Investigation of behavioural and physiological responses to fin-clipping in zebra fish
Lead Research Organisation:
University of Edinburgh
Department Name: Biological Services
Abstract
Zebra fish are extensively used as research animals. Use of zebra fish as experimental animals is increasing, mainly because zebra fish have particular characteristics that make them very suitable for genetic research on embryo development and are becoming popular as a replacement for mammals in biomedical, pharmacological and genetic research. Genetic analysis requires a tissue sample, which in zebra fish is obtained through fin clipping, i.e. the surgical removal of part of the caudal fin. This procedure is carried out under general anaesthesia but without the provision of pain relief following the procedure. Thus, there is potential for fish undergoing this procedure to experience pain once they wake up. Recent studies have provided anatomical, neurophysiological and behavioural evidence for the capability of different species of fish (including zebra fish) to experience pain. Tailfin clipping has been proven to induce pain-associated responses in various fish species. However, the potential of fin clipping to be painful and therefore produce changes in the behaviour and physiology of zebra fish has not been investigated. As large numbers of zebra fish undergo this procedure every year we hope this study will help refine this procedure if required and improve fish welfare in research.
By video-recording and analysing the behaviour and measuring physiological parameters (such as operculum/respiratory rate and non-invasive stress hormone -cortisol- measurement) of tailfin-clipped fish and comparing these parameters with appropriate control fish, we would identify potential behavioural and physiological indicators that might reflect a painful response to the injury.
We will also develop behavioural tests which would assess if fin clipping evokes an emotional response in zebra fish, manifested as changes in motivation and attention. These tests will measure, for example, the fear response to a novel object and the use of enrichment items such as an opaque cover, both of which have been reported to be decreased in fish suffering from pain.
The final part of the study would aim at investigating the effect of painkillers (i.e. morphine) on the behavioural and physiological pain indicators identified in the first part of the study, in order to validate that these indicators are indeed caused by pain.
The results from this study could potentially influence future policies for tissue collection for genetic analysis and lead to the development of refinements for this procedure, for example with the use of appropriate pain relief. Pain indicators identified during this study could be used in further studies and could also be integrated into pain assessment protocols, monitoring systems and as parameters for assessing painkiller efficacy. Findings from this study could also be implemented in other potentially painful procedures which would result in improving the welfare of laboratory fish worldwide.
By video-recording and analysing the behaviour and measuring physiological parameters (such as operculum/respiratory rate and non-invasive stress hormone -cortisol- measurement) of tailfin-clipped fish and comparing these parameters with appropriate control fish, we would identify potential behavioural and physiological indicators that might reflect a painful response to the injury.
We will also develop behavioural tests which would assess if fin clipping evokes an emotional response in zebra fish, manifested as changes in motivation and attention. These tests will measure, for example, the fear response to a novel object and the use of enrichment items such as an opaque cover, both of which have been reported to be decreased in fish suffering from pain.
The final part of the study would aim at investigating the effect of painkillers (i.e. morphine) on the behavioural and physiological pain indicators identified in the first part of the study, in order to validate that these indicators are indeed caused by pain.
The results from this study could potentially influence future policies for tissue collection for genetic analysis and lead to the development of refinements for this procedure, for example with the use of appropriate pain relief. Pain indicators identified during this study could be used in further studies and could also be integrated into pain assessment protocols, monitoring systems and as parameters for assessing painkiller efficacy. Findings from this study could also be implemented in other potentially painful procedures which would result in improving the welfare of laboratory fish worldwide.
Technical Summary
The zebrafish are housed in a purpose built Home Office accredited fish facility. The fish are housed in techniplast(TM) tanks at stocking densities of 3-10 fish per tank. Individual fish may be identified with the aid of elastomer tags (NorthWest Marine Technology Inc.). Fish husbandry practices (water parameters, nutrition and health surveillance) are in accordance with published guidelines (http://zebrafish.org, RSPCA 2010).
Zebrafish will be filmed following tail fin clipping. The fish will be anaesthetised by immersion in MS222 (tricaine methanesulfonate). The caudal (bi-lobed) part of the tail fin will be resected using a scalpel blade or sharp scissors. The fish will then be returned to holding tanks and either housed singly (as is standard procedure following fin clipping for identification purposes) or in groups (in order to assess changes in social behavior which might be associated with pain). The behaviour over the following 6-12 hours will be recorded and analysed with the aid of computer tracking software. The same recordings will be used to measure physiological parameters (such as operculum/respiratory rate). For the cortisol assay, the tank water will submitted for cortisol level assays after 24 hours from fin-clipping.
A second group of fish will undergo a similar procedure as described above but will be provided with analgesics (opioids e.g. morphine or NSAID e.g. fluribiprofen) systemically in bathing water or topically on the wound. To validate this model the behaviour of the above fish will be compared to three control groups:a group of fish anaesthetised but without undergoing fin clipping, a group of fish anaesthetised and fin-clipped and a group of fish exposed to analgesics but without fin clipping.
Similar treatment groups will be used in the behavioural tests using environmental enrichment or novel objects.
Reference:
RSPCA report (2010) Guidance on the housing and care of zebrafish, Danio rerio
Zebrafish will be filmed following tail fin clipping. The fish will be anaesthetised by immersion in MS222 (tricaine methanesulfonate). The caudal (bi-lobed) part of the tail fin will be resected using a scalpel blade or sharp scissors. The fish will then be returned to holding tanks and either housed singly (as is standard procedure following fin clipping for identification purposes) or in groups (in order to assess changes in social behavior which might be associated with pain). The behaviour over the following 6-12 hours will be recorded and analysed with the aid of computer tracking software. The same recordings will be used to measure physiological parameters (such as operculum/respiratory rate). For the cortisol assay, the tank water will submitted for cortisol level assays after 24 hours from fin-clipping.
A second group of fish will undergo a similar procedure as described above but will be provided with analgesics (opioids e.g. morphine or NSAID e.g. fluribiprofen) systemically in bathing water or topically on the wound. To validate this model the behaviour of the above fish will be compared to three control groups:a group of fish anaesthetised but without undergoing fin clipping, a group of fish anaesthetised and fin-clipped and a group of fish exposed to analgesics but without fin clipping.
Similar treatment groups will be used in the behavioural tests using environmental enrichment or novel objects.
Reference:
RSPCA report (2010) Guidance on the housing and care of zebrafish, Danio rerio
Planned Impact
Zebrafish are increasingly used as a model for biomedical research. Some may perceive the use of fish instead of rodents as a replacement or refinement on their experiments. However in the last decade these perceptions are changing with publications on pain perception in fish (Braithwaite & Boulcott 2007) and consensus meetings on the use of fish in research (Norecopa 2005 & 2009, www.norecopa.no). Despite zebrafish being a popular animal model the majority of literature on pain research so far is on larger fish species such as Salmonids.
The results from this study would contribute to building a body of evidence regarding the welfare implications of fin clipping in zebra fish.
The results from our study will provide information regarding the individual variation, sensitivity and validity of behavioural and physiological parameters and their usefulness as pain indicators as part of monitoring and assessment protocols.
The findings from this pilot study and subsequent studies will enable us to provide a more scientific based advice to our policy makers. One such example is the recommendations for severity classification of procedures in fish for the current European directive (Hawkins et al 2011).
Outcomes from this study will also influence current practices in zebrafish research within our institution, such as future policies for tissue collection for genotyping and the development of refinements of existing procedures (i.e. use of analgesic drugs). This is also expected to have an impact in the wider research community, following dissemination of our results through peer reviewed publications, meetings and relevant bodies such as Institute of Animal technology.
The findings in this project will have a wider impact beyond the welfare of fish in research. Behavioural parameters found to be of use in identifying pain can be used in Aquaculture and pet trade. The use of analgesics both in research and veterinary practice is sporadic, based often on anecdotal information and in large fish species. Any analgesic found to be of use in zebrafish could be used by both communities.
References:
Braithwaite V.A. and Boulcott P. (2007). Pain perception, aversion and fear in fish. Diseases of Aquatic Organisms. 75: 131-138.
Hawkins P, Dennison N., Goodman G., Hetherington S., Llywelyn-Jones S., Ryder K. and Smith A.J. (2011). Guidance on the severity classification of scientific procedures involving fish: report of a working group appointed by the Norwegian Consensus Platform for the Replacement, Reduction and Refinement of animal experiments. Laboratory Animals 45: 219-214.
The results from this study would contribute to building a body of evidence regarding the welfare implications of fin clipping in zebra fish.
The results from our study will provide information regarding the individual variation, sensitivity and validity of behavioural and physiological parameters and their usefulness as pain indicators as part of monitoring and assessment protocols.
The findings from this pilot study and subsequent studies will enable us to provide a more scientific based advice to our policy makers. One such example is the recommendations for severity classification of procedures in fish for the current European directive (Hawkins et al 2011).
Outcomes from this study will also influence current practices in zebrafish research within our institution, such as future policies for tissue collection for genotyping and the development of refinements of existing procedures (i.e. use of analgesic drugs). This is also expected to have an impact in the wider research community, following dissemination of our results through peer reviewed publications, meetings and relevant bodies such as Institute of Animal technology.
The findings in this project will have a wider impact beyond the welfare of fish in research. Behavioural parameters found to be of use in identifying pain can be used in Aquaculture and pet trade. The use of analgesics both in research and veterinary practice is sporadic, based often on anecdotal information and in large fish species. Any analgesic found to be of use in zebrafish could be used by both communities.
References:
Braithwaite V.A. and Boulcott P. (2007). Pain perception, aversion and fear in fish. Diseases of Aquatic Organisms. 75: 131-138.
Hawkins P, Dennison N., Goodman G., Hetherington S., Llywelyn-Jones S., Ryder K. and Smith A.J. (2011). Guidance on the severity classification of scientific procedures involving fish: report of a working group appointed by the Norwegian Consensus Platform for the Replacement, Reduction and Refinement of animal experiments. Laboratory Animals 45: 219-214.