Subjects and husbandry

Eight month old female zebrafish (D. rerio) of AB strain (n = 126; mean size 0.92 g ± 0.08) were randomly selected from the University of Liverpool aquarium in-house breeding project for the experiments. Females were used to prevent any confounding effects of gender on behavior and physiology as has been found in studies conducted in our laboratory20 and elsewhere21,22,23,24,25. Stock fish were maintained in a semi-closed recirculation system in 10 L tanks (Aquatic Habitats, Florida USA) at 27 ± 1 °C, with constant aeration on a 14:10 h light:dark cycle. Fish were selected at random, netted carefully into a 3 L tank and transferred from stock tanks to a semi-closed recirculation system consisting of two parallel rows of glass tanks (20 × 30 × 20 cm; n = 1 fish per tank). Each tank was fitted with an identical, external laminated printout of a green plant background; this allowed the easy detection of the focal fish by an in house tracking system26 due to the enhanced contrast provided by the green background. All tanks were supplied with filtered water (NH 3 = < 0.01 mg/L, NO 2 = < 0.01 mg/L, NO 3 = < 5 mg/L) maintained at a temperature of 27 ± 1 °C, under a 14:10 light:dark regime with aeration provided by an aerated 200 L biological filter with one third of the water replaced weekly. Fish were acclimatized in their individual experimental tank for two weeks prior to experimentation and fed twice daily ad libitum with a commercial tropical ornamental flake (TetraMin, Tetra, Melle, Germany). All fish used in experiments had fed consistently when food was presented for at least seven days prior to experimentation commencing. Fish were in chemical (through shared water) and visual contact with adjacent tanks so had social contact until the evening prior to experimentation when two opaque pieces of plastic were placed in between tanks to visually isolate the test individuals and the inflow was turned off 30 min prior to the commencement of experiments. All methods were performed in accordance with the relevant guidelines and regulations. A schematic representation of the procedures and timings used in two experiments is shown on Fig. 1.

Figure 1 A schematic representation of Experiment 1 and Experiment 2. Video recordings were made at each time point: pretreatment (−40 min), followed by anaesthesia and treatment (fin clip, PIT tag or subcutaneous injection of acetic acid) at 1, 2, 3 and 6 h after treatment. Control zebrafish were undisturbed and sham treated individuals were anaesthestised and handled. In experiment 2, one analgesic drug at one dose was dissolved in the tank water (bupivacaine at 0.25, 0.5 and 1 mg/L; flunixin at 2, 4 and 8 mg/L; lidocaine at 5 mg/L and morphine at 3 and 48 mg/L) 30 min prior to the first pre-treatment recording. Full size image

Experiment 1: The impact of common procedures on the behaviour of zebrafish

The effect of laboratory procedures on the behaviour of female zebrafish was tested against control (undisturbed) and sham handled groups (anaesthetized and handled in a similar manner and time frame but no invasive treatment). We did not perform any sham injections of non-painful saline since previous studies demonstrate there are no differences between control and sham injected fish27,28,29,30 (these studies demonstrate the injection itself does not significantly affect subsequent behavior). Fish were randomly assigned to one of seven treatment groups (n = 7 for each group; n = 49 total): Control; Sham; five noxiously-stimulated groups (1–3) injected subcutaneously with either 1%. 5% or 10% acetic acid into the lips (concentrations used in previous studies)15,29; 4. PIT tag insertion through the abdomen; and 5. Fin clip where 40% of the caudal fin was removed using sterile surgical scissors as described in The Zebrafish Handbook (http://zfin.org/zf_info/zfbook/chapt7/7.8.html). Control fish were left undisturbed for the duration of the experiment whereas after the first behavioral recording all treatment fish were carefully netted and transferred to a 1 L beaker containing 500 ml of aerated water dosed with benzocaine (0.033 g l-1; Sigma-Aldrich Co., UK) where fish were anaesthetized. Benzocaine was used as it has short lasting analgesic properties and fish were taken to deep plane anesthesia where they were unconscious during the noxious treatments shown by a lack of reflex responses31. After anesthesia the sham treatment group were handled (netted, placed in wet paper towels and returned to their home tank) for the same duration as the other procedures but no invasive procedure applied. During anesthesia the acetic acid groups were injected with the appropriate concentration of acetic acid using a sterile gastight syringe and needle (34 g; Hamilton; Bonaduz, Switzerland). Acid was injected subcutaneously into the frontal lips using 2 ɥl per lip. PIT tag treatment fish were orientated upside down and a sterile 20 gauge needle used to make an incision in the abdominal cavity so that a 4 mm PIT tag (Loligo systems; Denmark) could be careful inserted. All fish were returned to their home tank after the procedure and allowed to recover from the anesthesia for 1 h before observations commenced.

Experiments were conducted at the same time each day (10 am) to prevent any diurnal effects. Behaviour was recorded for 25 minute periods at the time points 40 minutes pre procedure, and then 1, 2, 3 and 6 hours after the procedure. These time points were chosen as fish subject to noxious stimulation usually show an initial adverse response up until 3 hours then recovery by 6 hours in acetic acid tests15,32. Two industrial IDS USB 3.0 colour video cameras (IDS; Obersulm, Germany) fitted with a 25 mm monofocal lens and connected to a computer (HP compact elite 8300; Palo Alto, Ca) running the fish tracking software26 were used to accurately track the movements of individual zebrafish. Cameras positioned above and to the front of the focal tank were used to track the 3D trajectories of fish. Cameras positioned dorsally were mounted on a sliding gantry 1.4 m above the two parallel rows of 9 tanks; this enabled the cameras to be moved from tank to tank without disturbing the fish. Cameras positioned to the side were attached to tripods 1.4 m away from the focal tanks and were moved manually between tanks. One tank on either side of the rows was randomly assigned to a treatment group with cameras moved the previous evening which allowed two fish per day to be recorded; as treatments were randomized this prevented any confounding effects of order in the experiment. Data files generated by the 3D tracking software were then processed with software in MATLAB26. This software allowed the extraction of 20 characteristics (Table 1) from an individual’s 3D coordinates. Videos were assessed blind until data had been collected then the identity of the fish was related to the data set.

Table 1 List of zebrafish behavioural characteristics extracted from the tracking and behavioural analysis software (those with abbreviated labels on Supplementary Fig. S8). Full size table

Experiment 2: The prevention of pain related behaviour via immersion analgesics

Out of the five invasive procedures, the fin clip resulted in some of the most consistent changes in behaviour without recovery at 6 h. Therefore, the fin clip was chosen as the procedure by which the efficacy of immersion analgesics would be tested. Three categories of analgesic including nonsteroidal anti-inflammatory drugs (NSAIDs), local anesthetics and opioids were tested at a range of doses and extrapolated from studies using fish or mammals14,15,27,28,29,31,32. The doses applied were: NSAID flunixin 2, 4, and 8 mg/L; opioid morphine 3 and 48 mg/L; local anaesthetics bupivacaine 0.25, 0.5 and 1 mg/L and lidocaine 5 mg/L.

Zebrafish were randomly assigned to each treatment group. At the start of the experiment (09:30) the water flow to the experimental tanks was switched off and the analgesic administered evenly throughout the tank. All analgesics were administered prior to the fin clip so that the effect of the drug on normal behaviour could be determined in the pre-treatment recording. Once the analgesic had been administered the same data collection methods as outlined in experiment 1 were carried out so that a behavioural profile could be obtained for the time points pre-treatment and 1, 2, 3 and 6 h afterwards.

Statistical analyses

Data were analysed using SPSS software (Version 24). A principal component analysis (PCA) was used to explore which characteristics were responsible for the differences between control fish and those experiencing a potentially painful intervention; the PCA was based on 20 characteristics extracted from the xyz coordinates of individuals. The control values consisted of all control values and all pre-treatment values from the other experimental groups. All ‘pain’ characteristics were taken from the 2, 3 and 6 hour time points from fin clip, PIT tag, and acid lip groups. PCA data had a Kaiser-Meyer-Olkin value of 0.781 and a Bartlett’s test of sphericity value of <0.0001 indicating the appropriate use of PCA to examine correlations. Data was rotated using direct Oblimin with Kaiser normalization. Once the three most important characteristics had been selected the data was then assessed for homogeneity of variance and normality. After transformation Experiment 1 data was normally distributed as assessed by Shapiro-Wilk’s test and displayed homogeneity of variance as assessed by Levene’s test. Data did not require transformation for experiment 2.

A mixed model ANOVA was used to investigate whether time, treatment, or their interaction had an influence on each of the behaviours in experiment 1 using repeated measures for individuals (results in Supplementary Table 2). Assumptions of sphericity were checked using Mauchly’s tests for the data for percentage time at bottom; none of the data sets exhibited sphericity and thus Greenhouse-Geisser (average speed, percentage tank explored) or Huynh-Feldt (percentage time spent at bottom of tank) corrections were applied based on estimated values of ε. Interactions were significant for all behaviours; for post-hoc analysis, therefore, simple effects of Time were determined, and separate one-way ANOVA tests conducted to examine simple effects of Treatment. In experiment 2 the fin clipped analgesic groups were compared with controls and with fin clip without analgesia since this allowed us to gauge the efficacy of the drug. If the drug was significantly different from control it was deemed not effective in preventing responses to the fin clip. Conversely, if the drug treated animals displayed behaviours that were similar to controls then the drug had been successful in preventing responses to fin clip. As described above, data sets failed the assumption of sphericity and therefore Greenhouse-Geisser (percentage time spent at bottom of tank) and Huynh-Feldt (average speed, percentage tank explored) corrections were utilised. The interaction of Treatment × Time was significant for average speed, and post-hoc analysis conducted as for experiment 1 (results in Table S3). However, for percentage time spent at bottom of tank and percentage tank explored, only the main effects of Time and Treatment were significant (Table S3). Post-hoc analyses were therefore conducted for each individual main effect separately, with Tukey (Treatment) and Bonferroni (Time) corrections as appropriate.

Creation of FBI

The FBI was designed to generate a scale in four broad categories, starting at normal and decreasing to an abnormal change in behaviour using generic labels for ease of reference: ‘Healthy’, ‘Ok’, ‘Unhealthy’ and ‘Abnormal’. We suggest these terms are easily understood and clear with Healthy and Ok not requiring concern, however, Unhealthy and Abnormal would require attention from carers and researchers. The timescales used to process acquired data from the videos were the latest 30, 20, 10 and 1 minutes, all updated once a minute in real time (SI Videos 1 and 2). The basic monitoring unit of 1 minute was thereby grouped into longer behavioural periods of 10, 20 and 30 minutes giving a range of timescales from very short (1 m) to relatively long (30 m). FBI is a combination of evaluations of “Activity” and of “Distance” travelled over these timescales providing a score which allows the behaviour of the fish to be scaled (Table 2). Activity was directly related to the parameter % tank explored and indirectly to % time spent in bottom of the tank and was calculated as the space utilization percentage (number of zones of the tank (1 to 9, out of 9) visited per minute with the horizontal and vertical axes of the tank each divided into three equal sections. Distance is the cumulative distance travelled in the timescale and relates directly to average speed. Not only are these significantly altered in the present study, these behaviours are known to be of scientific relevance (e.g.12,13,14,33,34,35,36,37,38,39 www.noldus.com/animal-behaviour-research/solutions/research-fish/stress-and-anxiety). Supplementary Fig. 1 illustrates FBI for a normal (untreated) fish monitored over 40 minutes separately from the 5 group experiments, giving latest 40, 30, 20, 10, 1 minute behaviour status (Supplementary Fig. 1(a)), plus a more detailed, deeper historical view of FBI over the 40 minutes with continuous 10 minute and 1 minute indicators (Supplementary Fig. S1(b)). Supplementary Fig S2 illustrates the data from which FBI is derived. Thus, the FBI is a live monitoring system to give a rating updated every minute and designed to characterize a subject’s behaviour as a combination of quantitative information and qualitative categories. As fish behaviour may vary considerably over different timescales, the FBI was designed to indicate fish responses to the stimuli over overlapping but independently evaluated timescales – short (1 min), medium (10 min) and long (30 min) - to provide a more complete picture of how behaviour varied. A medium timescale was considered significant for alerting to compromised welfare deviations requiring intervention (with e.g. analgesics, veterinary advice or the cessation of the experiment) since zebrafish can become immobile or swim erratically over the short term period (1 min) but then return to normal when behaviour is assessed over 10 minutes. Supplementary Fig. 3 compares medium timescale (10-minute) detailed FBI, updated every minute, for 2 healthy and 2 treated fish over 3 periods of 30 minutes. For comparative evaluation of behaviour using the method, top level 30 minute FBI was analysed and presented, for example as circled on Fig. 2 (Assessment of each treatment group over the experimental period can be seen in Supplementary Fig. 7).

Table 2 When combining the parameters “Distance” and “Activity” from zebrafish behaviour a score is calculated that is normalised to the range 0 to 1, where 1 represents normal pre-treatment or initial behaviour. Based upon the score we assigned categories that can easily be interpreted by human carers from Abnormal, Unhealthy, Ok to Healthy for the Fish Behaviour Index (FBI). Full size table

Figure 2 Examples of the Fish Behaviour Index (FBI; ranging 0 to 6) output before (1) and after (2, 3 h) treatment for 5 individual fish from 5 groups: (a) Control (b) Sham (c) Acid Lip (d) PIT (e) Fin Clip treated zebrafish over 3 hours in total (represented by 1, 2 and 3 at the end of each row), using 4 timescales each, from the latest 30 mins (circled) to the latest 20 mins, 10 mins and 1 min. Gradation of score (FBI) from green (Normal/Healthy), blue (Ok/Healthy), yellow (Unhealthy) to pink (Abnormal) provides a means of assessing how the subject’s behaviour differs from normal. Blue circles highlight Normal to Ok ratings whereas green circles refer to Unhealthy ratings and red circles highlight Abnormal ratings. Full size image

FBI analysis was additionally applied to several blinded tests including live testing of the FBI where zebrafish were subject to treatments (e.g. 10% acid, chronic stress) and some additionally given analgesia in real time (Table 3). Time points were selected at random and all 5 points were included for one subject. Qualitative comparisons were made of 10 min FBI with the 30 min indicator and with comments made by an independent observer (with knowledge of zebrafish behaviour) of the videos to validate FBI assessment. Videos of tests with analgesics administered (plus one without) were also assessed blind by a trained observer (trained in behavioural changes associated with invasive procedures) and related to the output of the FBI to verify that automated assessment correlated with human interpretation of zebrafish behaviour.

Table 3 Details of live FBI tests where female zebrafish were subject to treatments without (A) and with analgesia (B). Full size table

Ethical statement

Experiments were conducted with approval from the Home Office, U.K. (license no. 40/3534) and the University of Liverpool’s Ethics committee. Minimum effective sample sizes were determined using Power analysis. At the end of the experiment after the 6 h behavioural recording, fish were euthanized using a schedule 1 method (concussion followed by brain destruction) and tissue harvested for use in other studies. All fish were treated humanely and care taken when carrying out the treatments. The interventions were chosen as they either represent a standard pain test, such as the acetic acid groups9,15,29 or they are routinely used for ID purposes and genomic screening such as the fin clip40 (zfin.org/zf_info/zfbook/chapt7/7.8.html) and PIT tagging41. The PIT tags used weighed 0.020 g equating to around 2% of the bodyweight of the individuals used in this study which is below the threshold weight of tags known to affect swimming performance42. Although there is evidence that benzocaine may be more aversive than metomidate43, benzocaine also acts as a local anaesthetic and so provides a period of short-term pain relief pre-operatively thus being the more ethical choice of anaesthetic during these painful treatments. The sham treated group controls for any stress associated with anaesthesia.