at 10,625 g)
 the DNA pellet was air dried and resuspended in 30 µl ddH2O.
 Water
 sample collection. Cortisol
 measurements were made following protocols from Ellis et al. (2004),
 adapted by Sebire et al. (2007, 2009). One hour post-manipulation fish
 were placed into individual 200 ml glass beakers containing 100 ml of
 freshly prepared reverse osmosis water. This water contained Instant Ocean
 salts (Aquarium Systems, UK) ensuring the same conductivity as the system
 water without background cortisol traces. The fish were left undisturbed
 in beakers, visually isolated by white dividers, for 30 min during which
 they released cortisol into the water. After 30 min the 100 ml water
 sample was collected using a serological pipette and split into two 50 ml
 Falcon tubes. Blank samples of 100 ml of RO water were also collected each
 week during the experimental period. 0.5 ml of methanol was added to each
 water sample prior to snap freezing in dry ice and storage at − 80 °C for
 extraction at a later date. The fish were euthanised by immersion in 168
 mg/L MS-222 until they no longer touch responsive, followed by
 decapitation. Their dry weight was recorded to within 0.0001 g to allow
 cortisol concentration to be expressed as ng cortisol/g fish per
 h. Cortisol extraction and quantification. Cortisol was extracted from the water samples by pumping it through Sep-pak Plus C18 solid phase extraction cartridges (Waters Ltd., UK) following the protocol developed by the Cefas Weymouth Laboratory (Ellis et al., 2004). Cartridges were primed with 5 ml of methanol followed by 5 ml of distilled water (dH20) and water samples were pumped through the cartridges at 5 ml/min. Each cartridge was washed with 5 ml of dH<sub>2</sub>0, air-dried, wrapped in Parafilm® and stored at −80 °C until elution with 5 ml ethyl acetate. For the quantification by radioimmunoassay (RIA, Sebire et al., 2007; Ellis et al., 2004; Scott et al., 1982) the eluted extracts were evaporated at 45°C under nitrogen and each residue was reconstituted in 500 µl of RIA buffer until assayed. The elution and the quantification were carried out in a blind manner to reduce bias when analysing the data. Experimental design All experiments presented here were approved by the Animal Welfare and Ethical Review Board at the University of Leicester, and were carried out under the authority of a UK Home Office project licence (PPL P8F9CCE8B). The experimental design and fish numbers for each group were determined by the NC3Rs Experimental Design Assistant EDA and power analysis. The raw data used in power calculations were taken from previous publications from the Norton laboratory. For example, the cortisol experiment was based upon published data from the Norton lab comparing wild-type zebrafish to a <i>linc5</i> mutant. Mean control = 20.0±3.9 pg/g, mean treatment group = 9.9±3.2 pg/g. (1-α) is 0.05 and power (1-β) is 80 giving an n value of 4. The outcome measure was radioimmunoassay recordings of cortisol concentrations in water as described in Breacker et al., 2017. The DNA concentrations presented here are based upon data presented in Breacker et al., 2017. The outcome measure was readings of concentration and purity using a spectrophotometer. The PCRs show representative data from experiments published in Breacker et al., 2017. The outcome measure was a photograph of stained bands on the agarose gel. No animals were excluded from the final data analysis. Animals were haphazardly selected for inclusion in each experimental group, and the experimenter was aware of the identity of the samples processed, apart from the cortisol experiment which was conducted blind. One week prior to experimentation, fish were caught and haphazardly distributed into 13.4 L tanks for sticklebacks (maximum capacity 27 sticklebacks) and 3.5 L tanks for zebrafish (maximum capacity 30 zebrafish). The tanks were located in central positions in large racks within the respective species’ aquarium rooms, ensuring that all tanks received similar illumination and were surrounded by other tanks on the sides. No enrichment was provided, as is standard procedure in our fish facility. Fish were fed <i>ad libitum</i> each afternoon at the end of the experiments (sticklebacks: defrosted bloodworms, zebrafish: Zebrafeed (Sparos)). No fish of either species died during these experiments, and all animals were killed by a Schedule 1 procedure at the end of this study. Statistical design. Statistical analyses were carried out using GraphPad Prism7. The DNA concentration was compared using a one-way ANOVA followed by Tukey’s post hoc test. The cortisol data were tested for normality using the using the Shapiro–Wilk test. Since it was not distributed normally, we analysed it using the non-parametric Kruskal–Wallis test followed by a Dunn’s multiple comparisons test comparing each treatment to the control group. This means that the data was not transformed in order to satisfy parametric assumption. All tables were prepared in Excel (Microsoft).
description: Please contact Dr. Norton if you require any further
 information.