Dear All,
The Animal Ethics Committee of The University of Adelaide has asked me to investigate whether there is any known way of reducing the stress/distress/pain caused to zebrafish by ENU treatment. For example, has anyone investigated the use of low levels of Tricaine together with ENU?
Any comments or suggestions with relevance to this subject would be very gratefully received.
Thank you for all the very helpful replies that I received about the use of ENU and relieving the stress to fish caused by ENU treatment. Since this information is important and since many of the replies were to me directly, I have edited the replies (to preserve anonymity) and am sending them back out to the zf community.
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I found during our mutagenesis that males were much less stressed if temperature was carefully regulated. We performed mutagenesis at 21C and kept mutagenesis cages in a water bath (if room temp is above 21, this can be done quite easily with a large tray and a bit of ice - once the water reaches 21 it is quite stable). After fish are in the rinse cages, they are placed in the fish room and allowed to equilibrate to the higher temp, over a period of several hours. Our survival was better than 90%, with no appreciable change in hit rate. Under these conditions, the fish are much less stressed.
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Tricaine is an anesthetic. Keeping the zebrafish under a "light" anesthesia is not going to do much for the pain/distress. The anesthetic would allow the animal to 'ignore' the pain but wouldn't do much to 'relieve' the pain.. What you really want is an analgesic to deal with the chronic pain that the ENU would induce. Any of the morphine derivatives are the drugs of choice when ENU is given to mice. However, I am not familiar with the literature enough to know whether the ENU and morphine derivatives interact in mice. Unless you are interested in ENU enhancing tumor formation in the nervous system, I wouldn't worry too much about the interactions. I don't think there are morphine receptors outside the nervous system. My literature search suggests that people also haven't used morphine derivatives in zebrafish.
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I would ask what evidence is there that the animals are experiencing any pain or stress from the doses of ENU that you may be using? Of course any handling of fish will in itself induce some stress, and there may be justification in trying to reduce this using tricaine of salt solutions (check the salmonid fish literature for stress reduction). The question is, are the chemicals you are using causing additional stress or pain, or how could one even attempt to assess this question? I may note that we have ... experience in chemical carcinogenesis research with rainbow trout, medaka, and zebrafish .... Exposure protocols have involved embryonic injection, i.p. injection, egg or fry water bath exposure, and dietary treatments. We have observed no behavioural changes that would suggest stress in the animals due to the chemical that is being used, unless we approach acute toxicity. I do not believe that an a priori presumption that steps must be taken to relieve pain or stress is justified, in the absence of some indication that pain/stress is actually occurring. In any event, I see no reason you could not use mild tricaine treatment since it should not interfere with a direct mutagen such as ENU; there would be mechanistic concern for premutagens requiring metabolic activation. We use tricaine for operations such as ip injection or partial hepatectomy with our trout where the potential for pain is obvious; there is no requirement by our animal welfare committee for such treatment for the great majority of our work involving dietary or bath exposures because there is no overt evidence of stress or pain.
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We have mutagenized without apparent stress, and 100% survival, with effective doses. There is no need for Tricaine. We do mutagenesis in dim light, moving slowly, no slammed doors, and gentle handling using mesh-bottom tanks. Fish do not seem stressed, and we obtained the desired high rates of mutagenesis.......It is very healthy to have respect for ENU, since a lab that was careless with it managed to kill a couple scientists. However, with proper precautions, doing a "dry run" with all solutions and materials is very helpful. Sodium hydroxide renders the stuff inactive within a fraction of a second, since it is luckily very unstable at high pH. It is important to not be at all rushed during the procedure. ....... I dissolved the stuff myself (it's very slow to go into water, and needs to be physically mashed with a small spatula against the side of the glass. I use it within the 1/2 hour of dissolution.
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We have been going through ethical committee applications for a screen we are setting up here. The committee were reasonably straightforward to deal with simply by quoting procedures from the zebrafish book. That seemed to suffice. The survival rate in males from ENU treatment seems to be very high especially with using Stresscoat afterwards.
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We found ......... that the best way to reduce stress on the fish during treatment is to keep everything very quiet (no banging doors etc.), shield the hood with cardboard, and transfer them slowly to the bathing solution after the treatment. That way I got most of the fish to survive (with the 3x 1h treatment protocol).
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