Automated monitoring of behaviour in zebrafish after invasive procedures

Abstract

Fish are used in a variety of experimental contexts often in high numbers. To maintain their welfare and ensure valid results during invasive procedures it is vital that we can detect subtle changes in behaviour that may allow us to intervene to provide pain-relief. Therefore, an automated method, the Fish Behaviour Index (FBI), was devised and used for testing the impact of laboratory procedures and efficacy of analgesic drugs in the model species, the zebrafish. Cameras with tracking software were used to visually track and quantify female zebrafish behaviour in real time after a number of laboratory procedures including fin clipping, PIT tagging, and nociceptor excitation via injection of acetic acid subcutaneously. The FBI was derived from activity and distance swum measured before and after these procedures compared with control and sham groups. Further, the efficacy of a range of drugs with analgesic properties to identify efficacy of these agents was explored. Lidocaine (5 mg/L), flunixin (8 mg/L) and morphine (48 mg/L) prevented the associated reduction in activity and distance swum after fin clipping. From an ethical perspective, the FBI represents a significant refinement in the use of zebrafish and could be adopted across a wide range of biological disciplines.

Welfare Challenges Influence the Complexity of Movement: Fractal Analysis of Behaviour in Zebrafish

“…Current definitions of animal pain suggest animals may respond by altering their behaviour and when animals reduce activity this may be an attempt to conserve energy to divert to the healing process but may also be similar to guarding behaviour where reduced use of a limb or area prevents further damage and pain [5]. Reduced activity is observed in mammalian responses to pain and thus the data presented here and in other studies using trout and zebrafish [23,30,31,62] suggests this response is evolutionarily conserved and underlies mechanisms to recover from injury. In the laboratory context prolonged changes in behaviour could potentially confound experimental studies [65] whereas in a natural setting abnormal behaviours may alter the risk of being detected by predators [23].…”

“…The reduction in behavioural complexity over the 6 h period, therefore, is likely to result from the tissue damage that accompanies this procedure. The potential for this procedure to be painful is also validated by another study from within our laboratory where the standard behavioural measurements (average speed, percentage tank explored and percentage time spent in the bottom half of the tank) of PIT tagged fish diverted from control behaviour to a similar degree to that observed in the 5% acid group across the 6 h experiment [62]. Previous studies have noted that in some species tagging is associated with infections, reduced feeding and increased mortality [67,68].…”

“…In this study the FD value of female zebrafish trajectories declined at a rate that scaled with the concentration of acetic acid. A previous study conducted in our laboratory [62] explored the effect of these same noxious interventions on more traditional behavioural measures including average speed (cm/s), percentage time spent in the bottom of the tank and amount of the tank explored. In that study the behavioural response of the 1% acid group was easily distinguishable from the two higher strengths as the higher strength groups had slower average speeds and spent more time on the bottom of the tank with less exploration: the difference between the 5 and 10% acid group, however, was much harder to discern but can be seen using FD analysis here.…”

“…This failure to avoid the top half of the tank by 10% Acid fish could be due to the severity of the pain being so great that it took priority over normal anti predatory or anxiety behaviour; this phenomenon has been seen in another fish species, the rainbow trout, where subcutaneous acid injection into the lips resulted in these fish not exhibiting normal fear or anti-predator responses [23,64]. The use of standard behaviour measures, therefore, required at least two behaviours (time spent in the bottom half of the tank and either average speed or percentage of tank explored) to identify a meaningful difference between the 5 and 10% acid groups [62] while here fractal analysis was able to differentiate the two groups with just one value. Current definitions of animal pain suggest animals may respond by altering their behaviour and when animals reduce activity this may be an attempt to conserve energy to divert to the healing process but may also be similar to guarding behaviour where reduced use of a limb or area prevents further damage and pain [5].…”

“But It’s Just a Fish”: Understanding the Challenges of Applying the 3Rs in Laboratory Aquariums in the UK

“…Many 3Rs-geared ambitions for zebrafish would be impossible without specialist scientific expertise. In the UK, for example, Lynne Sneddon and colleagues have published influential data from numerous experiments focused explicitly on the possibility of deriving 3Rs interventions from them, for example, in areas such as analgesia research [81,[120][121][122][123], enrichment [97] and automated welfare monitoring [124]. Academic researchers have also led in the development of protocols with direct 3Rs implications, for instance, concerning the genotyping of zebrafish by means of fin clips on very young larvae (3dpf) [125].…”