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Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg(elavl3:H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine’s anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch.

Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg( elavl3 :H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine's anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ohnesorge, Wilzopolski, Steinfath, Lewejohann, Banneke and Heinl.)

Simple Summary: Zebrafish (Danio rerio) younger than five days post fertilization are not protected by legislation, and protocols for their euthanasia are poorly explored. In the present paper, we assess the euthanasic efficacy of anesthetic overdose, hypothermic shock, and electrical stunning on zebrafish at <12 h post fertilization, and 1 and 4 days post fertilization utilizing laboratories in different countries. Based on the survival/recovery rates 24 h after treatment, the most effective methods were an overdose with either clove oil or lidocaine with ethanol, and electrical stunning. For the oldest larvae, signs of aversion during treatment demonstrated that all the tested anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited anesthetic treatment was lidocaine hydrochloride, 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method is new and needs further assessment in other laboratories to draw firm conclusions. Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at <12 h post fertilization (hpf), 24 hpf, and 4 dpf. Based on the survival/recovery rates 24 h after treatment, the most effective methods were clove oil, lidocaine with ethanol, and electrical stunning. For 4 dpf larvae, signs of aversion during treatment demonstrated that all anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited euthanasic treatment was lidocaine hydrochloride 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol, which euthanized both embryos and larvae in an efficient and stress-free manner. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method needs further assessment in other laboratories to draw firm conclusions. [ABSTRACT FROM AUTHOR]

Tricaine, or MS-222, is the most commonly used chemical anesthetic in zebrafish research. It is thought to act via blocking voltage-gated sodium channels, though its mechanism of action, particularly at the neuronal level, is not yet fully understood. Here, we first characterized the effects of tricaine on both body balance and touch responses in freely swimming animals, before determining its effect on the neural activity underlying the optokinetic response at the level of motion perception, sensorimotor signaling and the generation of behavior in immobilized animals. We found that the standard dose for larvae (168 mg/L) induced loss of righting reflex within 30 seconds, which then recovered within 3 minutes. Optokinetic behavior recovered within 15 minutes. Calcium imaging showed that tricaine interferes with optokinetic behavior by interruption of the signals between the pretectum and hindbrain. The motion sensitivity indices of identified sensory neurons were unchanged in larvae exposed to tricaine, though fewer such neurons were detected, leaving a small population of active sensory neurons. We then compared tricaine with gradual cooling, a potential non-chemical alternative method of anesthesia. While neuronal tuning appeared to be affected in a similar manner during gradual cooling, gradual cooling induced a surge in calcium levels in both the pretectum and hindbrain. This calcium surge, alongside a drop in heartrate, is potentially associated with harmful changes in physiology and suggests that tricaine is a better anesthetic agent than gradual cooling for zebrafish laboratory research.

Tricaine (TMS) is usually used as an anesthetic in the transportation of aquatic animals. However, when consumed, the numerous residual drug residues found in fish can cause a high risk to human health. Therefore, it is important to develop a rapid and sensitive method for the detection of this drug in food. Here we have developed a novel lateral-flow immunochromatographic strip (LF-ICS) method for the detection of TMS in fish samples, using a very effective anti-TMS monoclonal antibody with a half-maximal inhibitory concentration value of 31.79 ng/mL. After the K2CO3 and coating antigen concentrations were optimized in phosphate buffered saline (pH 7.4), the LF-ICS was used to determine TMS levels in fish samples spiked with varying concentrations of the drug. Following this, the values for the visual limit of detection and the cut-off for TMS were found to be less than 0.1 and 1 μg/mL, respectively. These results indicated that our developed LF-ICS is applicable for on-site screening of TMS in fish samples.

Research with anesthetics in fish has been extensively explored over the years. The use of Tricaine (MS-222) as an anesthetic is widely known as an ideal anesthetic for fish intended for consumption. It is a synthetic drug derived from benzocaine, however, this drug is not available in the Brazilian market. Tambaqui (Colossoma macropomum) is a freshwater fish natural from the Amazon River and Orinoco River with a very characteristic flavor, it is responsible for a large part of the fish production in the Pará State. This study aimed to evaluate the anesthetic efficacy by monitoring the cardiac function of tambaquis submitted to different concentrations of tricaine: 100 mg.L−1, 150 mg.L−1, 200 mg.L−1, and 250 mg.L−1, for behavioral evaluation during the anesthetic induction and return phase. For the electrocardiogram, the animals were subjected to the same doses in other to record the electrocardiographic data during anesthetic induction and recovery for a period of 10 min. The MS-222 has a potential risk of causing electrocardiographic changes even at the lowest supported doses. • This is the first electrophysiological study of C. macropomum exposed to tricaine. • The high doses of tricaine of tambaqui can cause alterations in the hemodynamics of these animals. • The tricaine altered the electrocardiogram of tambaqui, during induction and recovery. • High concentration of tricaine caused cardiovascular risks to animals. [ABSTRACT FROM AUTHOR]

Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at

Tricaine (TMS) is usually used as an anesthetic in the transportation of aquatic animals. However, when consumed, the numerous residual drug residues found in fish can cause a high risk to human health. Therefore, it is important to develop a rapid and sensitive method for the detection of this drug in food. Here we have developed a novel lateral-flow immunochromatographic strip (LF-ICS) method for the detection of TMS in fish samples, using a very effective anti-TMS monoclonal antibody with a half-maximal inhibitory concentration value of 31.79 ng/mL. After the K2CO3 and coating antigen concentrations were optimized in phosphate buffered saline (pH 7.4), the LF-ICS was used to determine TMS levels in fish samples spiked with varying concentrations of the drug. Following this, the values for the visual limit of detection and the cut-off for TMS were found to be less than 0.1 and 1 μg/mL, respectively. These results indicated that our developed LF-ICS is applicable for on-site screening of TMS in fish samples. [ABSTRACT FROM AUTHOR]

Zebrafish are often euthanized by overdose of anaesthesia. However, fish may have aversion towards some anaesthetics, and protocol efficacy varies between species. Using wild type adult Danio rerio, we assessed time to loss of opercular beat, righting, and startle reflexes during induction of anaesthetic overdose by either tricaine (0.5 g/L or 1 g/L), benzocaine (1 g/L), 2-phenoxyethanol (3 mL/L), clove oil (0.1%), isoeugenol (540 mg/L), lidocaine hydrochloride (1 g/L), or etomidate (50 mg/L). Initial screening demonstrated that benzocaine and buffered lidocaine hydrochloride achieved the fastest loss of reflexes. The rapid induction times were confirmed when retesting using larger batches of fish. The fastest induction was obtained with 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO3, in which all adult zebrafish lost reflexes in less than 2 min. Next, we monitored signs of distress during benzocaine or buffered lidocaine hydrochloride overdose induction. The results indicated that buffered lidocaine hydrochloride caused significantly less aversive behaviors than benzocaine. Finally, we tested several buffers to refine the lidocaine hydrochloride immersion. The most efficient buffer for euthanasia induction using 1g/L lidocaine hydrochloride was 2 g/L NaHCO3 with 50 mL/L 96% ethanol, inducing immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviors during treatment.

Although daily variations in drug pharmacokinetics have been reported for a variety of teleost species, the influence of this daily variation on the cortisol response following anaesthesia remains poorly understood. To address this, two experiments were performed. The first experiment described the daily patterns of cortisol and glucose secretion in tilapia (Oreochromis niloticus). The second experiment investigated how the timing of anaesthetic administration (specifically at mid‐light [ML] or at mid‐dark [MD]) affects the induction and recovery times and plasma cortisol and glucose levels of juvenile Nile tilapia exposed to benzocaine, clove oil or tricaine methanesulphonate (MS‐222). The results revealed that the effect on the stress response associated with the moment when anaesthesia took place (ML or MD) varied according to the treatment (p < 0.05). Cortisol levels were significantly higher at ML for MS‐222 (ML = 116.23 ± 25.55; MD = 48.25 ± 22.33 ng/dl) (p < 0.05) and clove oil (ML 59.73 ± 14.27; MD 38.26 ± 12.07 ng/dl) (p < 0.05), whereas no significant differences were found between ML and MD cortisol levels for the control treatment (ML = 72.91 ± 18.42; MD = 64.80 ± 10.68 ng/dl) (p > 0.05) or in the benzocaine‐treated group (ML = 38.7 ± 4.90; MD = 38.60 ± 3.69 ng/dl) (p > 0.05). The highest plasma cortisol level in ML was found in the MS‐222‐treated group. All the tested anaesthetics had similar cortisol levels at MD (p > 0.05). [ABSTRACT FROM AUTHOR]

Summary: The purpose of this study was to evaluate the anaesthesia effects of eugenol and MS‐222 sedatives applied on juvenile Lateolabrax maculatus during simulated transport. In experiment 1, the juveniles were divided into two groups, with seven concentrations tested on each group (eugenol [4, 6, 8, 10, 12, 14 and 16 mg/L] and MS‐222 [20, 30, 40, 50, 60, 70 and 80 mg/L]). Induction and recovery times were recorded. The time for anaesthesia was shortened, and the time for complete recovery was prolonged as the anaesthetic concentration increased. The optimal transport concentration for each anaesthetic tested was 6 mg/L of eugenol and 30 mg/L for MS‐222. In experiment 2, the 5‐hr simulated transport test showed that the survival rate of L. maculatus juveniles with anaesthesia was 100%, and without anaesthesia, survival was 60%. After 24 hr of recovery following transport, the fish showed 100% survival for the group with added anaesthetic and 40% for the group without added anaesthetic. Compared to the non‐anaesthetized groups, the anaesthetized transport groups showed significant increases in the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (p < 0.05). The levels of AST, ALT and alkaline phosphatase (AKP) were significantly higher in the MS‐222 transport group than in the eugenol transport group (p < 0.05). The levels of AKP were significantly higher in the non‐anaesthetized transport group than in the anaesthetized group (p < 0.05). According to the present experiment results, eugenol was an efficient anaesthetic in L. maculatus, and we recommend eugenol instead of MS‐222 as an anaesthetic for the short‐time transport of L. maculatus. [ABSTRACT FROM AUTHOR]

Abstract The efficacy of four anesthetic agents was evaluated in the lambari-bocarra Oligosarcus argenteus. Adults (weight, 11.3 ± 3.3 g; length, 10.2 ± 1.1 cm) were exposed to tricaine (25, 50, 75, 100, 125, 150, and 175 mg L−1), benzocaine (25, 50, 75, 100, and 125 mg L−1), clove oil (25, 50, 75, 100, and 125 mg L−1), and menthol (25, 50, 75, 100, and 125 mg L−1) for inducing deep and surgical anesthesia. The anesthetic effect was evaluated by measuring time to induction stages, recovery time, appetite return, and 96-h mortality rate. For all anesthetics, the induction time decreased as their concentration increased. The anesthetics had no effects on fish mortality, and appetite return occurred before 24 h. The tricaine induced deep and surgical anesthesia in 3 min at concentrations ranging from 100 to 175 mg L−1 and from 150 to 175 mg L−1, respectively. The highest concentrations of tricaine, 150 and 175 mg L−1, provided shorter induction times and recovery times were not different when using concentrations ranging from 100 to 175 mg L−1. The benzocaine concentrations between 75 and 125 mg L−1 and 100 and 125 mg L−1, respectively, provided deep and surgical anesthesia induction times below 3 min. Recovery time was superior at benzocaine concentrations of 50 to 100 mg L−1. Clove oil effectively induced anesthesia at lower concentrations, with 50 and 75 mg L−1 inducing deep and surgical anesthesia. The recovery times were not different when using concentrations ranging from 25 to 125 mg L−1. All menthol concentrations tested effectively induced deep anesthesia. The menthol concentration of 75 mg L−1 effectively induced surgical anesthesia in 3.62 min, longer recovery times at 25 and 50 mg L−1. In conclusion, the tested anesthetics effectively induced anesthesia in lambari-bocarra, with deep anesthesia being induced at concentrations of 100 and 75 mg L−1 for tricaine and benzocaine, respectively, and 50 mg L−1 for clove oil and menthol. For surgical anesthesia, the recommended concentrations are 150, 100, 75, and 100 mg L−1 for tricaine, benzocaine, clove oil, and menthol, respectively. Highlights • This work evaluates the use of tricaine, benzocaine, clove oil and menthol at different concentrations in lambari-bocarra. • Lowest effective concentration for induction in surgical anesthesia was of the clove oil and the highest with the tricaine. • For deep anesthesia, natural anesthetics had the lowest effective concentration in relation to synthetic anesthetics. • Prolonged recovery times were observed with the use of natural anesthetics compared to tricaine. • Anesthetics had no effect on fish mortality during the tests and up to 96 h of observation, return of appetite before 24 h. [ABSTRACT FROM AUTHOR]

Anesthesia plays a critical role in prevention of mechanical injuries and manipulation stress in fish. Commercially reared fish are subjected to confinement and handling including netting, weighing, sorting, vaccination, and transport. In research, fish may also undergo surgical procedures ranging from tagging, blood sampling, and small incisions for invasive procedures. In these situations, treatment with anesthetic agents may be necessary to ensure the welfare of the fish. In this review, we provide information on optimal dosage and effects on fish physiology of three widely-used anesthetics worldwide: clove oil, tricaine methane sulphate, and 2-phenoxyethanol. These anesthetics are effective and have been widely used with many species of fish. [ABSTRACT FROM AUTHOR]

Highlights • MS-222 induces a dose-dependent increase in mortality during 256-cell stage. • Abnormal cartilage development as well as changes in noggin expression were perceived at 50% epiboly stage. • Locomotor deficits were also detected at 50% epiboly and associated with changes in noggin expression. • Developmental-related genes were upregulated to some extent following exposure during the 1–4 somites stage. • The use of MS-222 must be carefully considered in some conditions to avoid its influence on scientific outcomes. Abstract MS-222 has been widely used as an anaesthetic in fish, thus, raising the need to infer about its toxicological safety during development. In this study, MS-222 toxicity in zebrafish embryos was evaluated after a 20-min exposure at different stages of development. Embryos exposed during the 256-cell stage displayed an increase in mortality, associated with defective early developmental pathways. Following exposure during the 50% epiboly stage, an increase in mortality and abnormal cartilage development, as well as changes in noggin expression were observed. Locomotor deficits were detected and associated with changes in early signalling pathways through the involvement of noggin. When exposed at the 1–4 somites stage, zebrafish were phenotypically normal, although presenting changes in the expression pattern of developmental genes. These findings indicate a teratogenic impact, independent of sodium channels that should be taken into consideration when MS-222 toxicity is discussed. [ABSTRACT FROM AUTHOR]

Abstract The purpose of this study was to evaluate the effectiveness of the alfaxalone formulation Alfaxan™ as an immersion anesthetic in tropical fish species compared to that of tricaine methanesulfonate (MS-222). 22 black spot barbs (Puntius filamentosis) measuring (mean±SD) 11.4 ±1.4 cm in body length and 22 peacock cichlids (Aulonocara spp.) (measuring 8.4 ± 1.6cm were anesthetized in water baths containing 100 mg/L of MS-222 buffered with 200 mg/L of bicarbonate or 5 mg/L of alfaxalone following a 2-week washout period. Time to maximum effect, recovery periods, self-righting, spontaneous swimming movements, opercular movements, and response to noxious stimuli were recorded. The following results are for the black spot barbs following MS-222 and alfaxalone anesthesia, respectively: mean times (±SD) to surgical anesthesia were 5.5 ± 2.11 and 3.27 ± 1.72 minutes and mean recovery times were 2.95 ± 0.9 and 9.14 ± 3.15 minutes. The peacock cichlid anesthetic protocols for MS-222 (20 of 22 cichlids) and alfaxalone (20 of 21 cichlids) produced the following results, respectively: mean times (±SD) to surgical anesthesia were 14.75 ±5.43 and 11.1 ± 9.84 minutes and mean recovery times were 3.6 ±0.82 and 22.4 ±11.3 minutes. Median recovery time from 5 mg/L alfaxalone was significantly longer (P < 0.001) in both species, by 5 minutes for black spot barbs and by 17 minutes for peacock cichlids. Variation in induction and recovery times between species was observed, with black spot barbs having significantly (P < 0.0001) faster induction times when treated with both drugs, and a faster recovery time from 5 mg/L alfaxalone. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the study was to determine the optimum concentration of MS‐222 for given size groups of pikeperch and water temperatures. The study considered three size groups of pikeperch (body weight [BW] 8.56, 15.72, 52.91 g), an MS‐222 water solution (50, 100, and 150 mg/L), and two temperatures (20 or 23°C). It was revealed that the optimum MS‐222 concentration depended largely on the size of the pikeperch and on water temperature. For fish with a BW <10 g the recommended concentration is 100 mg/L but only at 23°C. For fish with a BW of 10–40 g at an immersion temperature of 20°C the safe MS‐222 concentration ranges from 100 to 150 mg/L. However, at a temperature of 23°C the recommended concentration of the anesthetic is 100 mg/L. Similarly for larger fish, that is, fish with a BW >40 g, the optimum MS‐222 concentration at a water temperature of 20°C is in the range of 100–150 mg/L, but at a temperature of 23°C is it approximately 100 mg/L. Additionally, when exposure to the anesthetic is shorter (several min), a concentration of 150 mg/L is also safe for juvenile pikeperch of this size. [ABSTRACT FROM AUTHOR]

OBJECTIVE To substantiate current AVMA guidelines for immersion euthanasia of goldfish (Carassius auratus) with tricaine methanesulfonate (TMS), determine whether immersion in propofol at 5 times its immersion anesthesia concentration for 30 minutes is sufficient for euthanasia of goldfish, and quantify the duration of myocardial contraction following immersion of goldfish in TMS and decapitation. DESIGN Prospective clinical trial. ANIMALS 36 healthy, adult goldfish. PROCEDURES Goldfish were randomly assigned to be immersed in I of 6 test solution treatments (n = 6/treatment): TMS (500 mg/L) for 15 minutes followed by placement in anesthetic agent-free water (TI5W), placement out of water (TI5A), or decapitation (TI5D); TMS (1,000 mg/L) for 15 minutes followed by placement in anesthetic agent-free water (TI5XW); TMS (500 mg/L) for 30 minutes followed by placement in anesthetic agent-free water (T30W); or propofol (25 mg/L) for 30 minutes followed by placement in anesthetic agent-free water (P30W). Any fish that resumed operculation in group TI5A was returned to anesthetic agent-free water. Times from onset of immersion to induction of anesthesia, cessation and resumption of operculation, and recovery (TI5W, TISA, TI5XW, T30W, P30W) or cessation of Doppler ultrasounds (TI5D) were recorded. RESULTS Overall, 5 of 6, 6 of 6, 6 of 6, 6 of 6, and 5 of 6 fish survived in the TI5W, TI5A, TI5XW, T30W, and P30W groups, respectively. Median time to cessation of Doppler ultrasounds in group TI5D was 77.5 minutes (range, 30 to 240 minutes). CONCLUSIONS AND CLINICAL RELEVANCE Timed immersion in test solutions (TMS at 500 mg/L or 1,000 mg/L or propofol at 25 mg/L) resulted in death in only 7% (2/30) of immersed goldfish. Myocardial contractions continued for up to 4 hours in decapitated goldfish. [ABSTRACT FROM AUTHOR]

Abstract: A system based on batch injection analysis (BIA) associated with amperometric detection at screen‐printed carbon electrode was used for the precise and rapid quantification of the anesthetics compounds benzocaine and tricaine in fresh fish fillets. Along this study, the best conditions for the BIA‐amperometry system were stablished for the rapid determination of these compounds. The results obtained demonstrate that the proposed method is an interesting alternative to the chromatographic methods, once it allows to perform rapid analysis (more than 300 injections per hour) with low limits of detection (3.02×10−8 mol L−1 for benzocaine and 3.19×10−8 mol L−1 for tricaine), using just 80 μL of sample for each analysis. Furthermore, it was possible to obtain high repeatability for both compounds analyzed, demonstrating good performance. The simple sample preparation developed in this study drastically reduced the amount of fat in the fish extract, favoring precision, as shown by the results of the recovery studies of both anesthetics contained in the fish samples (values above 99 % for both analytes). [ABSTRACT FROM AUTHOR]

This experiment was devised to evaluate the effects of tricaine methanesulfonate (MS-222) on blue tilapia, Oreochromis aureus at five different salinities (0, 8, 16, 20, 24 ppt) and four different tricaine concentrations (200, 300, 400, 500 mg l-1). Even though, a body of literature exist about the tricaine usage on fish, not much information is present on tricaine with salt. The requirement time to anaesthetize fish depends on intensity of tricaine concentration and salinity. Induction time of fish decreased as tricaine concentrations increased. When exposed to any of tricaine concentrations, fish entered a deep state of anaesthesia (induction time ranged between 0.19 and 2.54 min). Recovery time was highest at 400-500 mg l-1 of tricaine as salinity increased. Tricaine + salt combination is strongly recommended to use in blue tilapia culture. Ideal tricaine concentration was 200 mg l-1 of tricaine at 8 ppt of salinity to reduce stress in blue tilapia. [ABSTRACT FROM AUTHOR]

The present study was conducted to assess an optimal dose of tricaine methansulfonate (MS-222) for rapid anesthesia and recovery in juvenile Yellow perch Perca flavescens. Yellow perch (7.0 g/fish) exposed to increasing concentrations of MS-222 (100, 150, 200, 250 and 300 mg/L) showed significant (P<0.05) decreased anesthetic induction and increased recovery time with increasing MS-222 dose. Among the doses tested, the 250 mg/L MS-222 dose showed the shortest anesthetic induction and recovery times. Blood glucose and hematocrit levels were significantly (P<0.05) lower in juvenile yellow perch exposed to the 250 mg/L MS-222 dose than the other MS-222 doses tested. Juvenile yellow perch (about 9.7 g/fish) exposed to 250/mg MS-222 combined with bicarbonate buffer at three ratios (w/w: 1:0.5, 1:1 and 1:2) showed significantly (P<0.05) reduced anesthesia induction and recovery times. The shortest times of anesthesia induction and recover were observed in treatments using the 1:1 MS-222 to buffer ratio. Analysis on serum biochemical parameters (e.g., osmolality, glucose, albumin, calcium, alanine transaminase, total protein and calcium) showed that use of sodium bicarbonate with MS-222 did not have any significant effect on the above measurements compared with those determined in fish exposed to MS-222 only. Anesthesia induction time and recovery time of yellow perch were significantly affected by body sizes. These results suggest that a brief exposure to a 250 mg/L MS-222: sodium bicarbonate dose may be optimal for minimizing handling disturbance in juvenile yellow perch. [ABSTRACT FROM AUTHOR]

The article offers information on role of Anaesthesia in fisheries research and aquaculture in order to reduce stress in fish for transportation, catching and high density. Also includes usage of Tricaine methanesulfonate in fish food approved by Food and Drug Administration; and studies of fish larvae to understand effects of anaesthetics in larvae.

The aim of this study was to evaluate the anaesthetic efficacy and the effect of sedation with tricaine ( TMS®), also known as MS-222, on secondary and oxidative stress parameters in juvenile tambaqui, Colossoma macropomum transported in hyperoxia for 2, 6 and 10 hr. Juveniles were placed in aquaria containing six different concentrations of buffered tricaine (150, 180, 210, 240, 270 and 300 mg/L) and the times for anaesthesia induction and recovery determined. Fish transported in hyperoxic conditions were investigated for glycemia, ionic concentration (K+, Ca++, Na+) and osmolality, haematocrit (Ht) and haemoglobin concentration (Hb), partial pressure of gases ( pCO2 and pO2), pH and bicarbonate concentration ( [ABSTRACT FROM AUTHOR]

Although tricaine methanesulfonate (MS-222) is often used to tranquilize fish, the guidelines for its use in sea bass, a brackish species, have not been established. The aim of the study reported here was to establish the tranquilizing concentration of MS-222, based on the time required for MS-222 residue elimination and withdrawal. Thirty-six fish (6/group) were immersed in different concentrations of MS-222 (0, 30, 50, 60, 70 and 90 mg/l) to evaluate the fish physiological behavior. After 200 fish were anesthetized at 90 mg/l, the fish achieved a healthy recovery within 72 h after the administration of saline. The 10 fish in the control group were subject to the same treatment without anesthesia, 3 out of 10 died. After 108 fish (54/group) were immersed in 30 or 60 mg/l of MS-222, the sedated fish were healthy during and after the 8 h of transport. However, all the 10 fish in the control group died within 3 days. By high-performance liquid chromatography, the residue of MS-222 was assessed. In the skinned muscle and liver, the elimination half-life was 5.54 and 5.27 h (30 mg/l) and 8.72 and 7.15 (60 mg/l), respectively, and the withdrawal time was at least 4.5 days at 30 mg/l and 7.5 days at 60 mg/l. [ABSTRACT FROM AUTHOR]

Tricaine mesylate (MS-222) is one of the most used anesthetics in fish. It can be absorbed by the human body via food consumption, with related detriments to human health. In this study, a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was developed for the determination of MS-222 in carp muscle and water samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). For cleanup procedure, multiplug filtration cleanup (m-PFC) method with n- Hexane delipidation was adopted. The extraction solvent, cleanup methods and sorbents were optimized. All method validation parameters were in the acceptable range. The dissipation behavior study was followed by the method development. Firstly, the anesthesia dose and time were optimized in application study. Secondly, carps were revived for different period of time with (experimental group) and without (control group) the oxygenation aeration treatment to compare the dissipation rate of MS-222. After being anesthetized for 6 h at 50 mg/L and 12 h of elimination, the concentrations of MS-222 in crap muscle and water of experimental group was lower than those of control group. After 36 h of elimination under oxygenation aeration, over 90% of MS-222 was dissipated in carp muscle. The results showed that the half-life of MS-222 in carp muscle was 6.2 h. The findings suggested that the commonly-used oxygenation aeration treatment in aquaculture production had potential effects in accelerating the dissipation of MS-222 in carp and water. In this study, three days of withdrawal period was recommended in carps after MS-222 administration under oxygenation aeration. [ABSTRACT FROM AUTHOR]

Tricaine methanesulfonate (MS-222) has been widely used in intensive aquaculture systems to control stress during handling and confinement operations. This compound is dissolved in the water tanks and, once it is present in the Recirculating Aquaculture Systems (RASs), MS-222 can reach the environment by the discharge of contaminated effluents. The present work proposes the implementation of the adsorption process in the RASs, using pyrolysed biological paper mill sludge as adsorbent, to remove MS-222 from aquaculture wastewater. Adsorption experiments were performed under extreme operating conditions, simulating those corresponding to different farmed fish species: temperature (from 8 to 30 °C), salinity (from 0.8 to 35‰) and different contents of organic and inorganic matter in the aquaculture wastewater. Furthermore, the MS-222 adsorption from a real aquaculture effluent was compared with that from ultrapure water. Under the studied conditions, the performance of the produced adsorbent remained mostly the same, removing satisfactorily MS-222 from water. Therefore, it may be concluded that the produced adsorbent can be employed in intensive aquaculture wastewater treatment with the same performance independently of the farmed fish species. [ABSTRACT FROM AUTHOR]

Hematological effects of etomidate and tricaine at concentrations causing general anesthesia or sedation of carp juveniles were compared. Recovery of body balance took similar time in the case of both anesthetics, but fish treated with etomidate were depressed for several hours, while those subjected to tricaine immediately regained normal behavior. Immediately after exposure anesthetics caused minor hematological changes; the only statistically significant effects (decrease of hematocrit and plasma glucose) occurred after sedation with etomidate. One week after exposure a significant increase in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration occurred in all groups of fish compared to the previous values, with slight increase in hemoglobin and decrease in red blood cell count. The mean corpuscular volume values tended to increase in all groups except for the control. Oxidative metabolic activity of phagocytes was significantly higher in both groups treated with etomidate compared to the control and tricaine groups. The results revealed that etomidate was less aversive and better prevented a delayed immunosuppressive effect of stress caused by handling and bleeding, while very quick complete recovery was the benefit of tricaine. These findings suggest that etomidate is better when long-term stress prevention is required, while tricaine is more useful for short-term immobilization of fish when rapid recovery of normal behavior is needed. [ABSTRACT FROM AUTHOR]

The Largemouth Bronze GudgeonCoreius guichenotiis a vulnerable and valuable fish in the upper Yangtze River of China. It is essential to keep it calm by using anesthetic in routine operations such as weight measurement, tag injection, and artificial spawning. This study was conducted to investigate the efficacy of different concentrations of MS-222 (tricaine methanesulfonate) as an anesthetic agent inC. guichenoti. The results showed that anesthesia time decreased significantly (P< 0.001) with increasing anesthetic concentrations. However, anesthetic concentrations had no significant effect on recovery time. In addition, anesthesia time had an obvious positive correlation with body weight. Overall, MS-222 was an effective anesthetic agent forC. guichenoti. Moreover, the optimal concentrations of MS-222 inC. guichenoti(average body weight =135.29 ± 2.23 g) were 100, 120, 160, and 180 mg/L. Received June 27, 2016; accepted September 30, 2016 Published online December 14, 2016 [ABSTRACT FROM AUTHOR]

Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO 2 nanoflake-mediated (MnO 2 NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO 2 NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent o -phenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO 2 NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo[3,4- b ]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F 445 /F 570 ) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC-MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.

Analytical methods based on multiplug filtration cleanup coupled with pulse glow discharge-ion mobility spectrometry and liquid chromatography tandem mass spectrometry were developed for the analysis of tricaine mesylate residue in fish and fish-raising water samples. A silica fiber holder and an appropriate new interface were designed to make the direct introduction of the fiber into the pulse glow discharge-ion mobility spectrometry introduction mechanism. The multiplug filtration cleanup method with adsorption mixtures was optimized for the determination of tricaine mesylate in fish samples. Good linear relationships were obtained by the two methods. For fish samples, limits of detection were 6 and 0.6 μg/kg by ion mobility spectrometry and liquid chromatography with tandem mass spectrometry, respectively. The matrix effect of the established liquid chromatography tandem mass spectrometry method was negligible for fish samples but that of the ion mobility spectrometry method was not. The two methods were compared. The ion mobility spectrometry system could be used a rapid screening tool on site with the advantage of rapidity, simplicity, and portability, and the liquid chromatography tandem mass spectrometry system could be used for validation in laboratory conditions with the advantage of lower limit of detection, stability, and precision. [ABSTRACT FROM AUTHOR]

Many kinds of anesthetics are common used in aquaculture and fisheries research to alleviate stress reponse and minimize physical injury of fish. Such as catching, measuring fish, taking photo, transportation, administration of vaccines, tagging, and surgery, etc. In this review, informations have been given about effective. doses and cost of the most common anesthetics which is used in aquaculture (tricaine methanesulfonate, benzocaine, 2-phenoxyethanol, quinaldine, metomidate, clove oil, sedanol). [ABSTRACT FROM AUTHOR]

Currently, there are no sedatives approved by the U.S. Food and Drug Administration that are considered effective, safe to a broad range of fish species, practical to use, and allow sedated fish to be returned to public waters immediately upon recovery. Availability of such a sedative is critical for many field-based fisheries activities and research, particularly when working with federally listed threatened and endangered species such as Pallid SturgeonScaphirhynchus albus. Therefore, we conducted an experiment to quantitatively compare induction and recovery times of Pallid Sturgeon sedated using tricaine methanesulfonate (MS-222), eugenol, or electrosedation (pulsed DC) and assess the fish’s hematological profile following sedation. Induction times varied significantly among the sedatives evaluated, of which electrosedation yielded the fastest induction times (0.2 ± 0.04 min, mean ± SE) followed by MS-222 (1.8 ± 0.19 min) and eugenol (2.3 ± 0.26 min). Times to recovery of equilibrium and responsiveness to tactile stimuli also varied, ranging from 1.4 ± 0.1 min for electrosedation to 4.7 ± 0.2 min and 6.4 ± 0.7 min for MS-222 and eugenol, respectively. Except for plasma osmolality, hematological variables (hematocrit, glucose, lactate, and cortisol) did not vary over a 6-h postsedation sampling period. Osmolality was lower in fish sedated with MS-222 and eugenol and higher in electrosedated fish compared with unsedated reference fish. Our results showed that all sedation protocols tested effectively sedated Pallid Sturgeon, all sedated fish recovered, and there was no delayed mortality associated with sedation. Received April 21, 2015; accepted December 21, 2015 Published online May 24, 2016 [ABSTRACT FROM PUBLISHER]

The efficacy of four anesthetic agents—tricaine methanesulfonate (MS‐222), clove oil, metomidate, and 2‐phenoxyethanol (2‐PE)—was studied in adult, juvenile (133.3 ± 1.5 mm, 27.5 ± 8.9 g; mean ± SE), and larval Alewives Alosa pseudoharengus. In an initial trial, wild‐caught adults were anesthetized to a state where they failed to respond to strong tactile stimuli when dosed with 87.5–112.5 mg/L MS‐222, 25–40 mg/L clove oil, 0.5–5.0 mg/L metomidate, and 125–550 mg/L 2‐PE. Optimal doses for anesthesia, defined as those providing the combined shortest induction and recovery times, were similar for larvae and juveniles: 75–100 mg/L MS‐222, 40 mg/L clove oil, 5–7 mg/L metomidate, and 500 mg/L 2‐PE. All juvenile fish survived 48 h postexposure to each optimal dose. In a longer (24 h) sedation experiment, juvenile Alewives were netted and exposed to low clove oil (2.5 and 5.0 mg/L) and metomidate (0.25 and 0.50 mg/L) doses, after which plasma cortisol was measured. Fish exposed to the clove oil treatments exhibited a cortisol stress response that was prolonged in the higher‐dose treatment. No cortisol stress response was observed in the metomidate treatments. Overall, optimal acute anesthesia doses for Alewives were similar to those reported for other species, and metomidate may be useful for longer‐term sedation. [ABSTRACT FROM AUTHOR]

Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy. [ABSTRACT FROM AUTHOR]

The article discusses a study which evaluates the effectiveness of tricaine concentrations for larvae and juveniles of the freshwater fish Lophiosilurus alexandri. Findings reveal that all concentrations tested can be safely used as anaesthesia for classes II and III of the fish, but concentrations above 280 milligrams (mg) were not recommended for class I animals. The association between the animal weight and anaesthetic doses is also noted.

The efficacy of anaesthetic tricaine methanesulfonate ( MS-222) was evaluated in four freshwater aquarium fish species, Zebrafish ( Danio rerio), Guppy ( Poecilia reticulata), Discu ( Symphysodon discus) and Green swordtail ( Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L−1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio - 75, 100 and 125 mg L−1, P. reticulata - 125, 150 and 200 mg L−1, S. discus - 75 and 100 mg L−1 and X. helleri - 125 and 150 mg L−1. The induction time generally decreased significantly with increasing concentration of MS-222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS-222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS-222 concentration. MS-222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species. [ABSTRACT FROM AUTHOR]

The effects of four anaesthetic agents, tricaine methanesulphonate ( MS-222) (112.5 mg L−1), 2-phenoxyethanol (400 μL L−1), clove oil (70 mg L−1) and benzocaine (65 mg L−1) on juvenile marbled spinefoot ( Siganus rivulatus) of three mean body weights (7.3 g, 19.1 g, 55.5 g) and at three temperatures (20, 25, 30°C) were evaluated. In addition, the relationship between body lipid content and efficacy of the four anaesthetic agents was evaluated in juvenile S. rivulatus. Times necessary for induction and recovery were recorded. Significant effects of temperature on induction and recovery times were observed. Induction and recovery times decreased with increasing water temperature. No uniform relationship between body weight of juvenile marbled spinefoot and anaesthetic efficacy was observed. Body fat content was positively correlated with induction time only when MS-222 was used but did not affect induction times of fish exposed to 2-phenoxyethanol, clove oil or benzocaine. Recovery times were generally longer for all fish containing more body fat. Results of the study show that anaesthetic efficiency increases with increasing water temperature but is not strongly affected by body weight for juvenile marbled spinefoot. In addition, body fat in fish affected the efficacy of the various anaesthetic agents tested in this study, generally slowing down recovery. [ABSTRACT FROM AUTHOR]

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1 μM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96 h compared to controls (DMSO vehicle; p <0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24 h to 96 h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4 h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100 mg l −1 MS-222 for 2–5 min) in the gill, liver, gonopodium, dorsal or tail fin ( p <0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4 h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1 h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethal ex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America. [ABSTRACT FROM AUTHOR]

The article discusses a study which describes a case of phaeohyphomycosis in a Queensland grouper, Epinephelus lanceolatus. It says that the fish was anaesthetized by tricaine-methanesulfate and exploratory coeliotomy was performed. It describes the examinations of the swim bladder and the swim bladder infection that may caused by haematogenous seeding of the gas gland capillary rete.

In this study, we investigated the effects of MS-222 as an anesthetic on hematological parameters including hemoglobin (Hb), erythrocyte number (RBCs), hematocrit (Hct), and white blood cells (WBCs) in sterlet, Acipenser ruthenus. For this purpose, the hematological parameters were assessed immediately and 24 h after anesthesia. The results showed that despite no mortality occurred after anesthesia in 5 mg/l MS-222, this chemical substance influenced some hematological parameters. The values of Hb, Hct, and RBCs decreased. Immediately after anesthesia, these values increased significantly 24 h after anesthesia. The WBC concentrations did not show significant changes immediately after anesthesia, while its values decreased significantly 24 h after exposure. Our results showed that MS-222 induces some physiological alternations in level of hematological indices. Understanding on these alternations in fish may help to improve management and animal welfare. [ABSTRACT FROM AUTHOR]

The present study was performed to determine the effectiveness of the Hargreaves test for the evaluation of nociception in frogs, more precisely to determine if cutaneous thresholds to a radiant heat stimulus would increase with analgesics following an abdominal laparotomy performed under general anaesthesia. Non breeding female Xenopus leavis frogs (3 groups (non-anaesthetized, anaesthetized with tricaine methanesulfonate (MS222), with or without an abdominal laparotomy) were used to evaluate the effectiveness of the Hargreaves test. Cutaneous thresholds were evaluated at baseline and following anaesthetic recovery (over 8 h) at six different body locations. Increased reaction times were observed in the gular area only at 1 h post-recovery following a MS222 bath immersion in frogs with (p < 0.02) and without the abdominal laparotomy (p < 0.002). In conclusion, the Hargreaves test does not provide an adequate test to evaluate nociception induced by an abdominal laparotomy and consequently cannot be used to evaluate analgesics in X. leavis frogs. [ABSTRACT FROM AUTHOR]

The use of anesthetics in Angelfish ( Pterophyllum scalare ) has not been fully explored. The aim of this study is to determine the lowest effective dose (LED) of three anesthetics (2-phenoxyethanol, clove oil and tricaine methanesulphonate (MS-222)) and their effect after multiple exposures at 24 and 48 h. Each agent was tested on ten juvenile angelfish at four different doses. Considering the effect criteria of complete anesthetic induction time within 3 min and recovery time within 5 min, LEDs were established at 800 mg L − 1 for 2-phenoxyethanol, 100 mg L − 1 for clove oil and 140 mg L − 1 for MS-222. Different anesthesia and recovery times should be considered when repeated exposures during consecutive days are performed on juvenile angelfish. [ABSTRACT FROM AUTHOR]

Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Cav channels affecting movement are also primarily neural, suggesting that muscle Nav channels are relatively insensitive to tricaine. These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia. [ABSTRACT FROM AUTHOR]

Responses to anaesthesia with essential oil ( EO) of Aloysia triphylla (135 and 180 mg L−1) and tricaine methanesulfonate ( MS222) (150 and 300 mg L−1) were assessed in silver catfish. Exposure to the anaesthetics elicited a stress response in the species. In the case of MS222, it was displayed as a release of cortisol into bloodstream, elevation in hematocrit and plasma ion loss. The EO presented cortisol-blocking properties, but increased haematocrit and disturbances of hydromineral balance were observed. Liver antioxidant/oxidant status of EO and MS222-anaesthetized silver catfish was also estimated. The synthetic anaesthetic induced lipoperoxidation, notwithstanding increased catalase contents, whereas the naturally occurring product was capable of preventing the formation of lipid peroxides, possibly due to combined actions of catalase and glutathione-S-transferase. Anaesthetic efficacy was also tested via induction and recovery times. Overall, the promising results obtained for the physiological parameters of the EO-treated fish counterbalanced the slight prolonged induction time observed for 180 mg L−1. As for 135 mg L−1, both induction and recovery times were lengthy; despite that, the EO was able to promote oxidative protection and mitigate stress. None of the MS222 concentrations prompted such responses concomitantly. [ABSTRACT FROM AUTHOR]

The pros and cons of using anaesthesia when handling fish in connection with experiments are debated. A widely adopted practice is to wait thirty minutes after anaesthesia before behavioural observations are initiated, but information about immediate effects of a treatment is then lost. This is pertinent for responses to acute stressors, such as acid injection in the acetic acid pain test. However, omission of anaesthetics in order to obtain data on immediate responses will compromise the welfare of fish and contribute to experimental noise due to stress. We therefore tested the effect of tricaine methanesulfonate on the behaviour of zebrafish. We predicted that tricaine (MS 222) would decrease swimming velocity and that the control fish would show an increased level of anxiety- and stress-related behaviours compared to the tricaine group. Following acclimatization to the test tank, baseline behaviour was recorded before immersion in either tricaine (168 mg l−1, treatment group, N = 8) or tank water (control group, N = 7). Latencies to lose equilibrium and to lose response to touch were registered. The fish was then returned to the test tank, and the latency to regain equilibrium was registered in anaesthetized fish. When equilibrium was regained, and at five, thirty and sixty minutes after the fish had been returned to the test tank, behaviour was recorded. The tricaine fish showed the following responses (mean ± sd): latency to lose equilibrium 22.6 s±3.9; latency to lose response to touch 101.9 s±26.8; latency to regain equilibrium 92.0 s±54.4. Contrary to our predictions, neither treatment caused a change in any of the behaviours registered. This indicates that tricaine has no effect on several commonly used behavioural parameters, and that it may be unnecessary to postpone behavioural observations to 30 min after anaesthesia. [ABSTRACT FROM AUTHOR]

Abstract: This study investigates the role of branchial and extrabranchial processes in acid–base regulation in the Pacific Hagfish (Eptatretus stoutii). Hagfish were injected with one of the following solutions: acid saline (250mM HCl [pH=0.60], 250mM NaCl), alkaline saline (250mM NaHCO3, 250mM NaCl, [pH≈8.43]) or control saline (500mM NaCl) in order to achieve an acid/alkaline/saline load of 6000μmol·kg−1. Using a custom designed hagfish compartmentalizing flux chamber, we partitioned flux of net acid or base equivalents and ammonia into the anterior (gill+skin) and posterior (skin+intestinal/renal/cloacal) components. We found that Pacific hagfish excrete H+ primarily via branchial mechanisms but base excretion occurs through extrabranchial mechanisms located in the posterior region. In addition, we demonstrate that hagfish are able to excrete ammonia via the skin although this flux was not involved in compensation from an acid–base disturbance. [Copyright &y& Elsevier]

Zebrafish are becoming one of the most used vertebrates in developmental and biomedical research. Fish are commonly killed at the end of an experiment with an overdose of tricaine methanesulfonate (TMS, also known as MS-222), but to date little research has assessed if exposure to this or other agents qualifies as euthanasia (i.e. a “good death”). Alternative agents include metomidate hydrochloride and clove oil. We use a conditioned place avoidance paradigm to compare aversion to TMS, clove oil, and metomidate hydrochloride. Zebrafish (n = 51) were exposed to the different anaesthetics in the initially preferred side of a light/dark box. After exposure to TMS zebrafish spent less time in their previously preferred side; aversion was less pronounced following exposure to metomidate hydrochloride and clove oil. Nine of 17 fish exposed to TMS chose not to re-enter the previously preferred side, versus 2 of 18 and 3 of 16 refusals for metomidate hydrochloride and clove oil, respectively. We conclude that metomidate hydrochloride and clove oil are less aversive than TMS and that these agents be used as humane alternatives to TMS for killing zebrafish. [ABSTRACT FROM AUTHOR]