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Abstract

Anaesthesia is used daily in fish experimental procedures; however, the use of an inadequate anaesthetic protocol can compromise not only the animal's welfare but also the reliability of results. The use of zebrafish (Danio rerio) in biomedical research has increased in the last decades, highlighting the importance of appropriate anaesthetic regimens for this species. This article reviews the main anaesthetic agents and protocols used in laboratory adult zebrafish, and some of the analgesic methods to be used in this species that still need more research. In addition, a systematized observation of signs is proposed to evaluate adult zebrafish welfare to reduce pain and distress.

Keywords

anaesthesia analgesia refinement welfare zebrafish

The use of zebrafish (Danio rerio) in biomedical research has increased. Indeed, the percentage of publications using zebrafish has almost tripled in the last decade^1,2 in several research areas. In biomedical research, the behavioural or physiological changes that occur when an animal is exposed to a stressful or painful event can lead to unreliable results. The use of anaesthetic, sedative, or analgesic drugs is essential for reducing stress and/or pain in fish since they are sentient animals capable of pain perception.^3–5 Studies have demonstrated that fish show changes in normal behaviour and in physiological responses after being subjected to noxious stimuli, which may be ameliorated by the use of analgesics.^6–8 Despite these evidences, the discussion about pain perception in fish is still ongoing among researchers.⁹ Nevertheless, there is an agreement regarding the importance of fish welfare and that efforts should be made to minimize painful and stressful conditions.^9,10

Although the use of anaesthetics is important to ensure zebrafish welfare, these drugs can also have side-effects;¹¹ and it is essential to establish an anaesthetic regimen (doses, combinations) that suits each research procedure so as to minimize collateral effects. This review summarizes the anaesthetic and analgesic drugs that are used in laboratory adult zebrafish, anaesthesia protocols, anaesthesia depth, and recovery. Also, during all experiments it is of major importance to monitor zebrafish welfare, and for this, we propose a score sheet to monitor distress and pain.

Anaesthesia

Anaesthesia and sedation (Table 1) are routinely used in husbandry, clinical procedures and research. Thus, it is important to understand the side-effects of these drugs and how they may affect project outcomes in research. Anaesthesia has been described to negatively affect cognitive functions or to cause neurotoxicity, however, these effects are dependent on individual animals' age, regimen of administration, dose, duration and type of anaesthesia.¹² These factors combined with a yet scarce knowledge regarding anaesthesia in fish, make it difficult to choose an appropriate anaesthetic protocol suitable for each procedure.

Table 1.

Anaesthesia stages in fish.

Stage of anaesthesia	Description	Physiological and behavioural signs	Clinical interest
0	Normal	Total equilibrium Normal muscle tone Normal reaction to visual and tactile stimuli Normal respiratory rate
I	Light sedation	Slight loss of reaction to visual and tactile stimuli	Can reduce stress and physical trauma during transport
II	Deep sedation	Slight decrease in muscle tone No reaction to visual and light tactile stimuli Small decrease in respiratory rate	Appropriate stage for close visual observation and for minimal manipulation, weighing and measuring
III	Light narcosis/ excitement phase	Partial loss of equilibrium/ weak responses to postural changes Decrease in muscle tone Increased reaction to visual and tactile stimuli Respiratory rate increased and/or irregular	Higher risk of physical injury or escape/jump from container or aquarium
IV	Deep narcosis	Total loss of equilibrium/lack of responses to postural changes No reaction to minor visual and tactile stimuli Respiratory rate decreasing to almost normal	Good plane for external sampling and blood sampling. Avoid painful procedures/analgesia may not be present. Suitable for imaging techniques
V	Light anaesthesia	Complete loss of muscle tone No reaction to painful stimuli Decrease in respiratory rate Decrease in heart rate	Minor surgical procedures: fin biopsies and gill biopsies
VI	Surgical anaesthesia	Absence of reaction to massive stimulation Respiratory rate very low Slow heart rate	Major surgical procedures
VII	Medullary collapse/ overdose	Flaccid muscle tone Apnoea – absence of respiratory rate, which can be followed in several minutes by cardiac arrest if anaesthesia depth is not decreased Eventual death	Appropriate for euthanasia

Adapted from Murray 2002¹⁷ and Pereira 2016.¹⁸

Anaesthesia in fish may be achieved by diluting the anaesthetics in water (inhalation anaesthesia), which induces a general anaesthesia with the drug being absorbed mainly through the gills but also through the skin in a few species.^3,13,14 In fish, anaesthesia effects may vary depending on the administration route, pH, temperature, salinity, oxygenation, nitrogenous compounds and other water conditions.³ Furthermore, anaesthesia depth and recovery depend on its duration, anaesthetic concentration, animal's body weight and metabolism, gill surface, fish health status, strain, age, and on the different particularities of fish species.^3,11,15 Thus, anaesthesia trials with small numbers of fish, i.e. pilot studies, must be performed to determine the optimal dosage and exposure time prior to the establishment of protocols. In addition, proper training and supervision of fish anaesthesia are essential to avoid complications that can lead to death. Not only should anaesthesia be carefully monitored but also complete fish recovery,^2,16 which has been disregarded in the literature.

Anaesthetic agents used in fish

The ideal anaesthetic agent should (i) be easy to administer and effective at low dose or exposure; (ii) be able to induce sedation or anaesthesia in less than 3 min with a minimum of stress; (iii) provide immobilization and effective analgesia throughout the procedure; (iv) induce a quick recovery from the anaesthetic stage within 5 min; and (v) induce no or minimal changes in physiology and behaviour during or after anaesthesia.¹⁵ Ideally, the anaesthetic should be affordable, easily available, practical to use, and safe for the operator.

A recent international survey showed that around 93% of the surveyed participants used tricaine methanesulfonate (MS-222) for zebrafish anaesthesia. The use of 2-phenoxyethanol, benzocaine, clove oil, isoeugenol, etomidate, and lidocaine was also referred.¹⁹ In the following, several anaesthetic agents are discussed.

MS-222

MS-222, classified as a local anaesthetic, is the most used inhalant anaesthetic in fish. MS-222 is highly absorbed through the gills and is administrated by bath, inducing general anaesthesia. Overall it is a safe anaesthetic for fish,²⁰ although there are some concerns regarding risks of overdose in deeper stages of anaesthesia and long duration procedures, mainly in small animals such as zebrafish.^2,14,20,21 The anaesthetic solution of MS-222 should be buffered before use due to its acidic nature, which may cause aversion, epidermal and corneal lesions, and physiological alterations in the fish.^14,22,23 Also, MS-222 can be toxic to humans.^14,24

Clove oil

Eugenol is the major constituent (70–90% by weight) of clove oil extracted from the plant Syzygium aromaticum. Clove oil and eugenol are used as inhalant anaesthetics, and must be mixed with ethanol to be soluble in a water bath. They show rapid induction times and consistent anaesthesia, however, fish recovery takes longer than with MS-222.³ Clove oil is efficient at a range of temperatures, easily available, and relatively inexpensive.³ Aqui-S is a similar product available in the market constituted of isoeugenol, another compound of clove oil, which is soluble in water. Both have been suitable for harvesting and fish transportation.^3,15 In general, there are equivocal evidence of carcinogenic activity of eugenol and isoeugenol, while methyleugenol, another clove oil constituent, is carcinogenic to rodents.²⁵

Metomidate and etomidate

Metomidate and etomidate are non-barbiturate hypnotic drugs, and both are used as inhalant anaesthetics in a water bath in fish. These agents induce quick anaesthesia induction and recovery but they should only be used for minor procedures, as they do not induce a surgical anaesthetic stage or analgesia.^3,15 Also, they alter fish physiology by suppressing cortisol production.^11,15

Lidocaine

Lidocaine hydrochloride, a local anaesthetic and analgesic,¹⁵ is used as an immersion anaesthetic in fish. It induces anaesthesia within one minute and the recovery is also rapid, taking about three to four times the induction period.¹⁵ A high dose of lidocaine seems promising as an anaesthetic agent for surgical procedures but has a low margin of safety in zebrafish.²⁰ Thus, the addition of another agent, such as propofol, may potentiate this effect and reduce the dosage.² Moreover, perioperative analgesia with lidocaine seem to improve zebrafish welfare.²⁶

Propofol

Propofol is a sedative-hypnotic anaesthetic drug used for the induction and maintenance of general anaesthesia.²⁷ Propofol can either be injected or used in an anaesthetic bath. It is rapidly metabolized, thus lacking any cumulative effects. Although propofol is highly lipophilic, it can induce anaesthesia in a rapid and smooth way. Moreover, the recovery from anaesthesia is also quick and complete.^2,28 Although the preliminary results seem promising² it may not be fully soluble in the water and more research is needed.

Ketamine

Ketamine is an injectable agent often used in mammals and induces a dissociative anaesthesia with some analgesia.^29,30 In fish, it can either be injected or used in an anaesthetic bath. The use of ketamine in fish depends largely on the species, since it could cause incomplete anaesthesia, apnoea, prolonged recovery and excitement in salmonid species.³ Ketamine has been revealed to be neurotoxic to zebrafish larvae,^31,32 and to interfere with the development of embryos.³³

Isoflurane

Isoflurane is a hypnotic volatile drug routinely used in mammal anaesthesia. Studies evaluating volatile drugs in fish are scarce, since the anaesthetic depth is difficult to control, causing an overdose.^3,20 Furthermore, anaesthetic preparation and anaesthesia should be conducted in a chemical fume hood for scavenging waste gas, and reducing the risk to the operator. These characteristics and the observed clinical effects render volatile anaesthetics less practicable in fish anaesthesia.^3,20

Anaesthesia in laboratory adult zebrafish

Laboratory zebrafish has emerged as a powerful vertebrate model system in research.^1,34 Despite this interest, research on welfare and refinement of procedures in this species are scarce. Pain and unexpected mortality due to incorrect anaesthesia in zebrafish can constitute a serious animal welfare issue, which increases data variability, imposing an important scientific and economical cost on research.

Table 2 provides a summary of the main anaesthetic agents used in adult zebrafish, and their effects. Immersion is the most common method used, especially in small fish, such as zebrafish, where other invasive routes are impractical. Protocols for fish anaesthesia usually include only one anaesthetic agent instead of a combination; however, some studies have demonstrated that a mixture of two types of anaesthetics can result in a safer and more effective anaesthesia.^2,21

Table 2.

Anaesthetic and analgesic agents tested in laboratory adult zebrafish.

Species	Anaesthetic or analgesic agent	Anaesthetic stage/ analgesia	Dose	Observations reported/ Comments	Reference
Zebrafish (Danio rerio)	MS-222	Stage V	100, 120, 140, 160, 180 and 200 mg/L Immersion	The achievement of light anaesthesia, induced by all doses, ranged from a mean time of 1 min to 2.75 min in an inverse relationship response to dose. The achievement of surgical anaesthesia, induced by all doses, ranged from a mean time of 3 min to 14 min in an inverse relationship to dose. Inter-individual variability to achieve anaesthesia present at all doses. The total recovery of equilibrium, and reaction to external stimuli in the recovery phase of anaesthesia occurred at approximately 3 min regardless of the dose.	Grush et al. 2004³⁵
		Stages III–IV	100 and 120 mg/L Immersion	Doses were unable to induce light anaesthesia in all subjects.	Huang et al. 2010²¹
		Stage V	140, 160, 180 and 200 mg/L Immersion	Doses induced light anaesthesia within 2 min.
			All doses (100–200 mg/L) Immersion	Time required to achieve respiratory arrest ranged from a mean time of 3 min to 12 min in an inverse relationship response to dose. Subjects took over 2.2 min to recover after showing sign of respiratory arrest.
		N/A	100 mg/L Immersion	Induction of behavioural aversion.	Readman et al. 2013²²
		Stage VI	150 mg/L Immersion	Surgical anaesthesia was achieved within a mean time of 4.2 min and recovery 2.3 min post-anaesthesia. No mortality occurred during anaesthesia or post-anaesthetic period. No distressful behaviours were observed during induction or recovery.	Collymore et al. 2014²⁰
		N/A	168 mg/L Immersion	One exposure had no effect on several behavioural parameters of anxiety and activity immediately, 5 min, 30 min and 1 h after recovery.	Nordgreen et al. 2014¹⁶
		N/A	150 mg/L Immersion	One exposure caused conditioned place aversion and 53% of the subjects completely rejected the previous preferred side of the tank.	Wong et al. 2014³⁶
		Stage V	75 mg/L Immersion	Light anaesthesia was achieved in less than 2 min. Time to recover was less than 1 min.	Chambel et al. 2015³⁷
		Stage V	100 mg/L Immersion	Light anaesthesia was achieved in less than 2 min. Time to recover was less than 1 min.
		Stage V	125 mg/L Immersion	Light anaesthesia was achieved within 1 min. Recovery occurred within 1 min.
		Stage V	150 mg/L Immersion	Light anaesthesia was achieved within 1 min. Recovery occurred within 1 min. Exposure for 30 min at this dose induced 50% of mortality.
		Stage V	200 mg/L Immersion	Light anaesthesia was achieved in less than 1 min. Recovery occurred within 1 min. Exposure for 30 min at this dose induced 100% of mortality.
		Stage V	250 mg/L Immersion	Light anaesthesia was achieved in less than 1 min. Recovery occurred within 1 min. Exposure for 30 min at this dose induced 100% of mortality.
		Stage VI	100 mg/L Immersion	No mortality occurred during anaesthesia or post-anaesthetic period.	Valentim et al. 2016²
Zebrafish (Danio rerio)	Clove oil	N/A	55 mg/L Immersion	One exposure caused some conditioned place aversion and only 19% of the subjects completely rejected the previous preferred side.	Wong et al. 2014³⁶
Zebrafish (Danio rerio)	Clove oil	Stage V	60, 80, 100, 120 and 140 mg/L Immersion	The achievement of light anaesthesia, induced by all doses, ranged from a mean time of 0.5 min to 1 min in an inverse relationship response to dose. The achievement of surgical anaesthesia, induced by all doses, ranged from a mean time of 2.5 min to 8.5 min in a negative exponential response to dose. The total recovery of equilibrium after anaesthesia occurred at approximately 5 min regardless of eugenol dose. The reaction to external stimuli in the recovery phase of anaesthesia occurred at approximately 8 min, regardless of eugenol dose.	Grush et al. 2004³⁵
Zebrafish (Danio rerio)	Metomidate hydrochloride	Stage I	2 and 4 mg/L Immersion	The time to recover from anaesthesia ranged from 2.8 min to 5.2 min.	Collymore et al. 2014²⁰
		Stage IV	6, 8 and 10 mg/L Immersion	Light anaesthesia occurred between 2.3 min and 4.3 min. No surgical anaesthetic plane was achieved during the 10 min of exposure. The time to recovery from anaesthesia ranged from 5.6 min to 10.4 min regardless of the dose. No distressful behaviours observed during anaesthesia. No mortality during or after anaesthesia.	Collymore et al. 2014²⁰
		N/A	13.5 mg/L Immersion	One exposure caused some conditioned place aversion and only 11% of the subjects completely rejected the previous preferred side.	Wong et al. 2014³⁶
Zebrafish (Danio rerio)	Etomidate	Stage IV	4 to 10 mg/L Immersion	Induction of light anaesthesia was achieved for doses of 4 mg/L and higher, and occurred in less than 1 min. The time to recovery varied from around 25 min for 4 mg/L to 60 min for the highest dose.	Amend et al. 1982³⁸
		Stage IV	8 to 10 mg/L Immersion	Doses higher than 8 mg/L induced mortality.
		N/A	3 mg/L Immersion	Exposure for more than 10 min and 20 min showed 5% and 20% of mortality, respectively.
		N/A	9 mg/L Immersion	Exposure for 120 s on three consecutive days induced 30% of mortality.
		N/A	15 mg/L Immersion	Exposure for 120 s on three consecutive days induced 95% of mortality.
		N/A	2 mg/L Immersion	No behavioural evidence of aversion was observed in the subjects exposed.	Readman et al. 2013²²
Zebrafish (Danio rerio)	Lidocaine hydrochloride	N/A	100 mg/L Immersion	Induction of behavioural aversion.	Readman et al. 2013²²
		Stage I	300 mg/L Immersion	Induction of light sedation that occurred within a mean time of 5.4 min with a great variability. The recovery from anaesthesia occurred at a mean time of 3.4 min with a great variability. No mortality occurred during anaesthesia or post-anaesthetic period.	Collymore et al. 2014²⁰
		Stage VI	325 mg/L Immersion	Induction of surgical anaesthesia at a mean time of 0.85 min with a great variability. The recovery from anaesthesia occurred at a mean time of 5.8 min with a great variability. No mortality occurred during anaesthesia or post-anaesthetic period.
		Stage VI/VII	350 mg/L Immersion	Induction of surgical anaesthesia at a mean time of 1.7 min, and some subjects experienced Stage VII – medullary collapse/overdose. The recovery from anaesthesia occurred at a mean time of 1.9 min with a great variability. 30% mortality observed during anaesthesia.
		Analgesic	0.1–2 mg/kg (IM)	Analgesic drug. No side-effects observed. Very efficient at 1 mg/kg.	Sneddon 2012¹¹
		Analgesic	2–5 mg/L* Immersion	Pre- and post-surgical administration for tail fin clipping. Reduction of pain-related behaviours.	Schroeder 2016²⁶
Zebrafish (Danio rerio)	Propofol	Stage V	2.5 mg/L Immersion	The dose induced light anaesthesia in 93.4% of the subjects but was unable to induce analgesia in 40% of the subjects. This dose showed an occurrence of 33% mortality.	Valentim et al. 2016²
Zebrafish (Danio rerio)	Propofol	Stage V	5 and 7.5 mg/L Immersion	Both doses induced light anaesthesia but were unable to induce analgesia in 36% and 18% of the subjects, respectively. Both doses showed an occurrence of 9% mortality.	Valentim et al. 2016²
Zebrafish (Danio rerio)	Propofol (P) combined with lidocaine (L)	Stage VI	2.5 mg/L (P) + 50 mg/L (L) Immersion	No mortality occurred during anaesthesia or post-anaesthetic period.	Valentim et al. 2016²
		Stage VI	2.5 mg/L (P) + 100 mg/L (L) Immersion	Occurrence of 23% mortality.
		Stage VI	2.5 mg/L (P) + 150 mg/L (L) Immersion	Occurrence of 9% mortality.
Zebrafish (Danio rerio)	Ketamine	N/A	2000 mg/L (acute exposure during 5 min and chronic exposure for 5 consecutive days) Immersion	Subanaesthetic dose produced behavioural abnormalities such as increased circling behaviour, decreased gill movement (ventilatory response to hypoxia), and decreased stress response to hypoxia (body pulses). The repeated administration of ketamine did not cause tolerance or sensitization to specific drug effects.	Zakhary et al. 2011³⁹
Zebrafish (Danio rerio)	Ketamine	Stage IV (at least)	8000 mg/L Immersion	Physiological anaesthetic dose inducing a deep level of unconsciousness.	Zakhary et al. 2011³⁹
Zebrafish (Danio rerio)	Isoflurane	Stage III	0.5 mL/L Immersion	Distressful behaviours were observed (twitching, erratic swimming, and piping). Signs of disorientation, difficulty maintaining buoyancy, rolling, and swimming upside down also occurred. Recovery from anaesthesia within a mean time of 4.2 min. 30% mortality during anaesthesia and 1 day post-anaesthesia.	Collymore et al. 2014²⁰
Zebrafish (Danio rerio)	Isoflurane (I) combined with MS-222 (M)	Stage III–IV; Stage V (10–20%)	50 mg/L (I) + 50 mg/L (M) Immersion	Light anaesthesia was induced by this dose in only 10% –20% of the subjects.	Huang et al. 2010²¹
		Stage IV	60 mg/L (I) + 60 mg/L (M); 65 mg/L (I) + 65 mg/L (M); 70 mg/L (I) + 70 mg/L (M); 80 mg/L (I) 80 mg/L (M) Immersion	The combination doses between 60 + 60 mg/L and 80 + 80 mg/L induced light anaesthesia within 1.5 min.
			All doses	Regarding all doses, time required to achieve respiratory arrest ranged from a mean time of 6 min to 55 min in a negative exponential response to dose. For all the combined doses the subjects recovered within 2 min after showing signs of respiratory arrest.

N/A: not applicable/not evaluated. All anaesthetic agents were administered by immersion, with exceptions: IM: intramuscular injection. *Paul Schroeder, personal communication, 19 July 2016.

Monitoring zebrafish welfare

In addition to the importance of a good anaesthetic protocol to ensure fish welfare during experimental procedures, animals should also be monitored for distress, discomfort and pain throughout the experimental protocol and maintenance. Fish have demonstrated to react consistently to noxious chemical stimuli and present reliable phenotypes of stress, fear, and anxiety.^40,41 Thus, the use of analgesic drugs in fish during and after painful experimental procedures should be considered, especially due to the fact that not all anaesthetic agents available for fish have proved to have adequate analgesic properties.³ The information concerning the use of analgesics in zebrafish is extremely scarce (Table 2). Furthermore, the available analgesics tested in fish are administered intramuscularly and/or by the intraperitoneal route,¹¹ which make it difficult to perform in small fish, such as zebrafish, or in a large number of animals.²⁶ Lidocaine has recently been described as a promising analgesic for zebrafish,²⁶ which is an important step in the development of analgesic protocols to be administered in a water bath.

In Table 3 we suggest a score sheet to monitor the signs of distress and pain in adult zebrafish. This table is an initial proposal of a score sheet to monitor zebrafish welfare, and, as such, it should be further investigated for each individual protocol and genetic background.

Table 3.

Proposal of a pain and distress score sheet for laboratory adult zebrafish.

	Score
Physical appearance*
Normal	0
Missing operculum, and missing fins repeatedly, indicating possible antagonist behaviours; darkening/inflammation of fin	1
Mild scoliosis/lordosis, tegument lesions, mucus production, over- or under-conditioned (obese or thin), abrupt colour change, especially blanching	2
General emaciation (low body to head ratio), general body deformities, missing or protuberant scales	3
Food consumption
Normal	0
Unresponsive to food during 1 day	1
Unresponsive to food during 2 days, not even live food	2
Unresponsive to food, more than 3 days, not even live food as artemia or rotifers (**)	3
Respiratory pattern
Normal	0
Piping or extremely low rate, almost no opercular movement	3
Swimming behaviour (**)
Swimming through the water column	0
Difficulties to control buoyancy and/or to maintain equilibrium	2
Systematic swimming on the surface or in the bottom of the tank	3
Activity (**)
Normal	0
Hyperactive (erratic swimming) or hypoactive	2
Lethargic, no reaction to external stimuli	3
Social behaviour (**)
Normal. Shoaling	0
Individual often isolated when group-housed	1
Individual always chasing or being chased by conspecifics	2
Individual does not respond to conspecific behaviours towards itself	3
TOTAL

Judgement: 0–1: normal; 2–8: monitor carefully. Consider veterinary treatment including analgesics and consider also to analyse water quality; 9–12: suffering, provide relief, consult the specialized veterinarian, consider euthanasia; 13–18: severe status, euthanasia, rethink experimental procedure. Euthanasia may be considered if a score of 3 is observed in any of the categories, except for behaviour-related categories (**), in which scores of 3 suggests repeated and close observation for a final decision regarding euthanasia. *Take in consideration animals' age.

Concluding remarks

The use of a suitable anaesthetic protocol able to produce anaesthesia with effective analgesia is an important refinement for painful procedures. Studies addressing the effects of anaesthetics in zebrafish are variable and lack important information such as the time during which the anaesthetic stage can be maintained without secondary effects, a clear description of the anaesthesia stage achieved with a certain dose, and data about recovery. This lack of information can have a negative impact on zebrafish welfare when investigators are not experienced in the use of zebrafish anaesthesia. In addition, analgesia in zebrafish is another topic that needs further investigation in order to refine analgesic protocols during experimental procedures and for postoperative pain management, thus ensuring zebrafish welfare and reliable data collection.

In general, MS-222 is a good anaesthetic, justifying its wide use in zebrafish,¹⁹ but further refinement could be valuable when long duration procedures are required.^11,21 Furthermore, two studies have described zebrafish aversion towards this anaesthetic.^22,36 Thus, finding other anaesthetic protocols are advisable, for example, the use of anaesthetic combinations to decrease individual concentrations and the risk of unwanted side-effects. Also, lidocaine seems to be the most promising analgesic to be used in zebrafish, however, its efficacy needs to be tested in different painful procedures and experimental situations.

Footnotes

Acknowledgements

The authors would like to thank Hugo Santos (BOGA, CIIMAR – Interdisciplinary Centre of Marine and Environmental Research), Ana Cristina Borges, Maysa Franco e Liliana Vale (IGC – Instituto Gulbenkian de Ciência), Petra Pintado e Fábio Valério (CEDOC – NOVA Medical School), and Sandra Martins (Champalimaud Foundation) for their critical comments on the scoring sheet table.

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 Postdoctoral Fellowship SFRH/BPD/103006/2014 funded by FCT, and by the Fellowship BI/CITAB/UTAD/VET/2015 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 Project UID/AGR/04033/2013.

References

Kalueff

Echevarria

Stewart

. Gaining translational momentum: more zebrafish models for neuroscience research. Prog Neuropsychopharmacol Biol Psychiatry 2014; 55: 1–6.

Valentim

Felix

Carvalho

Diniz

Antunes

. A new anaesthetic protocol for adult zebrafish (Danio rerio): propofol combined with lidocaine. PLoS One 2016; 11: e0147747.

Neiffer

Stamper

. Fish sedation, analgesia, anesthesia, and euthanasia: considerations, methods, and types of drugs. ILAR J 2009; 50: 343–360.

Sneddon

. Pain in aquatic animals. J Exp Biol 2015; 218: 967–976.

Jones

. Fish sentience and the precautionary principle. Anim Sent 2016; 1.3: 10.

Sneddon

Braithwaite

Gentle

. Do fishes have nociceptors? Evidence for the evolution of a vertebrate sensory system. Proc Biol Sci 2003; 270: 1115–1121.

Sneddon

Braithwaite

Gentle

. Novel object test: examining nociception and fear in the rainbow trout. J Pain 2003; 4: 431–440.

Mettam

Oulton

McCrohan

Sneddon

. The efficacy of three types of analgesic drugs in reducing pain in the rainbow trout, Oncorhynchus mykiss. Appl Anim Behav Sci 2011; 133: 265–274.

Rose

Arlinghaus

Cooke

. Can fish really feel pain? Fish Fisheries 2014; 15: 97–133.

10.

Iwama

. The welfare of fish. Dis Aquat Organ 2007; 75: 155–158.

11.

Sneddon

. Clinical anesthesia and analgesia in fish. J Exot Pet Med 2012; 21: 32–43.

12.

Jevtovic-Todorovic

Absalom

Blomgren

. Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar. Br J Anaesth 2013; 111: 143–151.

13.

Ferreira

Schoonbee

Smit

. The uptake of the anaesthetic benzocaine hydrochloride by the gills and the skin of three freshwater fish species. J Fish Biol 1984; 25: 35–41.

14.

Carter

Woodley

Brown

. A review of tricaine methanesulfonate for anesthesia of fish. Rev Fish Biol Fisheries 2011; 21: 51–59.

15.

Ross

. Anaesthetic and sedative techniques for aquatic animals, Oxford: Blackwell Publishing, 2008.

16.

Nordgreen

Tahamtani

Janczak

Horsberg

. Behavioural effects of the commonly used fish anaesthetic tricaine methanesulfonate (MS-222) on zebrafish (Danio rerio) and its relevance for the acetic acid pain test. PLoS One 2014; 9: e92116.

17.

Murray

. Fish surgery. Semin Avian Exot Pet Med 2002; 11: 246–257.

18.

Pereira

Introduction to anaesthesia and surgery in fish. In: Oliveira

Bernardo

Robalo

(eds). Practical notions on fish health and production, Sharjah: Bentham Science Publishers, 2016, pp. 127–182.

19.

Lidster K, Readman GD, Prescott MJ and Owen SF. International survey on the use of zebrafish in research. (Under submission).

20.

Collymore

Tolwani

Lieggi

Rasmussen

. Efficacy and safety of 5 anesthetics in adult zebrafish (Danio rerio). J Am Assoc Lab Anim Sci 2014; 53: 198–203.

21.

Huang

Hsieh

Chen

. Combined use of MS-222 (tricaine) and isoflurane extends anesthesia time and minimizes cardiac rhythm side effects in adult zebrafish. Zebrafish 2010; 7: 297–304.

22.

Readman

Owen

Murrell

Knowles

. Do fish perceive anaesthetics as aversive? PLoS One 2013; 8: e73773.

23.

Davis

Stephenson

Noga

. The effect of tricaine on use of the fluorescein test for detecting skin and corneal ulcers in fish. J Aquat Anim Health 2008; 20: 86–95.

24.

Berstein

Digre

Creel

. Retinal toxicity associated with occupational exposure to the fish anesthetic MS-222. Am J Ophthalmol 1997; 124: 843–844.

25.

Center for Veterinary Medicine (CVM). Concerns related to the use of clove oil as an anesthetic for fish, Rockville: CVM, 2007.

26.

Schroeder P. Exploring suitable analgesics in zebrafish – a combined approach. In: 13th FELASA Congress. Brussels, Belgium, 13–16 June 2016, p. 32.

27.

Steinbacher

. Propofol: a sedative-hypnotic anesthetic agent for use in ambulatory procedures. Anesth Prog 2001; 48: 66–71.

28.

GholipourKanani

Ahadizadeh

. Use of propofol as an anesthetic and its efficacy on some hematological values of ornamental fish Carassius auratus. Springerplus 2013; 2: 76.

29.

Karmarkar

Bottum

Tischkau

. Considerations for the use of anesthetics in neurotoxicity studies. Comp Med 2010; 60: 256–262.

30.

Kohrs

Durieux

. Ketamine: teaching an old drug new tricks. Anesth Analg 1998; 87: 1186–1193.

31.

Kanungo

Cuevas

Ali

Paule

. Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expression in zebrafish. J Appl Toxicol 2013; 33: 410–417.

32.

Burgess

Granato

. Sensorimotor gating in larval zebrafish. J Neurosci 2007; 27: 4984–4994.

33.

Felix

Antunes

Coimbra

. Ketamine NMDA receptor-independent toxicity during zebrafish (Danio rerio) embryonic development. Neurotoxicol Teratol 2014; 41: 27–34.

34.

Oliveira

. Mind the fish: zebrafish as a model in cognitive social neuroscience. Front Neural Circuits 2013; 7: 131.

35.

Grush

Noakes

Moccia

. The efficacy of clove oil as an anesthetic for the zebrafish, Danio rerio (Hamilton). Zebrafish 2004; 1: 46–53.

36.

Wong

von Keyserlingk

Richards

Weary

. Conditioned place avoidance of zebrafish (Danio rerio) to three chemicals used for euthanasia and anaesthesia. PLoS One 2014; 9: e88030.

37.

Chambel

Pinho

Sousa

. The efficacy of MS-222 as anaesthetic agent in four freshwater aquarium fish species. Aquacult Res 2015; 46: 1582–1589.

38.

Amend

Goven

Elliot

. Etomidate: effective dosages for a new fish anesthetic. Trans Am Fish Soc 1982; 111: 337–341.

39.

Zakhary

Ayubcha

Ansari

. A behavioral and molecular analysis of ketamine in zebrafish. Synapse 2011; 65: 160–167.

40.

Maximino

. Modulation of nociceptive-like behavior in zebrafish (Danio rerio) by environmental stressors. Psychol Neurosci 2011; 4: 149–155.

41.

Roques

Abbink

Geurds

van de Vis

Flik

. Tailfin clipping, a painful procedure: studies on Nile tilapia and common carp. Physiol Behav 2010; 101: 533–540.

Anaesthesia and analgesia in laboratory adult zebrafish: a question of refinement

Abstract

Keywords

Anaesthesia

Anaesthetic agents used in fish

MS-222

Clove oil

Metomidate and etomidate

Lidocaine

Propofol

Ketamine

Isoflurane

Anaesthesia in laboratory adult zebrafish

Monitoring zebrafish welfare

Concluding remarks

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

References