Maintenance of colonies of established genetically altered animals, not used in other procedures
Objectives and predicted benefits of the project
Describe the objectives of the project (for example, addressing certain scientific unknowns, or scientific or clinical needs).
The research group uses zebrafish as a model to investigate chemosensory computations, functional development of the brain, and neural circuits mechanisms underlying neurological disease. A number of genetically modified and wild-type zebrafish lines are bred and maintained in the zebrafish facility at the institute.
In the management of such a colony, humane killing of zebrafish will be necessary, either to prevent overpopulation, to reduce lines that are no longer of scientific interest, to remove individuals that are getting old or to remove individuals with signs of disease or injury so that the health of the colony is maintained.
As of today, the group has around 200 zebrafish lines that are housed and maintained in the zebrafish facility. It is estimated that up to 40 000 fish may need to be euthanized during a 4-year period which is the duration of this application.
What are the potential benefits likely to derive from this project? Explain how science could be advanced, or humans, animals or environment may ultimately benefit from the project. Where applicable, differentiate between short-term benefits (within the duration of the project) and long-term benefits (which may accrue after the project is finished).
The current regulation lists a number of methods approved for euthanasia of fish. These methods are not always useful for tropical fish with the size of zebrafish. In addition, there are scientific publications that present evidence that the use of ice water (below 4 °C) is a quicker, less stressful and more humane euthanasia method for zebrafish. In this application we apply for permission to use ice water as a method for humane euthanasia of zebrafish.
Tropical fish are not able to compensate for sudden transitions to low-temperature water. When zebrafish are placed in ice water (2 – 4 °C), hypothermal shock is induced and the fish lose consciousness within a few (0 – 5) seconds. For comparison, an overdose with an anesthetic agent in the water appears to be more stressful and the process lasts longer. This is supported by several scientific publications. The potential benefits include better welfare of laboratory animals with a reduced stress preceding death and a faster death, lower risk of chemicals exposure for users of the facility, and reduced production of chemical waste.
Predicted harms
In what procedures will the animals typically be used (for example, injections, surgical procedures)? Indicate the number and duration of these procedures.
No procedures will be done on the fish other than the euthanasia.
What are the expected impacts/adverse effects on the animals, for example pain, weight loss, inactivity/reduced mobility, stress, abnormal behaviour, and the duration of those effects?
The procedure is to euthanize the animals
What species and numbers of animals are expected to be used? What are the expected severities and the numbers of animals in each severity category (per species)?
Species
Total number
Estimated numbers per severity
Non recovery
Mild
Moderate
Severe
Zebra fish (Danio rerio)
40000
40000
0
0
0
What will happen to the animals kept alive at the end of the procedure?
Species
Estimated numbers of animals to be reused, to be returned to habitat/husbandry system or to be rehomed
Reused
Returned
Rehomed
Please provide reasons for the planned fate of the animals after the procedure.
The euthanised animals are transferred to the freezer after confirmation of death and eventually disposed in biological waste.
Application of the Three Rs
1. Replacement
State which non-animal alternatives are available in this field and why they cannot be used for the purposes of the project.
The group has established a set of experiments that can be done on harvested tissues (e.g. brain explants), and such experiments are considered as replacement alternatives. However, it is a relative replacement, since living animals are needed to produce intact brains - there are no cell culture methods that can resemble the complexity of the network in an intact brain. It is necessary to keep a colony of zebrafish in order to collect brain tissue post mortem and to perform behavior experiments in vivo described in other FOTS applications. Euthanasia of zebrafish is necessary as a part of the management of the colony.
2. Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce the number of animals to be used, and principles used to design studies. Where applicable, describe practices that will be used throughout the project to minimise the number of animals used consistent with scientific objectives. Those practices may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
An important step in minimizing the number of animals that need to be euthanized is to limit the number of fish lines to those that are of scientific interest and avoiding excess production of each line. Evaluating the fish lines is a continuous process, and once it has been decided to eliminate one or more lines, the breeding of these lines will stop and the reduction will take place as soon as possible. Sperm freezing and in vitro fertilisation has been implemented in the laboratory to restrict the number of lines being actively maintained in the facility.
3. Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals. Describe the mechanisms to take up emerging refinement techniques during the lifetime of the project.
We are housing our fish in a room of 70 m2 size, which is air-conditioned and light cycled 14/10. We have a state of the art zebrafish housing system that is composed of 650 fish tanks. Each tank has an individual and a continuous supply of water. The water is based on reverse osmosis (RO) water which is filtered and UV-disinfected before it is supplied to the fish tanks. It is possible to adjust the amount of water to be recycled and the amount of freshly produced RO water. The housing system sends electronic alarms to the group members in case of a technical malfunction. This housing system automatically tests and adjusts water quality regularly. The group also does manual testing of tank water to keep control of the values of nitrogen compounds, temperature, pH, and conductivity. Moreover, we hired experienced technical staff to feed, health check, and maintain our fish on a regular basis. We believe all these measures ensure the well-being of our fish. The fish are housed in stable groups and feed three times per day. During one of the feedings, the fish are given live Artemia, which is considered an environmental enrichment.
During the euthanasia procedure, it is important to avoid stress in the fish to ensure that the process is fast.
Therefore, the fish is kept undisturbed in their home environment for as long as possible before being transferred to the euthanasia setup.
We have gained extensive experience for hypothermic euthanasia in our laboratory and have standardised protocols.
Explain the choice of species and the related life stages.
Zebrafish after 5 days post fertilisation will be euthanised using this method.
Project selected for Retrospective Assessment
Project selected for RA?
No
Deadline for RA
Reasons for retrospective assessment
Contains severe procedures
Uses non-human primates
Other reason
Explanation of the other reason for retrospective assessment
Additional fields
National field 1
Field will not be published.
National field 2
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National field 3
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National field 4
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National field 5
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Project start date
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Project end date
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Project approval date
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ICD code 1
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ICD code 2
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ICD code 3
Field will not be published.
Link to the previous NTS version outside the EC system