Abstract

Fifty-nine coldwater and warmwater fish ranging in weight from 2-35 kg were injected intramuscularly with the hypnotics alphaxalone-alphadolone (Saffan) and metomidate HCl (Marini) and the non-depolarizing muscle relaxant gallamine triethiodide (Flaxedil) using a new laser-aimed underwater dart gun. Alphaxalone-alphadolone produced sufficient sedation for easy netting within 5-20 minutes at doses between 0.3 and 0.5 ml/kg, with induction being somewhat faster in warmwater species. The pattern of induction was similar with metomidate but required doses of 40 to 60 mg/kg. The muscle relaxant gallamine triethiodide showed promise as a practical agent for capture and handling of large fish by virtue of its smooth induction of paralysis at doses between 1 and 3 mg/kg and its reversible supplementation with orally applied metomidate HCl.

Introduction

Capture and transport of fish, particularly specimens weighing several kilograms or more, is often attended by netting trauma and delayed stress-related disease and mortality (Tytler and Hawkins 1981). There is frequently a need to move large fish maintained for display, research or as broodstock in aquaculture, yet present methods allow few options other than netting followed by immersion in a water bath containing a dissolved or suspended anesthetic such as tricaine methanesulfonate (MS 222) or 2-phenoxyethanol. For large, aggressive fish maintained in oceanaria, neither operation is realistic, and husbandry options are correspondingly limited.

Recent development of laser-aimed underwater dart gun technology now makes possible the administration of sedative, immobilising and other drugs to fish and aquatic mammals in a manner analogous to remote injection techniques used for land animals over the last thirty years. Remote sedation of several coldwater elasmobranchs using this equipment has been described (Harvey et al 1987).

Choice of antibiotics for administration using the new technology is relatively straightforward and can be based on the substantial body of literature dealing with the treatment of fish and marine mammal disease. The choice of immobilising drugs for fish is, however, made difficult by an almost total lack of published information on the effects of intramuscular injection of these agents into fish. The subject is touched upon briefly in a recent review dealing with anesthesia of fish in general (Ross and Ross 1984), and sedation of captive sharks using ketamine/xylazine applied by pole syringe has been reported (Stoskopf et al 1985). The only substantive treatment is a report of experiments in which intramuscular injection of various sedative and immobilising drugs followed benzocaine-induced oral anesthesia (Oswald 1978).

In this paper we summarize the results of two years of experimentation with remote intramuscular injection of sedative and immobilising drugs uncomplicated by prior capture of the fish. The experiments took place at several locations including large oceanaria, fisheries research facilities and fish farms and were all performed under field conditions. The fish studied include representatives of warm and cold water teleosts and elasmobranchs, and range in size from a few kg to over 35 kg. The steroid anesthetic alphaxalone-alphadolone acetate (Saffan) was chosen on the basis of previous work showing it to be an effective hypnotic agent when administered intramuscularly in rainbow trout (Oswald 1978). Metomidate hydrochloride (Marinil; Hypnodil) was selected by virtue of its ready solubility in water and its proven efficacy as a fish anesthetic when administered as an immersion bath (Anon 1980; Escoubet 1982). Gallamine triethiodide (Flaxedil) has been used extensively in immobilisation of terrestrial warmblooded animals and appeared to hold special promise for fishes by virtue of its effectiveness in immobilising crocodilians (Woodford 1972).

Methods

Drugs were delivered using the Aquadart system (Marine Technology Ltd.). Metomidate and gallamine were delivered in volumes under 2.0 ml in the 3 ml dart provided with the gun, while the larger volumes of alphaxalone-alphadolone necessitated using the larger, 10 ml dart. Both darts inject pneumatically upon impact and remain attached to the fish after jettisoning of the lightweight spear shaft that carries them to the target. Healthy captive adult specimens were treated at the following locations: The Vancouver Public Aquarium (pacu Colossoma nigripinnis, dogfish Squalus acanthias, jack Caranx fuscus, sablefish Anoplopoma fimbria, carp Cyprinus carpio); Sea Life Park, Waimanalo, Hawaii (brown stingray Dasyatis lata, spotted eagle ray Aetobatus narinari, yellowtail Seriola dumerili, ulua Caranx stellatus, blacktip reef shark Carcharhinus limbatus, snapper Lutjanus boharl; Pacific Undersea, Gardens,Victoria, B.C. (rockfish, Sebastes caurinuv, sablefish, Anoplopomafimbria, skate Raja binoculata); Fisheries and Oceans Canada, West Vancouver Laboratory (coho salmon Oncorhyrichus kisutch and chinook salmon O. tshawyacha) and Sea Spring Farms, Genoa Bay, B.C. (O. tshawyacha).

Fish held in display aquaria (Vancouver Aquarium) and 3 m diameter outdoor fiberglass tanks (Fisheries and Oceans Canada) were shot by an operator standing outside the tank and submerging the dart gun so that the laser aiming device emitted its beam under water. Fish were shot from a distance of 4-7 feet in the thickest part of the dorsal musculature after momentary location of the red laser spot on the target area. Fish held in large display aquaria (Pacific Undersea Gardens; Sea Life Park) or in submerged net pens (Sea Spring Farms) were shot by a diver wearing scuba equipment and approaching within 4-7 feet. In both cases the accuracy provided by the laser aiming device was such that misses were exceptional and resulted from hurried shots at animals out of range.

Heavily scaled or sluggish fish in confined areas (eg. pacu at the Vancouver Aquarium; skate at Pacific Undersea Gardens) were shot using barbless needles which were retained long enough for injection and dispersion of the drug, Large, active specimens in large water volumes were shot using darts provided with lightly barbed needles, and these were withdrawn from sedated fish through a small incision made with a pointed No. 11 scalpel blade after removal of one or two scales. Chlortetracycline hydrochloride ointment (Aureomycin) was pressed into the wound.

In some cases the dosage of immobilising drug administered intramuscularly was sufficient to cause disorientation or partial loss of equilibrium but insufficient to permit netting without a struggle, In such cases 1-5 ml metomidate HCI, 20 mg/ml in tank water, was administered orally using the Power Syringe (Marine Technology Ltd.) a device that delivers up to 5 ml of fluid at high pressure upon pulling the trigger. The Power Syringe could be used from outside or within the water so long as the end of the probe was placed near or within the buccal cavity of the sedated fish.

Metomidate HCl and gallamine triethiodide were made up in 0.6% NaCl and used within 2 days; alphaxalone-alphadolone was used directly from the vial supplied by the manufacturer.

Results

In most cases fish seemed unaware of the presence of the laser spot on their bodies and did not anticipate the approach of the spear shaft and dart. The aluminum alloy shaft used to propel the dart to the target travels so rapidly through the water that, even at a distance of 6-7 feet the fish have no time to avoid it or to react to the sound of the pneumatic gun being fired underwater. Reaction to impact of the dart was commonly a swift startle response followed by rapid swimming that rarely lasted more than a few seconds. Most fish resumed normal swimming thereafter until the drug began to take effect. There were some exceptions to this rule: Sluggish elasmobranchs like the skate responded hardly at all to impact, while rapidly schooling, powerful fish like the yellowtail were often successful at rubbing the dart loose on rocks. The low mass of the alloy shaft prevented any visible tissue damage from impact, and the only sign of physical damage apart from the puncture wound was a transitory blackening of the area of skin surrounding the injection site in fish administered metomidate hydrochloride.

The degree of sedation or anesthesia obtained using the three intramuscular agents was difficult to relate to classical schemes describing levels of anesthesia obtained in fishes exposed to water-borne drugs (Jolly 1972), possibly because such schemes are based on the response of fish already stressed by net capture. Our scheme for describing the drug effects was as follows:

 Stage 1. Disorientation. Slowing of swimming; bumping into tank windows and walls. Avoids net.

 Stage 2. Partial loss of equilibrium; settling or rising to surface. Can be netted but still responsive to touch.

 Stage 3. Complete loss of equilibrium and cessation of swimming. No response to touch. Marked depression of ventilation.

There were no marked differences in symptomatic response to the three drugs used; in most cases there was a smooth progression through the three stages of sedation/anesthesia, with the process taking longer with lower doses. Results of the various drug tests are presented in Table 1.

Table 1 Response of a variety of fish to remote intramuscular injection of alphaxalone alphadolone, metomidate hydrochloride and gallamine triethiodide

Legend for Table 1.

1.  Estimated body weight in kg.

2.  met = metomidate hydrochloride. gall = gallamine triethiodide alf alphaxalone-alphadolone.

3.  mg/kg (metomidate and gallamine); ml/kg (alphaxalone-alphadolone).

4.  Deepest stage of sedation or anesthesia achieved within 1 hour.

5.  Drug delivered using two or more darts approximately 10 min apart.

6.  Exact weight; animal weighed while anesthetized.

Insofar as was possible under field conditions, fish were monitored for resumption of normal behaviour and feeding after treatment. In nearly all cases, feeding was resumed within a few days to two weeks, with the large uluas and yellowtails the slowest to recover. Only one fish died of an obvious drug overdose (Sebastes caurinus, injected with gallarnine at 30 mg/kg). Large specimens of ulua, yellowtail, snapper and pacu required post-recovery bath treatments of malachite green or furacin (Herwig 1979) to discourage fungal infestation at the site of barb removal; all wounds closed within two weeks of injection.

Discussion

Remote underwater delivery of drugs to fish and other aquatic animals is an important alternative to present methods necessitating prior capture and stress. Physiological response of fish to capture and netting has been well documented, and it seems safe to assume that, so long as the sedative or immobilising agent itself is not a stressor, remote injection for capture and treatment of fish will eliminate a major cause of disease and mortality in their husbandry. Results of the present study appear to support this assumption, although longer terrn, more closely controlled post-recovery observation of the fish is needed. Some idea of the promise of remote underwater sedation may, however, be gained from comparison with the results of standard netting techniques carried out during the removal of fish from the reef tank at Sea Life Park: when four uluas were netted and transported by the usual method, three did not survive (unpublished observations). It is also worth noting that the response to sedative/immobilising drugs in fish appears different when the drugs are applied intramuscularly to un-stressed fish rather than as a bath for net-caught animals; hence the requirement for a new scheme of stages of anesthesia.

Confidence in the response to a given dose of an immobilising drug must be built on the assumption that the animal received the entire volume of the drug. Hardware currently available for remote delivery of drugs to land animals has evolved around the need to accomplish this in the face of different sizes and shapes of animals with different skin/hair coverings and behaviours. The situation is analogous with aquatic animals, and the lightly barbed dart used in most of the present experiments with the Aquadart was developed to ensure injection of the drug at a depth of approximately 2.0 cm with solid anchoring of the barb under the dermis. This configuration should place the site of injection within the white muscle in most cases. We were unable to detect any pattern of efficacy that might be related to injection in the less vascularised white or the more heavily vascularised red muscle; the rapid onset of effects in many of the treated fish suggests that absorption from the injection site is adequate. Occasionally a fish succumbed to the effects of the drug extremely rapidly: a 13 kg pacu, for example, received 10 mg gallamine triethiodide and became incapable of normal swimming within one minute. The same pattern was occasionally seen with the other drugs, and suggests that in such cases injection was into a heavily vascularized region. We tested completeness of injection by shooting several coho salmon and yellowtail with darts filled with fluorescein or methylene blue dye; no leakage into the surrounding water was detected with these fast and powerful swimmers as subjects.

Two of the agents tested in the present study, metomidate hydrochloride and alphaxalone-alphadolone, are hypnotics with well-described effects in mammals and birds (Green 1982). We found them to be effective but not entirely predictable sedative/immobilising agents in freely swimming fish. Alphaxalone-alphadolone (Saffan), was effective in inducing Stage 2 sedation at doses between 0.3 and 0.5 ml/kg in most of the coldwater forms including coho salmon, sablefish, dogfish, common carp and skate; at higher doses (1.5 ml/kg) profound anesthesia was attained within 5 minutes in dogfish, followed by resumption of normal swimming within 5 hours. The therapeutic index thus appears acceptably broad. Saffan also gave impressive results in some of the warmwater fishes tested, although the results were less predictable. The blacktip reef shark Carcharhinus limbatus was, for example, easily netted within ten minutes of injection at 0.4 ml/kg, yet of the three brown rays tranquillized, the first two (0.3 ml/kg) were easily removed from the water within 15 min, while the third (0.8 ml/kg) required oral injection of metomidate HCI (20 mg/ml) in order to be brought under control. This sort of inconsistency is mirrored in the results for the pacu treated with Saffan: doses between 0.25 and 0.8 ml/kg appear unrelated to the speed or depth of sedation. Observations made during experiments in which fishes of the same species were shot in succession at the same location strongly suggest that animals treated first succumb much more rapidly to a dose of drug that is completely ineffective on a similar fish shot thirty minutes later and consequently already agitated by the activity in the enclosure. This frustrating inability to tranquillize certain excited animals is well documented in land animal immobilisation (Haigh, 1982).

Results using metomidate hydrochloride were similar to those with alphaxalone-alphadolone. 40-60 mg/kg was found adequate for Stage 2 sedation in coldwater forms, with recovery from Stage 3 100 mg/kg) taking several hours. Practical application of the result with a 9 kg female broodstock chinook salmon should be of interest to aquaculturists: this fish was shot by a diver in a net cage (42 mg/kg), given oral metomidate while still weakly swimming, then removed and transported to freshwater facilities where she eventually spawned normally. The fish appeared markedly less stressed in the freshwater holding facilities when compared to animals caught and transported by conventional methods. For large warmwater fish, however, metomidate was as unpredictable as alphaxalone-alphadolone, a pattern that can be clearly seen in the results with the pacu. The three large uluas captured with this drug at Sea Life Park again illustrate the need for relatively large doses of the drug with large, excited fish; for these fish and the yellowtail, the effect of doses as high as 80- 100 mg/kg was profound enough for removal of the fish (Stage 2 within 2-5 min) but appeared completely worn off within 30 min. It is worth noting that, for a fish such as the 35 kg ulua, a dosage of 100 mg/kg represents 3.5 g of drug, a dangerous amount of drug for humans to be handling in injectable form, even when applied in three doses as was the case in the present study. Propoxate, an analogue of metomidate shown to have excellent hypnotic effects in fish (Ross and Ross 1984) should be tested as a potentially safer agent in large animals.

Some of the problems of unpredictability and high doses experienced with metomidate and alph ax alone- alphadolone may be minimised with the muscle relaxant gallamine triethiodide (Flaxedil). Gallamine is a non-depolarising muscle relaxant no longer widely used for immobilisation of warm-blooded animals but of some interest for capture of polkilotherms (Harthoom 1976; Woodford 1972). The six rockfish treated with gallarnine responded predictably to doses between I and 4 mg/kg, although development of sedation was slow, taking up to 2 h in the larger specimens. In the two warmwater fishes tested (pacu), a dose of 1.4 mg/kg produced Stage 2 sedation within 10 minutes in one fish, while a dose of 1.3 mg/kg had the same effect within one minute in another fish. This second animal was the only one treated with gallamine to show any degree of excitation (occasional swimming to surface) and remained paralysed for three hours. Clearly the effects of this drug are not entirely predictable. Both rockfish and pacu were easily netted while making weak swimming movements on their sides; all the rockfish and one of the pacu recovered normal swimming activity within 30 minutes (pacu) and 90 min (rockfish). Effects on ventilation were inconsistent; ventilation frequency dropped in all fish, but, in the one pacu presumably injected in a highly vascularized area, ventilation was elevated thirty percent for two hours before falling to a normal rate. There was little excitation or muscle twitching in most of the fish as the drug took effect, in marked contrast to preliminary trials with the depolarizing muscle relaxant succinylcholine chloride, where loss of motor activity was marked by unpredictable and violent contractions of swimming musculature and where breathing was almost invariable fatally arrested. In both the pacu and rockfish it was possible to produce true sedation/anesthesia by applying metomidate orally to a semi-paralysed fish; this technique allowed fast-acting reversible analgesia for barb removal or transport and removed any reservations about handling an animal that has lost voluntary control of movement but not the capacity to feel pain. This technique would appear to combine humane considerations with efficacy, and is a very easy way of managing a partially or wholly paralysed fish. At doses of 1.5 mg/kg the risk of human toxicity in case of inadvertent injection should be less than for comparably effective doses of alphaxalone and metomidate, although the peculiar conditions of underwater remote injection in the field dictate extreme care, whatever the agent used. The paralytic action of gallamine triethiodide can be reversed in mammals by administration of the anticholinesterase neostigmine methylsulfate in conjunction with atropine (Green 1982); preliminary trials with tilapias (Oreochromis mossambicus ) paralysed with gallamine and injected with neostigmine show that paralysis can be reversed in teleosts as well.

While reliable equipment for remote delivery of drugs to aquatic animals is now available, much research needs to be done to identify sedative/immobilising drugs that are effective for a wide variety of species and that are easy to obtain as well as safe and convenient to use. The history of remote sedation of land animals is one of continued improvement of sedative/immobilising agents, and this process will have to be repeated with aquatic animals. Remote injection itself can, of course, be applied to delivery of antibiotics, vaccines and other non-sedative agents, and here again the development of underwater remote injection can follow terrestrial precedent. Thus while sedation of aquatic mammals is often problematical due to complications associated with diving physiology, non-stressful administration of antibiotics to captive pinnipeds has already been achieved using a 6 cm barbless needle in the system described in the present report (unpublished results).

Acknowledgements

The authors thank the management and staff of Sea Life Park (Waimanalo, Hawaii), The Vancouver Public Aquarium, Pacific Undersea Gardens and Sealand of the Pacific (Victoria) and Fisheries and Oceans Canada (West Vancouver) for their cooperation and donation of experimental subjects. We are particularly grateful for the assistance of P.A. Bruecker (Vancouver Aquarium), A. Bolz (Sealand) and E,M. Donaldson (West Vancouver Laboratory). The studies reported here were supported by funding from the Science Council of British Columbia.

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