Comparative study of tricaine methanesulfonate (MS-222) and eugenol as euthanasia agents in zebrafish ( Danio rerio ) as an experimental model

Abstract

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In experimental procedures inevitably leading to the sacrifice of animals, suitable measures should be taken to minimise their pain and suffering as much as possible, as well as to prevent any modification or masking in the experimental results obtained. An overdose of anaesthetic is the method of euthanasia most employed in fish, since it is effective and easy to apply. Our objectives were to compare the efficacy of eugenol and of tricaine methanesulfonate (MS-222) as euthanasia agents in zebrafish, and to make a histological evaluation of the possible effects derived from their application. The concentrations established for eugenol were 0.25 and 0.35 mg/mL, and those for MS-222 were 0.25 and 0.50 mg/mL, for both the buffered solution and the non-buffered one. Eugenol turned out to be a stronger euthanasia agent than MS-222 in zebrafish, presenting with significantly shorter euthanasia times. However, the exposure of the fish to euthanasia doses of eugenol triggered branchial alterations, in addition to serious lesions and changes in their nerve tissue. The results obtained with MS-222 also revealed a marked branchial alteration derived from its use. In this respect, the addition of a buffer to the MS-222 solution enhanced the effectiveness of the drug, with significantly shorter euthanasia times being achieved than with the non-buffered solution, and diminished the severity of the lesions described. We therefore determined that the buffered MS-222 solution is the most effective, reliable and safest method of euthanasia for use in research on zebrafish.

Keywords

Eugenol euthanasia fish MS-222 refinement

Introduction

The zebrafish (Danio rerio) has become a general experimental model in basic and applied research due to its multiple technical and practical advantages for studying biological processes.^1,2

In accordance with conditions specified in the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes,³ as well as in current regulations, in procedures that inevitably lead to the sacrifice of animals, adequate steps have to be followed in order to minimise their pain and suffering. Therefore, methods of euthanasia should satisfy a series of fundamental criteria in relation to the animal’s welfare. They should thus be painless and rapid, involving a minimum of immobilisation, and be reproducible and safe without altering or masking the experimental results obtained.^4,5

An overdose of anaesthetic diluted in the medium is one of the methods of euthanasia most employed in zebrafish, since it is effective and easy to use.^5–7 A considerable number of anaesthetics for use in research are currently available. The ideal anaesthetic should be soluble in water, easy to prepare and administer, chemically stable during a reasonable amount of time, and biodegradable.^7–10

Eugenol (4-alil-2-methoxyphenol) together with isoeugenol (4-propenyl-2-methoxyphenol) are the principal active components of clove oil (90–95%), a natural product obtained by distilling the flower of the clove tree (Syzygium aromaticum).¹¹ Its anaesthetic properties are derived from its effects on the ionic channels of the neural cells related to nociception, neuronal peak generation and synaptics.¹² Eugenol is highly lipophilic and is quickly absorbed through the gills and skin.¹³ Although it is a widely used anaesthetic in research involving fish, it is known that eugenol increases cortisol as a response,¹⁴ and it could be potentially toxic to the fish brain.¹⁵

Tricaine methanesulfonate (3-aminobenzoic ethyl ester, methanesulfonate), also known as MS-222, belongs to the local anaesthetic family and is the one most used in fish.¹⁶ Its mechanism of action consists of blocking the sodium channels and thus the action potentials of the membrane. However, how it acts as an anaesthetic is not entirely understood,¹⁷ and despite its popularity as an anaesthesia and euthanasia agent, more knowledge of it is required, as some instances of aversion to this drug have been described.¹⁸

The reviews on euthanasia agents in fish supply data such as their toxicity and their physical and chemical statistics. However, it is their safety margins, induction times, doses and possible physiological alterations that need to be established, since that would be an important step towards refinement.^8,9,19

Our objectives were to compare the efficacy of eugenol and of MS-222 as euthanasia agents in zebrafish, and to make a histopathological evaluation (structural and ultrastructural histopathology) of the possible effects from their use that may alter and/or mask the experiment results.

Methods

Humane care and use of animals

The experimental phase of the study was carried out in the Experimental Animal Service at Córdoba University (Spain), a centre registered as an establishment for the husbandry, supply and employment of animals for experimentation and other scientific purposes. All the applicable national, international and/or institutional guidelines for the care and use of animals were followed. All procedures complied with the instructions of the Animal Experimentation Committee at Córdoba University (04/05/2018/076), following the indications of Directive 2010/63/EU.⁴

Animals and husbandry

The minimum number of animals necessary to carry out the statistical study was considered, as well as to enable their histopathological evaluation. Thus, 90 zebrafish (Danio rerio) were used (45 males and 45 females), aged around 16 weeks, with a mean weight of 0.56 ± 0.14 g and a mean furcal length (the distance from the front end of the fish to the slit of the caudal fin) of 4.17 ± 0.24 cm. The animals were supplied by the Córdoba University Experimental Animal Service. All the animals were clinically healthy (i.e. free from both physical and behavioural abnormalities). The parameters considered in their selection were normal colouring of the body, gills and eyes, absence of skin lesions and body conformation anomalies, normal respiratory rate and natural swimming pattern.²⁰ The health monitoring protocol ensured the absence of viral, bacterial and parasitic pathogens determining DNA or RNA of these agents by polymerase chain reaction or reverse transcription polymerase chain reaction according to each case.

The animals were kept under a photoperiod of 16 hours of light and 8 hours of darkness. They were maintained in a water recirculation system with filtering, with a capacity to measure the water quality parameters, germicide ultraviolet radiation and modules controlling light and temperature. The animals were kept in groups of five inside this system at a density of one fish per litre of water for 14 days prior to the experiment. This low density was chosen to facilitate handling and ensure water quality. The water conditions were as follows: pH 7–8, conductivity between 300 and 500 µS/cm, temperature 26 ± 1°C, hardness 50–250 mg CaCO₃, ammonia (NH₃/NH₄⁺)<0.1 mg/L, nitrite (NO₂^–)<0.3 mg/L and nitrate (NO₃^–)<25 mg/L. The animals were fed three times a day with a combination of dry feed (Tropical Supervit Premium®) and frozen Artemia spp.

Experimental design and euthanised solution

The euthanasia agents employed were 100% pure MS-222 and 99% pure eugenol. To buffer the MS-222 solution, bicarbonate (NaHCO₃) was applied at double the concentration tested of MS-222.²¹ To enable eugenol solubility, the latter was previously dissolved in 96% pure ethanol (C₂H₆O) at a ratio of 1:9 (eugenol:ethanol).²² All these chemicals were supplied by Sigma–Aldrich(St Louis, MO).

The study concentrations established for eugenol were 0.25 and 0.35 mg/mL, and for MS-222 they were 0.25 and 0.50 mg/mL, for both the buffered and non-buffered solutions. These doses were established from those described by several authors in relation to employing two or three times the anaesthetic dose as a method of euthanasia.^5,22–24 The fish were allocated per sex in groups of five for each concentration in duplicate (five males and five females). The total number of animals in treatment groups was 60 (30 males and 30 females).

The remaining 30 animals were included in three groups in accordance with the treatments established of 10 fish each (five males and five females). Two groups were exposed to the highest doses of ethanol and bicarbonate used – 3.15 mg/mL and 1 mg/mL, respectively – under similar conditions to those of euthanasia, and there was also a negative control group. Those animals were sacrificed by decapitation, with prior sedation, which was performed by immersion in MS-222 at a dose of 0.05 mg/mL⁷ buffered with 0.1 mg/mL bicarbonate in the negative and ethanol control groups. Sedation times were 181.26 ± 21.06 seconds. The fish were decapitated in order not to interfere with the results. This method of euthanasia can be used when fish are unconscious.⁴ Each euthanasia process was performed individually.

Onset of euthanasia

The euthanasia time is that defined as being the period from the moment that the fish comes into contact with the euthanasia solution until its opercular movements have finally ceased.²⁵ To determine this, the following indicators were observed: the swimming axis, body mobility, response to external mechanical stimulation, and rate and frequency of the opercular movement.⁵ The exposure to the euthanasia agents was also timed.

Histological examination (structural and ultrastructural histopathology)

Sample collection and processing

A histopathological study was undertaken of all the animals. Before collecting the samples, any possible external anomaly was evaluated: erythema, ulceration, erosions, scale loss, nodules, exophthalmos, ocular opacity, aberrant pigmentation and so on. The samples taken from all the fish after their necropsy were gills, nerve tissue (spinal cord), liver, kidney, heart and intestine. All reagents used for sample processing were supplied by Sigma–Aldrich.

Optical microscopy

The histological study was performed in the microscopy section of the Central Research Support Service at Córdoba University. In order to examine them under the optical microscope, the organs were fixed in formaldehyde buffered at 10% at room temperature, dehydrated on a rising scale of ethanol and soaked in paraffin. The first sections of each block (4 µm) were stained with hematoxylin and eosin for their morphometrical and histopathological study. The cuts were observed and photographed in an Ortholux microscope (Ernst Leitz GMBH, Wetzlar, Germany).

Transmission electron microscopy and scanning electron microscopy

For their examination under the electron microscope, the samples collected were fixed in 2% glutaraldehyde in a 0.1 M solution of phosphate buffer (pH 7.4) at 4°C all night, and then they were fixed again in osmium tetroxide in a 0.1 M solution of phosphate buffer (pH 7.4) for 30 minutes. After dehydration on a rising scale of alcohol and after being placed in araldite, semi-fine and ultrafine cuts were made with an Ultratome (LKB, Bromma, Sweden). The semi-fine cuts were stained with toluidine blue, whereas the ultrafine ones were double-stained with uranyl acetate and lead citrate. They were studied and photographed under a transmission electron microscope (TEM; JEM-1400; JEOL, Tokyo, Japan) and under a scanning electron microscope (JSM-6300; JEOL), respectively.

The lesions found were classified according to the degree of alteration: 0, no alteration; 1, slight alteration; 2, moderate alteration; and 3, severe alteration.

Statistical analysis

The results obtained were analysed using IBM SPSS Statistics for Windows v16.0 (IBM Corp., Armonk, NY). The non-parametric Kruskal–Wallis test was used to determine differences in euthanasia times and in the histopathological analysis. In all the cases, a p-value of <0.05 was taken as being significant.

Results

Onset of euthanasia

The euthanasia times for the different groups appear in Table 1. The increase in the concentration of the euthanasia solutions applied to the fish represented a significant drop in the euthanasia times, both in eugenol and in MS-222, and in the buffered MS-222 solutions (p < 0.01).

Table 1.

Euthanasia times of zebrafish exposed to different preparations of eugenol (0.25 and 0.35 mg/mL), MS-222 (0.25 and 0.50 mg/mL) and buffered MS-222 (0.25 and 0.50 mg/mL).

Drug	Doses (mg/mL)	Euthanasia time (s)
Drug	Doses (mg/mL)	Mean*	Standard deviation
Eugenol	0.25	242.0	15.7
Eugenol	0.35	103.0	13.9
MS-222	0.25	616.0	40.7
MS-222	0.50	372.4	36.8
Buffered MS-222	0.25	312.5	57.6
Buffered MS-222	0.50	175.0	42.9

The mean ± SD are depicted.

*Significant difference (p < 0.05) between all the groups.

Eugenol at 0.25 mg/mL achieved shorter euthanasia times (p < 0.01) than non-buffered MS-222 at 0.25, 0.50 and buffered 0.25 mg/mL. Buffered MS-222 at 0.50 mg/mL gave a shorter euthanasia time than 0.25 mg/mL eugenol (p < 0.01). However, the shortest euthanasia time was achieved with 0.35 mg/mL eugenol (p < 0.01).

In all the MS-222 concentrations studied, the fish reached the euthanasia phase in a shorter time with the buffered MS-222 solution than those euthanised with non-buffered MS-222 (p < 0.01).

Histological examination

Macroscopic observations

None of the animals in the control groups exhibited macroscopic alterations during dissection. In the treated groups, there was general evidence of a marked reddening of the gill arches, this being accentuated in the highest concentrations. The rest of the organs had an apparently normal aspect in these groups.

Structural and ultrastructural observations

The principal histopathological findings described in the zebrafish exposed to the different eugenol and MS-222 concentrations, as well as those of the control groups, are shown in Table 2, in which the control groups are expressed jointly, since no lesions were found in them.

Table 2.

Principal histopathological findings described in zebrafish exposed to euthanasia concentrations and in the control groups.

	Control (N = 30)	Eugenol (mg/mL)		MS-222 (mg/mL)		MS-222 buffered (mg/mL)
	Control (N = 30)	0.25 (N = 10)	0.35 (N = 10)	0.25 (N = 10)	0.50 (N = 10)	0.25 (N = 10)	0.50 (N = 10)
Gills
Hyperaemia/inflammation	–	+	++	++	+++	++	++
Oedema	–	–	+	++	+++	++	++
Haemorrhage	–	+	++	++	+++	+	++
Tissue degeneration	–	–	+	++	+++	+	+
Nerve tissue
Oedema	–	++	+++	+	++	+	++
Neural degeneration	–	+	+++	++	+++	+	++
Necrosis	–	++	+++	–	–	–	–
Heart
Oedema	–	+	++	+	+	–	+
Liver
Glycogenic degeneration	–	–	+	+	++	+	+
Intestine
Oedema	–	–	–	–	–	–	–
Kidney
Glomerulopathy	–	–	++	+	+	–	–

The different degrees of anatomopathological alterations are depicted.

–: no alteration observed; +: slight alteration; ++: moderate alteration; +++: severe alteration. Different signs indicate significant differences (p < 0.05).

Gills

The fish group exposed to 0.35 mg/mL of eugenol displayed gill alterations. Both the filaments and the primary lamellae were seen to be swollen, with interdigitations (Figure 1, B1). An active, general hyperaemia was also observed, with numerous cells of an exudative and inflammatory nature, and abundant haemorrhagic areas (Figure 1, B2). Furthermore, the components forming the respiratory barrier showed a slight disruption (Figure 1, B3).

Figure 1.

Histopathological examination of gills and nerve tissue of zebrafish. Control group (a), eugenol group (0.35 mg/mL) (b), MS-222 group (0.50 mg/mL) (c) and buffered MS-222 group (0.50 mg/mL) (d). Bars: OM 100 µm; SEM 10 µm; TEM 10 µm. Gills: Aspect of the gill filaments (Fi) with chondrocyte (Cd) and lamellae (La) with capillaries (Cp) (A1–A3). Disrupted gill filaments (Fi) and lamellae (La) with desquamation and hyperaemia (B1, C1). Gill filaments (Fi) and lamellae (La) structured with oedema and hyperaemia (D1). Gill filaments (Fi) and lamellae (La) with abundant infiltrative cells (In) (B2, C2). Gill filaments (Fi) and swollen lamellae (La) (D2). Hyperaemic lamellae (La) with capillaries (Cp) with collapsed lumina (B3, C3). Hyperaemic (Hi) lamellae (La) with capillaries (Cp) with dilated lumina (D3). Nerve tissue: An aspect of the spinal cord. Radicular neurons (N) (A4, A5, D4, D5). Spinal cord with radicular neurons (N) with tigrolysis processes (Ti) (B4, C4). Radicular neurons (N) with a vacuolised cytoplasm (Va) and nuclear picnosis (Np) (B5, C5).

The group exposed to 0.50 mg/mL of MS-222 presented a strong gill alteration, with total loss of its normal structure. A marked disruption was detected, with the total destruction of the lamellae, a notable hyperaemia, oedema, exudation and extensive haemorrhage. The images obtained correspond to a grave, vascular-toxic shock (Figure 1, C1, C2 and C3).

In the buffered 0.50 mg/mL of the MS-222-treated group, the severity of the gill alterations was milder than that of the group exposed to 0.50 mg/mL of non-buffered MS-222, so that both the filaments and lamellae maintained practically normal structures. In the gill filaments, the capillaries showed themselves to be hyperaemic, and oedema was found in the interstitial tissue (Figure 1, D1). The primary lamellae were swollen, with a slight destruction of the respiratory tissue (Figure 1, D2). An increase in capillary lumina was evidenced (Figure 1, D3).

Nerve tissue

In the group treated with 0.35 mg/mL of eugenol, it was the nerve tissue that exhibited the greatest disruption. The radicular neurons of the spinal cord were those most affected, with significant general oedema and ballooning degeneration in the motor neurons, and with a loss of Nissl granules (Figure 1, B4). The neurons showed areas of extensive necrosis due to protein aggregation, with a strong densification of the cytoplasm, and general vacuolisation of the membrane system, presenting irregular nuclei, dilatation of the nuclear envelope and denaturation of the chromatin. No neuroglia mobilisation was observed (Figure 1, B5).

In the group treated with non-buffered MS-222 (0.50 mg/mL), a diffuse oedema was observed, affecting both the neurons and the neuropil. Under the OM, the neurons displayed a tigroid degeneration, with a homogenisation of the perikaryon and a clear increase in the nuclear basophilia (Figure 1, C4). Some serious ultrastructural alterations were also recorded (Figure 1, C5), such as the densification of the neuronal cytoplasm due to protein aggregation. The whole vacuolar system exhibited an evident vacuolisation, a prominent nuclear picnosis, with the presence of a denatured chromatin, with large granules of heterochromatin, and a structural loss of the nucleole.

In the group treated with buffered MS-222 (0.50 mg/mL), the nerve tissue had a diffuse oedema in the radicular neurons of the spinal cord, but the Nissl bodies were maintained. Some neurons showed a certain tigroid degeneration, with a homogeneisation of the perykarion (Figure 1, D4 and D5).

Other tissues

The images obtained of the liver, kidney, heart and intestine of the different groups are displayed in Figures 2 and 3. Hardly any modifications stand out in the different groups for the rest of the tissues. In all of them, the liver kept the structure of the hepatic cords, and a certain movement of the cellular glucogen was noted in the hepatocytes. On examining the kidneys, in the groups exposed to eugenol (0.35 mg/mL), a glomerulopathy was observed, with delation in the Bowman capsule. The nephrons remained apparently normal in all the groups.

Figure 2.

Histopathological examination of zebrafish liver and kidney. Control group (a), eugenol (0.35 mg/mL) group (b), MS-222 (0.50 mg/mL) group (c) and buffered MS-222 (0.50 mg/mL) group (d). Bars: OM 100 µm; TEM 10 µm. Liver: Apparently normal hepatic cords, polyedric morphology with central nucleus and clear cytoplasm. An aspect of the apparently normal hepatocyte (He), with cytoplasmic organoids, reticules and mitochondria. Apparently normal pancreatic parenchyme (Pa) (A1–D1, A2–D2). Kidney: Apparently normal renal parenchyme, with tubules (Tu), glomerules (Gl) and abundant microvilli (Mi) in the apical zone of the cell. An aspect of the renal glomerule (GI) with basal membrane (Mb), podocytes (Po) and apparently normal endothelial cells (Ce) (A3–D3, A4–D4).

Figure 3.

Histopathological examination of zebrafish heart and intestine. Control group (a), eugenol (0.35 mg/mL) group (B), MS-222 group (0.50 mg/mL) (C) and buffered MS-222 group (0.50 mg/mL) (D). Bars: OM 100 µm; TEM 10 µm. Heart: An aspect of the myocardium with cardiac fibres (Fc) with apparently normal striae (Es) (A1–D1, A2–D2). Intestine: an aspect of the enterocytes (En) with abundant apparently normal microvilli (Mi) (A3–D3, A4–D4).

With regard to the heart, only the group exposed to 0.35 mg/mL of eugenol showed oedema in the interstice of the myocardium, with no alterations in the rest of the groups. Finally, no notable changes were found in the intestinal mucosa in any of the groups.

Discussion

In this study, the use of ethanol as a solvent, bicarbonate as a buffer and MS-222 as a sedative agent must be taken into account, since they can alter the results obtained. Regarding the toxicity of ethanol in adult zebrafish, Gerlai et al.²⁶ exposed zebrafish to a dose of 7.89 mg/mL for one hour – a much longer time and a higher dose than ours – and they found no abnormalities in the fish’s posture or motor patterns, such as paralysis or seizures, suggesting that this dose is not harmful to the fish. On the other hand, Agues-Barbosa et al.,²⁷ who exposed zebrafish to 3.95 mg/mL for 30 minutes, noted an excitatory effect on locomotion, which disappeared after chronic exposure for 22 days. Since we did not find any histopathological alterations in the fish of the control ethanol group, we can dismiss this solvent as being responsible for the lesions found in fish exposed to eugenol. Second, the bicarbonate effect has also been rejected, since no lesions were found in the bicarbonate control group, as bicarbonate only increases aquarium alkalinity and is commonly used in aquaculture.²⁸ Finally, neither was the effect of MS-222 as a sedative agent considered for the same reason, that is, the absence of alterations in the histopathological analysis.

Regarding the gill lesions, it should be noted that the lamellar epithelium is highly vascularised and structurally adapted to gaseous exchange.²⁹ For that reason, the swelling in the primary lamellae and the appearance of haemorrhages and hyperaemic areas in the animals exposed to eugenol and MS-222 led us to believe that these changes are produced with the aim of increasing the functional surface of the gills in order to extract more oxygen from the medium.³⁰ In this respect, insufficient branchial ventilation is a well-documented undesirable side effect of euthanasia.³¹ Furthermore, Dong et al.³² reported that exposure to MS-222 and eugenol regulated the metabolic pathways and triggered an immunoreaction in the gills of Asian sea bass (Lateolabrax maculatus). In their study, Wang et al.³³ concluded that administering MS-222 and eugenol to that species could harm the morphological structure of the gills, induce apoptosis, interfere in osmoregulation and modulate immunity functions. In fact, in the fish sacrificed with non-buffered MS-222, an important branchial disruption was highlighted. The parts mainly affected were the branchial arch mucosa and the principal lamellae, especially the primary ones. These grave alterations could be attributed to the marked acidic nature of MS-222, as they were not present in the groups exposed to MS-222 buffered with bicarbonate. Other studies have demonstrated that MS-222 causes swelling in the erythrocytes, which impedes the absorption of oxygen in the gill lamellae.^34,35

Regarding the alterations in the nervous system, eugenol caused more damage than MS-222. In fact, eugenol at euthanasia concentrations (0.35 mg/mL) caused serious neuronal lesions, fundamentally in the radicular neurons of the spinal cord. The results obtained can be explained by the fact that eugenol is distributed throughout the nerve tissue (it is a highly lipophilic substance)¹¹ and by its effects on ion channels in neuronal cells.¹² Moreover, Barbas et al.¹⁵ verified that eugenol did not manage to promote central nervous system depression in tambaquis (Colossoma macropomum), and that it could have a potentially toxic effect on fish brains, given that it caused an intense neuronal excitability, with seizure patterns on electroencephalogram. On the contrary, MS-222 blocks sodium channels mostly in the muscles and, to a lesser degree, the potassium channels in nerve membranes,³⁶ which would explain the lesser damage in nerve tissue found in the fish exposed to this anaesthetic.

On assessing all the groups studied, it was proved that eugenol presented a shorter euthanasia time, following by buffered MS-222, which agrees with other studies comparing eugenol with other anaesthetics commonly used in fish.^22,37,38 In the case of MS-222, in all the concentrations studied, the fish were euthanised in a shorter time with the buffered MS-222 solution than those with non-buffered MS-222. As for buffering MS-222 with bicarbonate, studies are conflicting regarding the euthanasia time. Wilson et al.²⁵ found that immersion in 0.25 mg/mL of non-buffered MS-222 triggered the cessation of the opercular beat in around 50 seconds – a much shorter time than ours. Moreover, Collymore et al.³⁹ reported a much longer mean time (500 seconds) up to the loss of the opercular beat at the same dose of MS-222, this time buffered. Thus, it might be thought that MS-222 loses its efficacy when it is buffered. Nevertheless, von Krogh et al.⁴⁰ established an average time that was much closer to that determined by us (120 seconds) for a buffered solution of 0.50 mg/mL of MS-222. We are unable to explain these euthanasia time differences between studies, which could be due to environmental factors (i.e. temperature, oxygen content, pH, hardness and salinity of water).¹⁶ For example, water at a low temperature can dramatically increase the potency of anaesthetic agents such as MS-222.⁸ Regardless of euthanasia time and due to the greater severity of the lesions, mainly in the gills, between MS-222 and buffered MS-222, it is clear that buffering MS-222 is necessary to ensure the animal’s welfare. In fact, Davis et al.⁴¹ showed that the use of unbuffered MS-222 causes serious epidermal and corneal damage in fish.

For both anaesthetic agents, we were able to observe that when the euthanasia dose was increased, the euthanasia time decreased. Iversen et al.⁴² describe similar dynamics in codfish (Gadus morhua) and in salmon (Salmo salar). Similarly, we determined that a higher euthanasia dose is related to more severe histopathological lesions. The latter is a key point for refinement, as it is important to note that a shorter euthanasia is not always better than a longer one, that is, a longer time with less stress could be better for zebrafish than a shorter time with more stress. In this context, it would be advisable not to use high doses. The same occurs between eugenol and MS-222, as we have seen, when although eugenol is more powerful than MS-222, it seems to be more aversive as our results indicate.

Euthanasia – ‘the act of inducing humane death in an animal’ – should cause minimal pain, stress and discomfort.⁵ Therefore, despite the extensive use of eugenol and MS-222 for fish euthanasia, more studies are necessary on their toxic effects, which should be carefully reviewed, since they generate uncertainty, that has a direct repercussion on ethical themes and on animal welfare.

Conclusions

Eugenol has proved to be a more powerful euthanasia agent than MS-222 in zebrafish, giving significantly shorter euthanasia times. However, exposing the fish to eugenol euthanasia doses caused gill modifications, as well as serious lesions in neuron groups and alterations in nerve tissue, which were comparatively much more severe than those described for MS-222. The results obtained with MS-222 also showed a marked gill alteration, resulting from its employment. In this sense, the addition of a buffer to the euthanasia MS-222 solution enhanced the effectiveness of the drug, with significantly shorter euthanasia times being obtained in zebrafish than with the non-buffered solution, and reduced the degree of severity of the lesions described. In view of the above, we determined that the MS-222 solution when buffered constituted a more effective, safer and innocuous method of euthanasia for use in research with zebrafish.

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) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Nahúm Ayala-Soldado

Rafael Mora-Medina

Antonio Jesús Lora-Benítez

Ana M Molina-López

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