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The stress response common to nearly all fish species when handled and disturbed is reviewed. Several stress-mitigating techniques are described and insights provided to illustrate how they work
in coolwater fish species. Some key handling precautions are discussed to help minimize discomfort to fish used in research or other procedures.
In the case of fish species occupying higher trophic levels than some of the classic lab species such as goldfish, carp, minnows and zebrafish, it is often problematical to find an entry reference for animal care literature. Yet, there are many species of fresh and saltwater fish whose predatory habits and ecological specialization makes them well-suited to research protocols where a more sensitive organism provides a clearer result. Species receiving some attention in this category include various salmon and trout, pike/pickerel, cod, flatfish species, and tuna/mackerel.
Increasingly, medical and environmental research is relying on fish as alternatives to more expensive and/or higher orders of research animals. Fish have been identified either as good models for responses in higher orders, e.g., in cancer research, or as the most sensitive indicator available of toxicity and low-level chemical factors (Wolke, 1984). This change in direction means researchers and animal care staff may find themselves in unfamiliar territory when it comes to routine handling of their animals. Fortunately, there exists a considerable body of useful information interspersed among the scientific literature relating to fish culture and stock rebuilding and replacement.
The sheer diversity of research procedures precludes fish care comment beyond broad generalities. However, nearly all fish protocols share a requirement for physically handling the animals. In this article, I will review some of the main handling considerations in research protocols using fish less tolerant of abrupt change. I hope users of fish in research, teaching, and testing will find some of the following information useful and thought provoking.
Fish respond to handling as a stress. Their stress response is practically
speaking, like our own; upon perception of the stress by the nervous system,
adrenaline is released into the bloodstream.
This hormone is followed
closely by other steroids such as cortisol, which prepare the fish for its
reaction, e.g., escape (Mazeaud and Mazeaud, 1981). The result is blood glucose
levels, red blood cell counts, heart and ventilation rates all increase, and
digestive processes may cease temporarily. Once triggered, even by a very
transient stress such as being dipnetted in air from one tank to another, the
sequence plays through in proportion to the severity and duration of the
stressor.
Although a fish may not suffer from temporarily increased blood glucose or red blood cell counts, other of the stress response components have significant drawbacks. Loss of appetite can persist following a handling stress (Wedemeyer, 1976), and normal reproductive functions may be suppressed for considerable time (Carragher and Sumpter, 1990). Adrenaline disturbs ion transport at the gill membrane, and both adrenaline and cortisol cause temporary changes in gill permeability which, in fresh water, results in dilution of the blood by excessive gain of water, and vice versa in normal seawater (Mazeaud et al., 1977; Folmar and Dickhoff, 1980). Consequently, blood levels of calcium, magnesium, sodium, and other vital electrolytes are pushed out of normal operating ranges for as much as 24 hours after a brief stress such as dipnetting (Wedemeyer, 1972). The burden of trying to restore physiological and metabolic order diverts precious energy, which may leave the fish less capable of fighting pathogens. Cortisol elevation itself suppresses immune system function (Barton et al., 1987; Maule et al., 1987). A frequent sign of poor handling practices in fish is subsequent infection.
Thus, it would appear if one can keep the fish from perceiving a disturbance when handling is necessary, many problems can be averted. This may not be as difficult as it sounds. In some instances, conditioning to handling has been accomplished by associating presentation of a dipnet with daily feeding. Actual netting out, when it occurs, is then only a minor departure from routine. A variation of this is to use a flashing light to signal feeding time; the light flashing before any other essential procedures then takes advantage of the conditioning and is not limited to a particular action.
Fish are more comfortable under low light and exposure conditions. Therefore, if they must be held interim in a small container, simply covering it to exclude light can be comforting. This observation, as well as numerous related considerations in working with fish is listed in Hubbs, et al., (1988). In chinook salmon undergoing simulated transport in small containers, exclusion of light reduced the hormonal response to stress by about 25 percent over unprotected fish (Wedemeyer, 1985). Many who work with fish find domestic plasticware such as pails and storage containers very useful. Simply covering the container when fish are inside will make an appreciable difference to the occupants.
Sedation is another option for reducing sensory awareness. A wide range of waterborne anaesthetics have been used in fish culture and research to induce various planes of anaesthesia (reviewed in Bell, 1967, and McFarland and Klontz, 1969). Most applications minimize stress while out of water for some reason, or to reduce pain and immobilize fish during a procedure. Sedation (light anaesthesia) which allows fish to maintain equilibrium, swimming, and breathing has been used in some cases to mitigate stress in fish transports, and has been suggested as a means to reduce metabolic rates over unsedated fish (Piper et al., 1982).
Deriving the most value from sedation, however, is a matter of timing and chemistry. Studies involving crowding, capture, dipnetting, and deep anesthesia, have shown that it is during the initial crowding stage when most of the fish's stress response is invoked (Strange and Schreck, 1978; Barton et al., 1980; Matthews et al., 1986). Simply adding sedative to the water in a transport container is therefore something of a catch-up effort. Other research has shown that while many anaesthetics are effective for rapid induction of deep anaesthesia, some of those most widely used, e.g., tricaine methane sulfonate (MS-222) and 2-phenoxy ethanol, have an excitatory effect during initial absorption by the fish (Davis et al., 1982; Barton and Peter, 1982), which largely defeats the purpose of calming fish which are to remain conscious.
Many established fish anaesthetics are central nervous system depressants, which contribute to the transient stress during induction. A few fish anaesthetics, however, are classed as hypnotics, and do not have an excitatory impact. These are etomidate and its analogue, metomidate. Initial studies with these compounds indicated their suitability for use in fish based on wide safety margins, lack of electrolyte disturbance, and minimal elevation of cortisol in striped bass and channel catfish (Davis et al., 1982; Limsuwan et al., 1983).
Kreiberg and Powell, (1991) assessed the value of metomidate for cushioning handling impact on chinook salmon, a species which can suffer loss of health and condition after seemingly minor handling procedures. Quietly introducing stock solution of metomidate to give a sedating dose in the fishes' home tank, we found that blood cortisol response to stress of mild crowding and dipnetting in air was negligible relative to untreated control fish (Figure 1).
FIGURE 1 In subsequent experience at our lab, we found that taking five minutes to pre-treat fish with metomidate introduced as benignly as possible into home tanks allowed for much improved collection and sampling processes. Both researchers and fish benefitted from this effort. Researchers experienced improved quality of work and fish enjoyed improved quality of life. Prospective users of fish chemicals and drugs should always familiarize themselves with local regulatory conditions and occupational health information before using any compound.
Further relief from handling stress can be achieved by keeping fish in their home element as much as possible. When transferring fish between containers, it may seem intuitive to use a dipnet to catch and move the fish. With some fish species however, there can be a heavy price for this convenience. Use of in-water transfers reduced 96-hr mortality from a level of 82 percent to 15 percent in American shad (Murai et al., 1979) and reduced long-term mortality from 52 percent to 15 percent in Atlantic salmon (Flagg and Harrell, 1990), over controls transferred in air by dipnet. Survival after dipnetting in air is a complex affair, depending on a variety of factors which vary from one instance to the next. There is a clear advantage to use in-water transfers, even if the benefit is less dramatic than reduced mortality. Nets may still be used to crowd and guide the fish into a scoop or other device, as the damage clearly occurs when the fish are no longer supported by their native element.
Said native element can also be improved upon for purposes of handling. Fish maintain their blood and tissues at a salinity intermediate between fresh and normal seawater. Therefore, they constantly expend some energy in this respect, except when in brackish water of similar osmotic pressure. In their gills, only a cell-layer about the thickness of a red blood cell separates vascular system contents from the outside environment. The provision of brackish water surroundings when fish are likely to be stressed has definite advantages, both in conserving energy for physiological emergency purposes, and in countering the electrolyte disturbances associated with stress-elevated adrenaline and cortisol levels described above. The presence of salts does not repress stress hormones, but softens their side-effects.
The utility of this improvement has been demonstrated in various fish species dwelling in fresh or salt water, as well as those which can inhabit both, including threadfin and American shad (Murai et al., 1979; Collins and Hulsey, 1963), blueback herring (Guest and Prentice, 1982), yellowfish and bass (Hattingh et al., 1975), muskellunge (Miles et al., 1974), rainbow and brown trout (Haswell and Thorpe 1982; Nikinmaa et al., 1983), chinook salmon (Long et al., 1977), and steelhead trout and coho salmon (Wedemeyer, 1972). In some cases, immediate improvements were striking: greatly improved survival following a transport procedure. In other cases, the benefits were most apparent in reduced disturbance of electrolyte balance. In all cases however, fish undergoing a stressful procedure with a coincident mild salt exposure came through the ordeal in superior physiological condition for coping with subsequent challenges. Undoubtedly, the experience itself was also less traumatic.
Using water of moderate salinity to mitigate stressful procedures with fish is effective regardless of whether the fish are coming from salt or fresh water, and likewise independent of the destination salinity. In cases where fish will be returned to fresh water, it will be beneficial if they can be given a recovery period of one to two days in brackish water. This serves to reduce osmotic stress and provides a reservoir for essential ions which the fish may need to replace. One should be careful however, not to take too simplistic an approach and attempt to create salinity with a single salt such as pure sodium chloride. Ocean seawater relies on several ions for its characteristic salinity. Therefore, it is best to provide as full a spectrum as possible. In fact, higher salinity created by only one ion may be harmful. Natural or artificial seawater provides the best mixture. A general recipe may be found in Spotte, (1970). Alternately, concentrations of single ions or salts reflecting their occurrence in natural seawater may serve (for sodium chloride, this will be of the order 0.5 to one percent in aqueous solution, to achieve a final salinity of about half that of normal seawater).
Choice of anaesthetic is often determined by a researcher based on the particular procedure for which a fish may be destined. Reviews of anaesthetics for fish such as McFarland (1960), Bell (1967), McFarland and Klontz (1969), and Ross and Ross (1984), provide a wide variety of options, often with species toxicity and safety margin information which help prevent needless stress and mortality to fish. However, regulatory acceptance of a drug may be the pivotal criterion for its selection, and in this respect MS-222 may continue to find relatively wide use in fish. It is worth noting that stock solutions of MS-222 are strongly acidic in soft fresh water, with potential for gill irritation to fish. In the absence of natural buffering, such as provided by normal seawater, it is valuable and considerate to buffer solutions with sodium bicarbonate (Allen and Harman, 1970) or sodium hydroxide (Wedemeyer, 1970). Other toxicity problems can arise with MS-222 and closely related benzocaine if the stock solution is exposed to sunlight.
Given the acute sensitivity of fish to their surrounding water temperature, it may be tempting to try to throttle back their metabolism, hence stress responses, by using a lower temperature for a procedure. Studies have shown, however, that if temperature is altered rapidly, coolwater fish species perceive it as a stress and respond with elevated cortisol levels and ensuing impacts (Wedemeyer, 1968; Strange et al., 1977; Barton and Peter, 1982). A broad rule of thumb is to restrict temperature changes to one to two Centigrade degrees per day, which allows physiological compensation to take place. Generally speaking, many of the other methods described above offer equivalent or better ways to minimize fish discomfort than does temperature manipulation.
Another anaesthetic currently in vogue due to its environmental acceptability (greenhouse effect notwithstanding) is carbon dioxide. It may be created cheaply and readily using vinegar and baking soda, sublimated dry ice, or supplied pure from commercial gas distributors. It is probably one of the crudest and least elegant methods of immobilizing fish. At high concentrations the gas is toxic and completely inhibits oxygen consumption (Shelton, 1970). Its anaesthetic effect at lower levels arises from its power to reduce the animal's capacity to extract oxygen from the environment (Black et al., 1954), and is not dissimilar to applying a choke-hold and causing a black-out. Coupled with inherent difficulties in maintaining control over exposure due to its volatility, it may serve humanitarian and more mundane purposes equally to explore other options, even though they may require greater initial effort.
The problem of humanely killing fish for which no other realistic option exists has, like the anaesthesia and handling issues, received little study dedicated to animal care concerns. Euthanasia, which in the strict sense connotes a painless peaceful death, is rather often employed in a euphemistic sense. The onus remains on the animal handler to be as well-informed as possible about the pharmacology and physiological impacts of a method of `euthanasia', which may mean going beyond blind adoption of a previously reported method. Use of carbon dioxide for example would not, in light of my foregoing comments, rate as a good method of euthanasia due to its violent induction and mode of action.
By contrast, use of established lethal levels of central nervous system depressants such as MS-222 and benzocaine would be preferable. These compounds are recommended in a handbook issued by the Canadian Council on Animal Care (Olfert et al., 1993) and the American Veterinary Medical Association (Andrews, et al., 1993). Research with MS-222 has shown that stress, as indicated by elevation of plasma cortisol prior to death, did not reach levels warranting humanitarian concern if an established lethal dose for the particular species was used (Strange & Schreck, 1978). As with reversible induction, use of appropriate buffering described earlier would avoid pH-related trauma.
A rapid stunning blow to the head is also considered to be a humane method of killing fish, although it is most suitable for larger fish, and is usually preceded by anaesthesia to quieten the fish. Suffocation, either by draining a tank or removing fish to a dry tub or pail, is not a humane alternative. In general, if fish are small, a lethal dose of appropriate anaesthetic is best. If the fish are large enough to permit efficient rapid manual stunning, prohibitive expense may be avoided by confining use of chemical anaesthetics to achieving sufficient sedation to allow the fish to be handled readily. If the stunning method is used, common sense would suggest that the task be placed in the most experienced hands available.
Euthanasia of fish relies on one or more of: oxygen starvation, direct depression of vital nervous system parts (e.g., by anaesthetics), or physical destruction of nervouse system parts. Whether a fish dwells in salt or fresh water thus will not affect greatly the method of euthanasia or its efficacy. Solubility of most common fish anaesthetics is not strongly affected by normal salinity levels, however, the tendency for larger species of fish to live in salt water does affect the choice of method. Euthanasia of fish should be treated with the same level of preparation applied to any other critical phase of a study or project, both for the sake of the animals and to minimize distress to staff who may participate.
In summary, fish represent such a wealth of evolutionary diversity and specialization that it is difficult to apply many broad animal care considerations to their uses by humankind. Many fish species do, however, perceive and respond to disruptive influences in a similar physiological manner, which opens the door to making such experiences less stressful for them. None of the various processes and mitigative options discussed here are especially new, and most of them are within the reach of practically anyone who keeps and looks after fish. Unfortunately, this information is not often found under one heading. The present survey is neither particularly thorough nor comprehensive, but it may prove helpful in increasing awareness of animal care considerations in research and other uses of fish.
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| Copies of this journal are no longer available for sale, but our other two journals, Society & Animals and the Journal of Applied Animal Welfare Science, are available and subscriptions are quite affordable. They can be ordered online via our secure order page. |