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The number of fish used in research has increased in the last decades. Anaesthesia is required when fish must be held immobile and it is crucial to promote fish welfare, because these vertebrates can show signs of stress and/or pain during handling, transport, tagging, sampling and invasive procedures. The use of an inadequate anaesthetic protocol can compromise not only the welfare of the fish, but also the reliability of the research results. Thus, the development of suitable anaesthetic regimes for each fish species is important. This article reviews the main anaesthetic and analgesic agents used in adult fish in a research setting.

Keywords

anaesthesia ethics welfare laboratory fish refinement

Introduction

Fish handling can elicit relevant physiological stress responses. Chemical restraint may increase the safety of fish handling by minimizing trauma and integument damage during minor procedures.¹ In addition to minimizing stress responses and movement, the use of anaesthetics is crucial during invasive or surgical procedures. Teleost fish not only possess nociceptors but are also capable of experiencing pain.^2–4 Thus, the European Union directive on the protection of animals used for scientific purposes regulates fish use starting at the independently feeding larval stage. The literature regarding anaesthetic use in fish remains scarce. Because of the high variability observed among fish species (size, natural habitat, behaviour and physiology), the choice of type, dose of and the length of exposure to anaesthetic or sedative drugs should be considered with utmost care. Thus, further research is needed to increase the knowledge about the effects of anaesthetics on fish used in research, to develop appropriate anaesthetic protocols and specific procedures for each species. This article complements our previous review regarding anaesthesia and analgesia in zebrafish⁵ by providing information about other adult fish species used in research. Anaesthesia in fish larvae and the non-pharmacological methods that are used to induce immobility are out of the scope of this review and are briefly mentioned in the supplementary material.

Anaesthetic agents used for research in adult fish

Anaesthesia is defined as a reversible state that is produced by an external agent and results in the loss of sensation through depression of the central nervous system (CNS). Anaesthetics may be local or general, depending on their application. General anaesthesia suppresses CNS activity and results in unconsciousness and a total lack of sensation; this state may be followed by different levels of analgesia and muscle relaxation. Anaesthesia and sedation are usually divided into several stages that reflect the anaesthetic depth.⁵ The most common anaesthetic technique used in fish is the addition of the anaesthetic agent to the water. This is inhaled through the gills and diffuses rapidly to the secondary lamellae and to the efferent arterial blood, thus taking a short route to the brain, where it acts.⁶ This is similar to inhalation anaesthesia in terrestrial animals. This route of administration is easy and does not require physical restraint while administering the anaesthetics. Note that physical restraint may activate the hypothalamo–pituitary inter-renal axis, thus inducing cortisol release, which in turn elicits secondary stress responses.¹ Using the immersion route, the water should be aerated during the induction and maintenance of, and recovery from, anaesthesia. To maintain oxygen saturation, it is better to add oxygen to the water compared with pure air, but hyper-oxygenated water should be avoided, as it depresses ventilation and produces hypercapnia and, potentially, life-threatening acidosis.

The most common anaesthetics, such as benzocaine, lidocaine, MS-222 and 2-phenoxyethanol, can be used to induce anaesthesia in fish via the immersion technique. In contrast, there are fewer drugs available that can be used as injectable anaesthetics. The need of physical restraint when injecting the anaesthetics, the lack of licensed drugs and the poor efficacy of several anaesthetics used in mammals limit the use of injectable drugs.⁶ In fish, the intraperitoneal route is the most common route of injection. Intravenous injection of fast-acting anaesthetics is possible in larger fish, while the intramuscular route of administration allows the injection of a low volume of anaesthetics; however, the latter may be efficacious in fish with a developed red muscle.⁶ Thus, the best route of administration will depend on the type of anaesthetic and the size and physiological characteristics of the fish.

The selection of anaesthetics to be used in fish is complex. The decision process is based on the quality of anaesthesia induction, maintenance and recovery. Ideally, the anaesthetic dose selected should not be aversive to the animal and should induce unconsciousness very quickly, to reduce stress. However, these two properties are not always easy to find in the same drug. The animal may experience less aversion for a longer time after the administration of one drug, compared to other agents that may induce a quick loss of consciousness, but with a higher degree of aversion within a short period. Thus, a compromise may have to be found between the stress level inflicted on the fish and the time that the animal is subjected to it.

Anaesthetics must preserve fish physiology within safety limits. Significant alterations in cardiovascular and respiratory parameters have serious implications for the course of, and recovery from, anaesthesia. Anaesthetics that reduce the heart rate may cause high mortality under long-term sedation.⁷ Anaesthesia should also allow a quick and full recovery, with fishes showing normal behaviours, such as normal swimming patterns in a water column and positive responses to food.^8,9

Anaesthetics are used when noxious stimuli and/or stressful situations are expected. Anaesthesia with analgesia is important when animal handling associated with a risk of trauma is present (invasive studies, such as surgery and fin clipping). Anaesthesia that does not require the analgesia component may be used when fish need to be immobilized, for example for imaging. Anaesthetics can be used at low doses to induce sedation and reduce stress during handling for transport, grading, some types of tagging and vaccination or other injections.¹⁰

As described for other species, when using an anaesthetic regimen for the first time, it is advisable to test in one animal a given drug and dose, to ensure that an appropriate depth of anaesthesia is attained, and that recovery is uneventful. The response of different species to anaesthetic agents varies considerably, and the dosages quoted in this chapter may need to be modified for use in a different species or strain. Each investigator should search the literature regarding the techniques and protocols that are tailored to their animal model and research objectives, to reduce and refine the procedures while ensuring the best animal welfare. The common side effects and complications associated with each individual anaesthetic should also be reviewed carefully.

In addition to zebrafish (Danio rerio), other fish species have been used for research purposes, such as goldfish (Carassius auratus; a model that has been used for the study of transcription, receptors and growth factors),¹¹ Nile tilapia (Oreochromis niloticus; which has been used in toxicological studies¹² and gene identification),¹³ Mozambique tilapia (Oreochromis mossambicus; which has been used in studies of social behaviour),¹⁴ medaka (Oryzias latipes; a model that has been used for studying sex- and age-related bone metabolism and non-alcoholic fatty liver disease),^15,16 Xiphophorus fish (platyfish and swordtails; models that have been used to study melanoma) and rainbow trout (Oncorhynchus mykiss; which have been used as a model of carcinogen-induced cancer, particularly liver cancer).¹⁷ Atlantic salmon (Salmo salar) is an economically important species and is studied in the setting of aquaculture-related research.^18,19 Table 1 (below) provides a summary of the main anaesthetic agents that are used for these fish species, focusing on their effects, the doses used to achieve different anaesthetic stages and the times that are necessary for induction of, and recovery from, anaesthesia. The anaesthetic stages are based on the descriptive table included in Martins et al.⁵ The information regarding anaesthesia for each fish species is scarce compared to that available for zebrafish.⁵

Table 1.

Anaesthetic agents tested in adult fish used in research.

Species	Anaesthetic agent	Anaesthetic stage	Dose	Observations reported/Comments	Reference
Goldfish (Carassius auratus)	2-Phenoxyethanol	Stage III	0.3 ml/l immersion	For water temperature at 20 ℃, only 47% of small fish (c. 2 g weight) reach loss of equilibrium and none of large fish (c. 9 g weight) reached this stage. For water temperature at 25 ℃, only 27% of small fish reached loss of equilibrium and only 33% of large fish reached this stage. For water temperature at 30 ℃, only 20% of small fish reached loss of equilibrium and none of large fish reached this stage.	Weyl et al. 1996⁸⁰
		Stage III–VI?	0.4 ml/l immersion	For water temperature at 20 ℃, 93% of small fish and 100% of large fish reached loss of equilibrium. For water temperature at 25 ℃, this concentration induced loss of equilibrium in all small and large fish. For water temperature at 30 ℃, only 40% of small fish reached loss of equilibrium and only 75% of large fish reached this stage.
		Stage IV–VI?	0.5 ml/l immersion	For water temperature at 20 ℃, 25 ℃ and 30 ℃, the dose induced loss of equilibrium in all small and large fish between 3 and 6 min. Time to recover ranged between 3.7 and 5.6 min depending on temperature.
	MS-222	Stage VI	150, 200, 300, 400 and 500 mg/l immersion	Fish were exposed at different salinities: 0, 8, 12, 14 and 16 ppt at 25.6 ℃. The lowest effective doses, i.e. anaesthesia induction in 3 min or less and a complete recovery within 5 min, were 300 mg/l at 0 and 8 ppt of salinity, and 200 mg/l at 12, 14 and 16 ppt of salinity. For both anaesthesia induction and recovery times, they decreased as concentrations and salinity increased.	Küçük and Çoban 2016⁸¹
	Propofol	Stage I	1 mg/l immersion		Balko et al. 2017⁸²
	Propofol	Stage VI	5 and 10 mg/l immersion	Anaesthesia was induced in less than 4 min for 5 mg/l and in less than 2 min for 10 mg/l. With this concentration, only one of eight fish had a positive response to supramaximal stimulation (insertion of a 25-gauge needle into the caudal epaxial musculature) The recovery time varied from 4 min for 5 mg/l and 5 min for 10 mg/l.	Balko et al. 2017⁸²
Marine medaka (Oryzias dancena)	Clove oil	Stage VI	50, 75, 100, 125, 150 and 175 mg/l immersion	Anaesthesia induction time decreased as both anaesthetic doses and water temperature (23 ℃, 26 ℃ and 29 ℃) increased. The exposure to 125 mg/l at 23 ℃, 100 mg/l at 26 ℃, and 75 mg/l at 29 ℃ were considered the optimal concentrations for inducing anaesthesia within 1 min. Smaller fish took less time than larger fish to recover from anaesthesia.	Park et al. 2011⁸³
Marine medaka (Oryzias dancena)	Lidocaine hydrochloride	Stage VI	300, 400, 500, 600, 700 and 800 mg/l immersion	Anaesthesia induction time decreased as both anaesthetic doses and water temperature (23 ℃, 26 ℃ and 29 ℃) increased. The exposure to 800 mg/l at 23 ºC, and to 700 mg/l at 26 ℃ and 29 ℃ were considered the optimal concentrations for inducing anaesthesia within 1 min. Smaller fish took less time than larger fish to recover from anaesthesia.	Park et al. 2011⁸³
Platyfish (Xiphophorus maculatus)	MS-222	Stage I	10 mg/l immersion		Guo et al. 1995⁸⁴
		Stage II	20 mg/l immersion
		Stage III	30 mg/l immersion
		Stage IV/V	40 mg/l immersion	Induction of complete loss of equilibrium.
	Etomidate	Stage V	2–24 mg/l Immersion	Light anaesthesia occurred within 1.4 min for doses of 2 mg/l and higher. The time to recovery varied from around 13 min for 2 mg/l to less than 60 min for 18 mg/l.	Amend et al. 1982⁸⁵
			3 mg/l immersion	Exposure for 40 min showed 30% of mortality.
			24 mg/l immersion	Exposure for 120 s induced mortality.
			9 mg/l immersion	Exposure for 120 s on three consecutive days induced 5% of mortality.
			15 mg/l immersion	Exposure for 120 s on three consecutive days induced 15% of mortality.
	Metomidate	Stage I	0.5 mg/l immersion		Guo et al. 1995⁸⁴
		Stage II	1 mg/l immersion
		Stage III	1.5 mg/l immersion
		Stage IV/V	2 mg/l immersion	Induction of complete loss of equilibrium.
	2-Phenoxyethanol	Stage I	110 mg/l immersion		Guo et al. 1995⁸⁴
		Stage II	220 mg/l immersion
		Stage III	330 mg/l immersion
		Stage IV/V	440 mg/l immersion	Induction of complete loss of equilibrium.
Salmonid species	Metomidate (Atlantic salmon (Salmo salar))	Stage V	1 mg/l immersion	Light anaesthesia was achieved within 12 min (water temperature 5 ℃).	Olsen et al. 1995⁸⁶
		Stage V	2 mg/l immersion	Light anaesthesia was achieved within 6 min (water temperature 5 ℃).
		Stage VI	3 mg/l immersion	Light anaesthesia was achieved within 4 min, but surgical anaesthesia was achieved in 39 min (water temperature 5 ℃).
		Stage VI	5 mg/l immersion	Light anaesthesia was achieved in 2 min and surgical anaesthesia was achieved in 8 min (water temperature 5 ℃).
	MS-222 (Rainbow trout (Oncorhynchus mykiss))	Stage V	40 mg/l Immersion	The dose induced loss of equilibrium within 5 min but showed high variable time of response.	Keene et al. 1998⁸⁷
		Stage VI	60, 80 and 100 mg/l immersion	The onset of surgical anaesthesia occurred between 2 and 6 min, decreasing with the dose.
			All doses (40–100 mg/l) immersion	Time to recover the equilibrium increased with the dose, and it occurred in less than 4 min.
	Eugenol (Clove oil) (Rainbow trout (Oncorhynchus mykiss))	Stage VI	20 mg/l immersion	The time to achieve loss of equilibrium occurred in 1.8 min, while the onset of surgical anaesthesia within 10 min. High inter-individual variability to achieve both stages.	Keene et al. 1998⁸⁷
		Stage VI	40, 60, 80, 100, 120 and 140 mg/l immersion	Loss of equilibrium occurred faster with the dose increase and in less than 1 min. The onset of surgical anaesthesia occurred faster with the dose increase and within 4 min. The response times to achieve loss of equilibrium and surgical anaesthesia tended to plateau at concentrations higher than 60 mg/l.
			All doses (20 – 140 mg/l) immersion	Times to recover the equilibrium were longer with the dose increase, and it varied from a mean time of 12 min to 18 min with a great variability. No mortalities occurred.
	Propofol (Rainbow trout (Oncorhynchus mykiss))	Stage V	10 mg/l immersion	Light anaesthesia was induced in less than 3 min at 12 ℃ and 17 ℃. Safe anaesthesia, but a gradual reduction on the respiratory rate occurred. No mortality during anaesthesia or recovery.	Gomułka et al. 2015⁸⁸
Tilapias	MS-222 (Hybrid tilapias)	Stage IV	200 mg/l Immersion	Deep narcosis stage was reached at the first exposure within 2.7 min. Fish exposed weekly for six consecutive weeks showed a reduction in the mean time to achieve deep narcosis at the second (2.3 min) and third exposure (2 min), reaching a plateau by the fourth exposure (1.95 min).	Smith et al. 1999⁸⁹
	Clove oil (Nile tilapia (Oreochromis niloticus))	Stage VI	100 mg/l immersion	The reduction of respiratory rate occurred within 2.5 min. The time to recover was 8.4 min	Simões et al. 2010⁹⁰
		Stage VI–VII	150 mg/l immersion	The reduction of respiratory rate occurred in 1.7 min, and exposure for 8.7 min induced cessation of the respiratory rate. The recover from anaesthesia occurred within 10 min.
		Stage VI–VII	200 mg/l immersion	The reduction of respiratory rate occurred in 2.1 min, and exposure for 3.8 min induced cessation of the respiratory rate. The recover from anaesthesia occurred within 10 min.
		Stage VI–VII	250 mg/l immersion	The reduction of respiratory rate occurred in 1.6 min, and exposure for 3.2 min induced cessation of the respiratory rate. The recover from anaesthesia occurred within 11 min.
		Stage VI–VII	300 mg/l immersion	The reduction of respiratory rate occurred in 1.4 min, and exposure for 2.3 min induced cessation of the respiratory rate. The recover from anaesthesia was long and occurred within 18 min.
	2-Phenoxyethanol (Nile tilapia (Oreochromis niloticus))	Stage IV–VI?	0.5 ml/l immersion	Anaesthesia achieved in less than 4 min.	Auperin et al. 1997⁹¹
	2-Phenoxyethanol (Nile tilapia (Oreochromis niloticus))	Stage IV–VI?	1 or 2 ml/l immersion	Anaesthesia achieved in less than 1 min.	Auperin et al. 1997⁹¹
	Propofol [Nile tilapia (Oreochromis niloticus)]	Stage IV–VI?	0.15 ml/l immersion	Anaesthesia was induced in 18 min and recovery of normal swimming occurred in less than 10 min.	Valença-Silva et al. 2014⁶⁰
		Stage IV–VI?	0.30 ml/l immersion	Anaesthesia was achieved around 7 min and full recovery around 12 min.
		Stage IV–VI?	0.60 ml/l immersion	Anaesthesia was induced around 6 min and total recovery in less than 20 min.

?: depth of anaesthesia not specified.

MS-222

Tricaine methanesulfonate, or MS-222, is an inhalational anaesthetic that is commonly used in fish.²⁰ Its major mechanism of action occurs via the inhibition of the entrance of sodium into excitable brain cells, thus blocking action potentials. However, this mechanism is still under study and is not fully understood.²¹ Although MS-222 is classified as a local anaesthetic, it induces general anaesthesia in an anaesthetic bath, as it is absorbed through the gills and partly through the skin of the fish (scaleless fish).²² The MS-222 solution needs to be buffered to prevent the decrease of the pH of the water.^22–24 Although it is considered safe for fish,²⁵ as observed for other anaesthetics, MS-222 can cause secondary effects, such as hypoxemia, hypercapnia, hypoglycaemia, increased levels of lactic acid, erythrocyte swelling, elevated haematocrit and changes in blood electrolytes, hormones, cholesterol, urea and inter-renal ascorbic acid.^6,26 In pikeperch (Sander lucioperca), MS-222 increased the level of glucose 10 min after its administration, and decreased the leukocyte count and the levels of ammonia, triacylglycerols and inorganic phosphate 24 h later.²⁷ In carp, oxygenation aeration treatment reportedly increased the dissipation rate of MS-222.²⁸ Species-specific differences have been detected regarding MS-222 aversion: medaka avoided MS-222, but carp and rainbow trout did not.²⁹ These results should be taken into consideration when selecting an anaesthetic agent. MS-222 can cause mucous-membrane irritation in humans,²² and one episode of MS-222-related retinotoxicity has been reported.³⁰ Thus, care should be taken during the handling of this substance.

Benzocaine

Benzocaine is a white, odourless and tasteless local anaesthetic that is chemically similar to MS-222, but is more economic in large-scale procedures.^6,31 Because of its high lipophilicity, benzocaine is almost insoluble in water and requires the preparation of a stock solution using an organic solvent, such as ethanol or acetone, before its application as an immersion anaesthetic. Benzocaine presents low toxicity in fish: it does not have a negative impact on growth or reproductive capacity,⁶ although it may have a negligible effect on food intake and growth in Atlantic salmon,³² and its safety margin may decrease at higher temperatures.⁶ Even though benzocaine is well tolerated by some fish species, it can cause avoidance behaviours in medaka, similar to the results obtained for MS-222.²⁹

Clove-oil compounds

Clove oil is extracted from the plant Syzygium aromaticum and its major constituent, eugenol, is used as an inhalational anaesthetic. This oil requires mixing with ethanol before its use in an anaesthetic bath, as it is insoluble in water, or it can be dispersed in water at higher temperatures by vigorous shaking and used to anaesthetize tropical-water fish.⁶ Eugenol has been used as a local and topical anaesthetic and analgesic in humans. In fish, it exhibits rapid induction times and provides consistent anaesthesia compared with other anaesthetics. However, it may be associated with longer recovery times¹ and increased mortality caused by ventilatory failure when higher doses are used.³¹ Eugenol is efficient at a wide range of temperatures and is easily available and relatively inexpensive. However, one study questioned the analgesic properties of eugenol in red pacu (Piaractus brachypomus).³³ Clove oil has been reported to change some physiological parameters, as manifested through the reduction of cardiovascular activity, the prolongation of cardiovascular recovery, the lowering of feed intake after anaesthesia, the development of electrolyte imbalances and the reduction of stress responses observed in some species.^34–40 As noted for other anaesthetics, clove oil may also alter biochemical parameters. In pikeperch, clove oil increased the levels of glucose 10 min after its administration and decreased the leukocyte count and the ammonia and triacylglycerol levels 24 h after anaesthesia induction.²⁷

Isoeugenol is another constituent of clove oil and is the active ingredient of Aqui-S®, which is soluble in water. A similar product, Aqui-S® 20E, contains 10% eugenol. Both products have been approved in some countries for use in fish for human consumption, because they present a zero-withdrawal period.⁴¹ However, during the transport of high-stock densities, Aqui-S® increased the levels of total ammonia in Nile tilapia and yellow perch (Perca flavescens), probably because of an increase in protein metabolism.⁴² In that study, the authors recommended the use of high levels of oxygen to avoid the mortality induced by ammonia toxicity.⁴² Clove oil has been considered to have some risks to users, as one of its compounds, methyl eugenol, is a suspected carcinogen.⁴³ The other compounds, eugenol and isoeugenol, are considered as skin sensitizers, and the use of proper personal protective equipment by operators, such as a laboratory coat and gloves, is essential.⁴⁴

Metomidate and etomidate

Metomidate and etomidate are imidazole-based non-barbiturate hypnotic drugs that act through the activation and modulation of the inhibitory gamma-aminobutyric acid type A (GABAA) receptors.^6,26 They are used as immersion anaesthetics in fish and produce rapid anaesthetic induction and recovery; however, special care should be taken when using these agents, as they do not induce analgesia or surgical anaesthesia.^1,6 These drugs suppress cortisol production and, consequently, the cortisol stress response, which should be considered when fish are used for research.^6,26 In fact, etomidate prevented the delayed immunosuppressive effect of stress and its use has been recommended when long-term stress prevention is required.⁴⁵ While both agents have the same mechanism of action, they are different compounds, which implies some differences in their effects. While etomidate has minimal effects on respiratory function or the cardiovascular system,⁴⁶ metomidate can induce respiratory depression and reduce blood circulation.²⁶ One hour after the induction of etomidate anaesthesia, the mean corpuscular haemoglobin increased in carp and, 1 week later, these fish also exhibited an increase in erythrocytes, erythroblastosis, erythrocyte damage, lymphopenia, neutrophilia, monocytosis and thrombocytosis,⁴⁷ which represent alterations in blood parameters that may affect research results. Recently, Readman et al.²⁹ showed that, apparently, medaka and rainbow trout do not avoid etomidate, while carp behave in the opposite way. Metomidate hydrochloride is commercially available under the name Aquacalm™ and is used for sedation and anaesthesia in ornamental finfish, but not in fish that are intended for human or animal consumption; it is also relatively expensive. The manufacturer of Aquacalm contraindicates its use in air-breathing fish, in which the achievement of complete anaesthesia causes death. Even in a transportation setting, sedation induced by metomidate did not decrease the stress levels in this type of fish.⁴⁸ Metomidate may enhance pigmentation, muscular tremors or involuntary movements in some species.⁴⁹

2-Phenoxyethanol

2-Phenoxyethanol is a colourless or straw-coloured oily liquid with slight solubility in water. For efficient use as an immersion anaesthetic, its solubility can be increased by adding ethanol. This agent has been used as a topical anaesthetic, bactericide and fungicide. 2-Phenoxyethanol can provide deep stages of anaesthesia and is relatively inexpensive. Nevertheless, it has no other great advantages over other drugs.^6,50 The use of 2-phenoxyethanol can elicit several side effects, such as respiratory and heart-rate decreases, blood pressure and blood oxygenation reduction, alterations in blood parameters and an increase in the plasma levels of adrenaline, glucose and cortisol.^50,51 However, in meagre (Argyrosomus regius), low concentrations of this drug did not raise plasma cortisol and glucose levels.³⁴ In carp, this agent increased the levels of alanine aminotransferase in blood plasma 24 h after anaesthesia,⁵² and raised the haematocrit and haemoglobin levels 1 h after the induction of anaesthesia. One week later, the levels of erythrocytes, erythroblastosis, erythrocyte damage, lymphopenia, neutrophilia, monocytosis and thrombocytosis were increased.⁴⁷ In pikeperch, 2-phenoxyethanol decreased the erythrocyte count, haematocrit, leukocyte count and ammonia and triacylglycerol levels 10 min and 24 h after its administration, and increased the mean corpuscular haemoglobin concentration 10 min after its administration.²⁷ Even though its complete mechanism of action remains unknown, this agent inhibits the N-methyl-D-aspartate (NMDA) receptors, which have been related to analgesic effects.^50,53 However, the analgesia induced by 2-phenoxyethanol may be incomplete.⁵⁴ Nevertheless, this agent seems to be suitable for the induction of the light sedation that is necessary for the transportation of juvenile Persian sturgeons (Acipenser persicus), which renders it important for aquaculture and restocking purposes.⁵⁵ Prolonged exposure to 2-phenoxyethanol has been reported to cause a neuropsychological syndrome in some handlers.^1,56

Propofol

Propofol is a GABAA receptor agonist and a sedative–hypnotic anaesthetic drug that can be injected or used as an immersion anaesthetic. Propofol induces a quick anaesthetic state and provides a rapid and complete recovery. It has been described as a safe and effective depressant drug in several fish species,^9,57,58 and as an agent that is useful for fish transportation.⁵⁵ Propofol maintains the ionic and respiratory homeostasis and prevents peroxidative damage in the organs of the silver catfish (Rhamdia quelen).⁵⁹ It has also been shown to be a promising anaesthetic at low doses in Nile tilapia, as it has no genotoxic effects.⁶⁰ However, it has no analgesic properties.⁶¹

Essential oils

New drugs have been tested to reduce the side effects of anaesthesia and to reduce the legal and budgetary constraints related to the use of anaesthesia. Essential oils (EOs) and their constituents are the main sedative and anaesthetic drugs that have been tested in fish as a natural alternative to the commonly used synthetic drugs. These include the EOs of Hyptis mutabilis,⁶² Aloysia gratissima,⁶³ Aloysia triphylla,⁶⁴ Lippia alba,^65–67 menthol⁶⁸ and Condalia buxifolia methanolic extract, among others.⁶⁹ For instance, the EO extracted from the leaf of H. mutabilis induced anaesthesia in approximately 21 min at a concentration of 344 mg/l in silver catfish, with no mortality observed; however, approximately 2 h were necessary for the achievement of total recovery in all animals.⁶²

In silver catfish, the EO of A. gratissima can induce a stage of surgical anaesthesia in 12–18.1 min at a concentration range of 300–900 mg/l (the achievement of the anaesthetic level was faster at higher doses). However, the time to recovery ranged between 12.4 and 19.8 min and was independent of the concentration of the drug.⁶³ Although the EO from A. gratissima can cause involuntary muscle contractions during the induction of, and recovery from, anaesthesia, it did not cause mortality in silver catfish.⁶³ Nevertheless, its use is not recommended in Brazilian flounder (Paralichthys orbignyanus), as it can cause mortality during transportation.⁷⁰ In silver catfish strains, the EO of A. triphylla (100–800 µl/l) induced deep anaesthesia in 1.2–11.4 min and recovery in 5.3–18.3 min, without mortality (higher concentrations decreased the time to anaesthesia induction, but resulted in longer recovery times).⁶⁴ Moreover, A. triphylla is recommended for use in Nile tilapia, as it caused a reduction in cortisol levels in this fish.⁷¹ However, it induced genotoxicity in gilthead seabream (Sparus aurata), which can be avoided by using low doses of the drug.⁷²

In silver catfish, the EO from L. alba induced anaesthesia in 1.25–8.75 min and recovery in 4.5–5.82 min in the concentration range of 100–800 µl/l,⁶⁵ without mortality. Another study showed that, in the same species, the EO from L. alba induced anaesthesia in approximately 6.4–25 min and recovery in 6.7–20 min in the concentration range of 100–300 µl/l.⁶⁷

Regarding the use of menthol in Nile tilapia, concentrations of 250 and 300 mg/l were necessary to induce a surgical stage of anaesthesia in 9 and 8.7 min, and recovery in 13.4 and 10.3 min, respectively,⁶⁸ After 10 min under menthol anaesthesia, an increase in the glucose plasma levels was reported, indicating the induction of stress. A prolonged exposure to the anaesthetic (20–30 min) caused mortality in 10–20% of the animals.⁶⁸ However, in other species, such as lambari (Astyanax altiparanae), menthol attenuated the stress response (50 mg/l was the most effective concentration that could be used to induce deep anaesthesia safely after 6 min of exposure).⁷³ Another study showed that 108–133 ppm of menthol were sufficient to anaesthetize common carp in 1–4 min, with recovery occurring within 5 min.⁷⁴

In silver catfish, the methanolic extract of C. buxifolia in the range of 1–120 mg/l was sufficient to induce only a light sedation stage, without increasing cortisol plasma levels.⁶⁹ Other EOs extracted from Origanum vulgare, Eugenia aromatica, Melaleuca alternifolia and Cinnamomum zeylanicum induced short-time anaesthesia and provided light sedation and/or surgical anaesthesia. Anti-stress, anti-genotoxic and geno-protective results were reported for the EO from C. zeylanicum in gilthead seabream.⁷²

The EOs of basil (Ocimum basilicum) and lemongrass (Cymbopogum flexuosus) were tested as anaesthetics for Nile tilapia; both are recommended for sedation at a concentration of 10–25 µl/l, and for anaesthesia at a concentration of 400 µl/l for basil EO and 600 µl/l for lemongrass EO.⁷⁵

In juvenile tambaqui (Colossoma macropomum), the EO of citronella (Cymbopogon nardus) at a concentration of 200 µl/l was sufficient to induce sedation, while the concentration range of 400–600 µl/l was adequate to induce deep anaesthesia. A concentration of 600 µl/l was sufficient to promote a rapid induction in less than 3 min and a recovery from deep anaesthesia in less than 5 min.⁷⁶ The EO of Nectandra grandiflora and the waxy extract of Spilanthes acmella were also tested in juvenile tambaqui. An extract of S. acmella at a concentration of 10 mg/l was sufficient to induce fast anaesthesia in less than 3 min and safe recovery in less than 5 min.⁷⁷ The sedation concentrations of 1 mg/l of S. acmella and 30 µl/l of N. grandiflora enhanced the protection against oxidative damage, mainly in the muscle and gills of fish. Thus, these EOs have the potential to be used as chemo-protectants during transport.⁷⁷ In tambaqui, the EOs of Myrcia sylvatica and Curcuma longa are recommended for long-term sedation and rapid anaesthesia. These EOs did not alter most biochemical parameters and potentially increased the antioxidant capacity in vital tissues, as the levels of lipid peroxidation were reduced.⁷⁸ EOs and other natural substances appear to be advantageous over synthetic anaesthetics based on their ability to reduce side effects and mitigate stress effects, as well as the fact that they are affordable.⁷⁹ However, the risks of mortality depend on the substance, species and time of exposure used; further studies are required to explore better the potential application and safety of these natural compounds.

Analgesia in adult fish

Analgesia refers to the relief of pain; however, as animals do not express pain in the same way as humans, this parameter can be difficult to evaluate in other animals,⁹² especially in fish. The arguments in favour of considering fish as sentient animals are related with studies of the anatomy, physiology, behaviour and pharmacological responses of fish.³ The presence of nociceptors in fish indicates the occurrence of nociception, as observed based on the motor responses of fish to noxious stimuli. During the performance of behavioural tasks, fish sought the analgesic agent when exposed to painful stimuli and learned to avoid a potentially noxious stimulus, indicating the presence of pain perception.^93,94 In addition, another study showed that fish that were subjected to noxious stimuli can prioritize behavioural responses depending on the situation, indicating the central processing of pain.⁹⁵ Other behavioural and physiological changes, such as the reduction in swimming and food intake, the increase in gill ventilation rate and the rubbing of the area affected by painful agents, are also indicators of pain perception in fish.⁹⁶ These changes were significantly reduced after the administration of an analgesic, such as morphine.⁹⁷

Depending on the anaesthetic agent used, analgesia in the true sense of the word is not always present. Thus, when painful procedures are performed, the use of an analgesic protocol should be considered. Few studies have addressed the effects of analgesics in fish species.

Opioids, non-steroidal anti-inflammatory drugs (NSAIDs) and local anaesthetics are three types of analgesic drugs that have been studied as potential analgesic agents in fish.²⁶ Opioid analgesic drugs exert their effects via the inhibition and modulation of pain signals.⁹⁸ Morphine is a pure mu-opioid receptor agonist with a great hypoalgesic effect that is commonly used in veterinary medicine.⁹⁹ As this type of opioids causes addiction in humans, its use is strictly controlled, which may limit their application. Butorphanol is a kappa-opioid receptor agonist and a mu-opioid receptor antagonist, which limits its analgesic properties and efficacy.⁹⁹ Buprenorphine is a partial mu-opioid receptor agonist with analgesic effects that result from a combination of both opioid and non-opioid mechanisms of action.¹⁰⁰ In addition, buprenorphine has a lower addiction potential compared with mu-opioid receptor agonists¹⁰¹ and is one of the most-used opioids in veterinary medicine.⁹⁹ Tramadol is a central analgesic that acts as a mu-opioid receptor agonist, although with weak affinity, and as an inhibitor of the reuptake of the monoamine neurotransmitter, thus achieving its analgesic effects through the synergy of these mechanisms of action.¹⁰²

NSAIDs are used in veterinary medicine and provide good and long-lasting analgesia. Ketoprofen is an NSAID that may cause gastrointestinal ulceration and nephrotoxicity.¹⁰³ Carprofen is an NSAID that provides good postoperative analgesia (up to 24 h) and exhibits reduced side effects.¹⁰⁴ Both opioids and NSAIDs have adverse side effects.

Lidocaine hydrochloride⁶ is a classic example of a local analgesic that is relatively inexpensive and is safe to handle. Low-dose lidocaine immersions can be recommended to aid perioperative analgesia in zebrafish, thereby improving welfare.¹⁰⁵ This drug can also be used as a general anaesthetic when applied in an immersion bath.¹⁰⁶ However, the observation of a slow recovery has led to the abandonment of the use of lidocaine alone as an immersion anaesthetic for fish.⁶ Nevertheless, low lidocaine doses have been used as an analgesic adjuvant in perioperative pain management, to allow the reduction of the dose of other anaesthetics used during general anaesthesia in other species.^107,108 This concept of balanced anaesthesia and analgesia had a major impact on the development of anaesthetic and analgesic protocols based on the combination of drugs with lower doses and reduced secondary effects. Our group has demonstrated this possibility for adult zebrafish anaesthesia⁹ and similar approaches may be adopted for other fish species.

Similarly, analgesic agents can provide better analgesia when different classes of drugs are used in combination (multimodal therapy), which allows the reduction of the dose of each analgesic agent, consequently causing less side effects.^109,110 The use of a combination of morphine, butorphanol, buprenorphine, ketoprofen, carprofen and lidocaine in salmonid species, chain dogfish and Koi carp has been reported. However, difficulties in assessing pain responses in these species, and the likely wide variability of the responses among species, severely hamper the elaboration of recommendations of effective dose rates. Furthermore, these drugs are injected via intramuscular, intraperitoneal and subcutaneous routes, which limit their use in the intra-operative period or in the short post-operative period without extra animal restraint and anaesthesia. The combination of different drugs exhibiting different action times is often used to improve analgesia therapy. For example, butorphanol induces a rapid onset of analgesia, but yields mild and short-duration analgesia; in contrast, the time-to-peak analgesic effect of buprenorphine is quite slow, but its analgesic duration can be quite long. When administered together, the short-term analgesia of butorphanol wanes as buprenorphine is reaching its peak effect, and the animal continues to benefit from an analgesic effect.¹¹¹

Table 2 summarizes the various analgesic agents and their efficacy in several fish species in the adult stage.

Table 2.

Analgesic agents tested in adult fish.

Species	Analgesic agent	Dose	Observations reported/comments	Reference
Atlantic salmon (Salmo salar)	Lidocaine	2 mg (0.1 ml/site of 2% solution) (local infiltration)	Used at the sites of incisions.	Kiessling et al. 2003¹¹²
Chain dogfish (Scyliorhinus retifer)	Butorphanol	0.25-5 mg/kg (IM)	Lack of efficacy.	Davis et al. 2006¹¹³
Chain dogfish (Scyliorhinus retifer)	Ketoprofen	1–4 mg/kg (IM)	Lack of efficacy.	Davis et al. 2006¹¹³
Goldfish (Carassius auratus)	Butorphanol	0.1, 0.2 and 0.4 mg/kg (IM)	The dose 0.1 mg/kg decreased the MAC. The higher doses increased MAC. Probably efficient at 0.1 mg/kg.	Ward et al. 2012¹¹⁴
	Ketoprofen	0.5, 1 and 2 mg/kg (IM)	All doses decreased MAC. Probably efficient at all doses.	Ward et al. 2012¹¹⁴
	Morphine	2, 20 and 40 mg/kg (IP)	Prevented rubbing behaviour after acetic acid administration.	Newby et al. 2009¹¹⁵
		40 and 50 mg/kg (IM)	Lack of relevant analgesic effect during a thermal noxious stimulation. Both doses decreased the impact of heat stimulation on behaviour in the home tank.	Nordgreen et al. 2009¹¹⁶
		5–50 mg/kg (IM)	No side effects observed. Very efficient at 5 mg/kg.	Sneddon 2012²⁶
		10, 20 and 40 mg/kg (IM)	All doses decreased MAC. Probably efficient at all doses.	Ward et al. 2012¹¹⁴
Koi carp (Cyprinus carpio)	Butorphanol	0.4 mg/kg (IM)	Attenuation of behavioural changes after surgery.	Harms et al. 2005¹¹⁷
	Butorphanol	10 mg/kg (IM)	Induced temporary buoyancy problems. Decreased respiratory rate. Food consumption returned to baseline values within 3 h after treatment. Efficient as an analgesic.	Baker et al. 2013¹¹⁸
	Ketoprofen	2 mg/kg (IM)	No impact on behavioural changes after surgery. Not efficient as analgesic.	Harms et al. 2005¹¹⁷
	Morphine	5 mg/kg (IM)	Induced temporary bouts of excessive activity. Decreased respiratory rate at some time points. The food consumption has not changed. Efficient to produce analgesia.	Baker et al. 2013¹¹⁸
	Tramadol	10, 30, 50, 80 and 100 nmol/g (IM)	The analgesic effect was dose-dependent. The higher the dose, the more quickly it acted.	Chervova and Lapshin 2000¹¹⁹
Rainbow trout (Oncorhynchus mykiss)	Buprenorphine	0.01, 0.05 and 0.1 mg/kg (IM)	Reduced activity. No impact on feeding or ventilation. Not efficient.	Mettam et al. 2011¹²⁰
	Carprofen	1, 2.5 and 5 mg/kg (IM)	Depressed activity. Reduced time to feed using 2.5 and 5 mg/kg. Increased ventilation.	Mettam et al. 2011¹²⁰
	Lidocaine	0.5, 1 and 2 mg/fish (SC)	Reduced time to feed using 1 and 2 mg/kg.	Mettam et al. 2011¹²⁰
	Lidocaine	0.1–2 mg/kg (IM)	No side effects observed. Very efficient at 1 mg/kg.	Sneddon 2012²⁶
	Morphine	5–50 mg/kg (IM)	No side effects observed. Very efficient at 5 mg/kg.	Sneddon 2012²⁶
Winter flounder (Pseudopleur- onectes americanus)	Morphine	40 mg/kg (IP)	Analgesic effect at 40 mg/kg, 50 min after injection.	Newby et al. 2007¹²¹

IM: intramuscular injection; IP: intraperitoneal injection; SC: subcutaneous injection; MAC: minimum anaesthetic concentration (a model to assess analgesic effect).

Adapted from Sneddon 2012.²⁶

Concluding remarks

The increasing importance of fish in research renders essential the identification of suitable anaesthetic and analgesic protocols for each species and experimental purpose. An optimal anaesthetic protocol should be stable during the entire experimental procedure and afford short recovery times and minor or no physiological and behavioural alterations. Studies addressing the effects of anaesthetics in adult fish used in research remain scarce (Table 1). The publications that are available on this topic frequently lack information regarding the anaesthetic stage achieved, side effects, anaesthesia duration, induction properties and the duration and quality of the recovery from anaesthesia. Because of the countless variables that can interfere with the efficacy of anaesthesia, it is always advisable to perform a pilot study before starting the experimental protocol. Regarding analgesia, and despite the limited information available on this subject (Table 2), morphine is probably the best-established agent, as it is efficient in several species and has a reduced number of side effects. Nevertheless, the use of pure opioids can be conditioned by legal restraints. The administration routes may also limit the use of analgesics. Except for the case of analgesic immersion, the fish must be immobilized/calmed with anaesthesia prior to the administration of analgesia. Thus, pain management in fish will benefit from further research regarding administration routes, dose frequencies and analgesic multimodal therapies.

Supplemental Material

Supplemental material for Anaesthetics and analgesics used in adult fish for research: A review

Supplemental Material for Anaesthetics and analgesics used in adult fish for research: A review by Tânia Martins, Ana Valentim, Nuno Pereira and Luis Marques Antunes in Laboratory Animals

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by the Post-Doctoral Fellowship SFRH/BPD/103006/2014 funded by FCT, and supported by the Post-Doctoral Fellowship BI/CITAB/UTAD/VET/2015 from the Centre for the Research and Technology of Agro-Environmental and Biological Sciences supported by: European Investment Funds by FEDER/COMPETE/POCI–Operacional Competitiveness and Internacionalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT–Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013.

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