The detection, assessment and alleviation of pain in laboratory zebrafish.
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
The use of fish in scientific experiments is increasing in the UK with nearly half a million fish reported in 2010. This rise in popularity means that large numbers of fish are subject to experimental procedures that are invasive causing tissue damage that would give rise to the sensation of pain in humans and mammals. We currently know relatively little on how to detect and assess pain in fish, yet to meet ethical standards we must assess the welfare state of an animal and provide pain relief where appropriate.
This project aims to clearly identify markers of pain by using non-invasive measures of behaviour and physiology in a model species, the zebrafish, during common practices such as fin clipping and invasive tagging where tissue damage often occurs. Behavioural changes will be tracked using sophisticated monitoring software to determine changes in activity, swimming, feeding, posture, face shape and social interactions. The data will be used to develop a simple classification tool with software that classifies fish according to whether they are behaving normally or exhibiting signs of stress or pain. This invaluable assessment tool will allow researchers and animal carers to accurately determine the state of the fish and allow interventions such as the use of pain killing drugs to alleviate pain.
Zebrafish are traditionally kept in uniform tanks with no physical enrichment (e.g. substrate, refuge) and little consideration as to the social context they are placed in. Healthy animals held under better husbandry conditions with resources that allow them to display their normal behaviour and provide them with sensory stimulation recover more quickly from any negative events that cause stress or pain. Therefore, this project will explore what zebrafish prefer using choice tests and implement the top preferences in their holding tanks to determine if the presence of enrichment dramatically improves the animal's welfare and consequently enhances recovery from pain.
Providing pain relief to animals that are subjected to invasive procedures is routine in mammals, however, relatively little is known about the effectiveness of pain killing drugs in fish. It is impractical to inject drugs into small species such as zebrafish and often experiments involve large numbers of fish. Therefore, we propose to investigate the use of a range of pain killing drugs by dissolving them in the fishes' tank water and determining whether they reduce the symptoms of pain. A range of doses will be explored to identify the most effective dose and drug that can be applied routinely during invasive experiments in fish.
Overall, this project will provide robust, new criteria to assess the level of pain experienced in fish by integrating different types of measurement, including behaviour and physiology. It will show how stress affects these measures of pain, and it will help us define holding conditions and pain-killing drugs that best protect fish the discomfort that they might experience when being used in experiments. We believe that the outcome of this work could have practical value to scientific community and should help refine and reduce the severity of experiments for an estimated 300,000 fish annually in the UK and possibly millions of fish worldwide.
This project aims to clearly identify markers of pain by using non-invasive measures of behaviour and physiology in a model species, the zebrafish, during common practices such as fin clipping and invasive tagging where tissue damage often occurs. Behavioural changes will be tracked using sophisticated monitoring software to determine changes in activity, swimming, feeding, posture, face shape and social interactions. The data will be used to develop a simple classification tool with software that classifies fish according to whether they are behaving normally or exhibiting signs of stress or pain. This invaluable assessment tool will allow researchers and animal carers to accurately determine the state of the fish and allow interventions such as the use of pain killing drugs to alleviate pain.
Zebrafish are traditionally kept in uniform tanks with no physical enrichment (e.g. substrate, refuge) and little consideration as to the social context they are placed in. Healthy animals held under better husbandry conditions with resources that allow them to display their normal behaviour and provide them with sensory stimulation recover more quickly from any negative events that cause stress or pain. Therefore, this project will explore what zebrafish prefer using choice tests and implement the top preferences in their holding tanks to determine if the presence of enrichment dramatically improves the animal's welfare and consequently enhances recovery from pain.
Providing pain relief to animals that are subjected to invasive procedures is routine in mammals, however, relatively little is known about the effectiveness of pain killing drugs in fish. It is impractical to inject drugs into small species such as zebrafish and often experiments involve large numbers of fish. Therefore, we propose to investigate the use of a range of pain killing drugs by dissolving them in the fishes' tank water and determining whether they reduce the symptoms of pain. A range of doses will be explored to identify the most effective dose and drug that can be applied routinely during invasive experiments in fish.
Overall, this project will provide robust, new criteria to assess the level of pain experienced in fish by integrating different types of measurement, including behaviour and physiology. It will show how stress affects these measures of pain, and it will help us define holding conditions and pain-killing drugs that best protect fish the discomfort that they might experience when being used in experiments. We believe that the outcome of this work could have practical value to scientific community and should help refine and reduce the severity of experiments for an estimated 300,000 fish annually in the UK and possibly millions of fish worldwide.
Technical Summary
The use of fish as experimental models has resulted in a significant increase in the numbers used in licensed procedures in the UK (+23% in 2010). Fish are subject to many routine procedures that result in tissue damage that would give rise to pain in mammals. For example, the removal of tissue in fin clipping for identification purposes and genetic screens; invasive tagging where tags are introduced into the body cavity; scraping of skin for disease screening; and invasive surgery with recovery. This project will identify robust behavioural and physiological indicators to detect and assess pain in a model species, the zebrafish, and use the data collected to develop a simple, cost effective monitoring tool. Video recordings of fish subject to four potentially painful procedures shall be compared with undisturbed and sham treated fish along with non-invasive physiological measures of stress (ventilation rate and water cortisol). An intelligent monitoring system will be produced that can classify individuals according to behavioural measures and will provide an unequivocal means of determining whether fish are behaving normally, are stressed or experiencing pain. This project will also determine what preferences zebrafish have for physical enrichments and social context using a choice chamber paradigm. Once the top preferences are identified these shall be investigated as a means of improving welfare and the recovery from pain by comparison with fish held under standard, barren conditions. Finally, a range of immersion analgesic and local anaesthetic drugs shall be tested to examine whether they effectively reduce the indicators of pain in zebrafish and what the effective dose is.
Planned Impact
Success in this project will provide new knowledge to improve the practical aspects of animal experimentation using fish, specifically in providing a greater understanding of what constitutes pain and suffering, and which experimental and husbandry procedures minimise suffering and promote coping strategies for pain. These refinement procedures can be adopted and implemented by others leading to improvement of conditions and management of invasive procedures during experimentation.
To our knowledge, no other research group is tackling this important subject and, therefore, the results from this project will be entirely novel. The development of an intelligent monitoring system that can unequivocally categorise fish as normal, stressed or in pain would substantially improve welfare assessment in our laboratory and allow researchers more time to conduct experiments. The development of an automated tool for the continuous monitoring of fish behaviour has commercial potential. Provided that the technical approach can be reduced to an inexpensive form with easy-to-use methodology for handling the resulting data, then any laboratory undertaking fish experimentation could monitor the animals used in invasive experimental manipulations. This would at least indicate the scale of likely problems, but if used widely could be used to monitor every specimen used in a particular experiment or procedure. Given that the resulting insights relate to the normal behavioural repertoire of the subjects, then this equipment could also be used by aquaculturists and hobbyists, greatly expanding the market. A prototype demonstration unit with embedded software and appropriate information extraction algorithms will be evaluated during the project. If appropriate this knowledge and know-how will be transferred to an SME (MHA-IES Ltd) who have previously been involved with technology transfer from the Centre for Intelligent Monitoring Systems (CIMS) research group and they are currently manufacturing equipment transferred by CIMS. The technology transfer will be covered by a licence agreement and will give MHA access to the IPR.
Cortisol is widely used as an indicator of stress and can be determined, even in low concentrations, by immunoassay. Automated "lab-on-a-chip" based tests offers a novel and more rapid approach to the analysis of cortisol. Other options include the development of aptamer probes (BBSRC project for this technology is underway) and of direct analysis by cathodic stripping voltammetry for aqueous detection (Dr Van den Berg in the School of Environmental Sciences, holds IP for this technology). This provides a suite of potential technical approaches to the continual and much less expensive forms of analysis for this important hormone and would present an instantaneous measure of status in experimental fish.
The University of Liverpool has an active IP exploitation model that provides University control and support to offer flexibility in exploiting the value of IP and maximising impact from the academic activity. This is supported by the University's IP exploitation partner: Impact Science. This project, as with all projects with commercial potential, will be discussed with Impact Science who will evaluate the IP potential, submit a proposal to the IP Operations Team and decide the best Exploitation Strategy usually by licensing by market the IP to companies who they believe would best develop the products in the target markets or by the creation of a spin-out company.
To our knowledge, no other research group is tackling this important subject and, therefore, the results from this project will be entirely novel. The development of an intelligent monitoring system that can unequivocally categorise fish as normal, stressed or in pain would substantially improve welfare assessment in our laboratory and allow researchers more time to conduct experiments. The development of an automated tool for the continuous monitoring of fish behaviour has commercial potential. Provided that the technical approach can be reduced to an inexpensive form with easy-to-use methodology for handling the resulting data, then any laboratory undertaking fish experimentation could monitor the animals used in invasive experimental manipulations. This would at least indicate the scale of likely problems, but if used widely could be used to monitor every specimen used in a particular experiment or procedure. Given that the resulting insights relate to the normal behavioural repertoire of the subjects, then this equipment could also be used by aquaculturists and hobbyists, greatly expanding the market. A prototype demonstration unit with embedded software and appropriate information extraction algorithms will be evaluated during the project. If appropriate this knowledge and know-how will be transferred to an SME (MHA-IES Ltd) who have previously been involved with technology transfer from the Centre for Intelligent Monitoring Systems (CIMS) research group and they are currently manufacturing equipment transferred by CIMS. The technology transfer will be covered by a licence agreement and will give MHA access to the IPR.
Cortisol is widely used as an indicator of stress and can be determined, even in low concentrations, by immunoassay. Automated "lab-on-a-chip" based tests offers a novel and more rapid approach to the analysis of cortisol. Other options include the development of aptamer probes (BBSRC project for this technology is underway) and of direct analysis by cathodic stripping voltammetry for aqueous detection (Dr Van den Berg in the School of Environmental Sciences, holds IP for this technology). This provides a suite of potential technical approaches to the continual and much less expensive forms of analysis for this important hormone and would present an instantaneous measure of status in experimental fish.
The University of Liverpool has an active IP exploitation model that provides University control and support to offer flexibility in exploiting the value of IP and maximising impact from the academic activity. This is supported by the University's IP exploitation partner: Impact Science. This project, as with all projects with commercial potential, will be discussed with Impact Science who will evaluate the IP potential, submit a proposal to the IP Operations Team and decide the best Exploitation Strategy usually by licensing by market the IP to companies who they believe would best develop the products in the target markets or by the creation of a spin-out company.