Cardiovascular responses of Chinook salmon (Oncorhynchus tshawytscha) during rapid anaesthetic induction and recovery

The effects of three anaesthetics on induction and recovery were compared in Chinook salmon (Oncorhynchus tshawytscha). Heart rate (HR), cardiac output (Q), dorsal aortic pressure (DAP) and stroke volume (SV) were measured in minimally disturbed salmon during 5 min anaesthetic inductions with approximately equi-potent concentrations of MS222 (100 ppm), metomidate (6-10 ppm) and AQUI-S (60 ppm). MS222 induction caused a steady decline in DAP only, while metomidate induction did not affect any cardiovascular variable. AQUI-S caused a biphasic response, and within 2 min had depressed HR, Q, DAP and SV by between 20 and 50%. In the final 3 min HR returned to pre-anaesthesia levels, and Q and SV climbed to greater than pre-anaesthesia levels. Blood samples taken pre- and post-anaesthesia showed all inductions caused hypoxaemia (oxygen partial pressure of dorsal aortic blood (PaO2): MS222 47 mmHg, metomidate 35 mmHg, AQUI-S 21 mmHg). Haematocrit and plasma adrenaline and noradrenaline levels increased slightly in AQUI-S treated fish only. Recovery was monitored for 6 h post-anaesthesia, and was similar for each anaesthetic. All cardiovascular variables had returned to control levels within 5 min with the exception of DAP, which was initially slightly elevated (up to 20%) but returned to control values within 30 min. Anaesthesia is usually preceded by handling. Netting prior to anaesthesia caused significant increases in HR, Q and SV, which masked any anaesthetic dependent effects. Recovery from anaesthesia combined with surgery was also generally anaesthetic independent and recovery was prolonged, compared to anaesthesia alone. These data suggest limiting fish handling/manipulation is more important in minimising cardiovascular disturbance than the choice of anaesthetic.     

The effect of MS 222 an anaesthetic on the peroxide metabolism enzymes in erythrocytes of freshwater and marine fish species

1. The effect of 70 mg/l and 35 mg/l MS 222 an anaesthetic on the enzymes: superoxide dismutase (SOD), catalase (C) and peroxidase (P) were estimated in erythrocytes of Cyprinus carpo, a freshwater fish and Dicentrarchus labrax, a marine fish. 2. The end of the summer, at 16 degrees C MS 222 in concentration 70 mg/l caused an enhancement of the SOD and peroxidase activities and a decrease of the catalase activity. 3. In the autumn at 22 degrees C SOD and peroxidase activities in erythrocytes of Dicentrarchus labrax are normally higher than at 16 degrees C. On the contrary MS 222 causes no significant modification of enzymatic activities measured, but an increase in the dispersion of the results. 4. At 13 degrees C in the spring, MS 222 has no immediate influence on the activity of these enzymes, whilst at the same temperature at the beginning of winter, SOD is the only one activated. 5. It seems that in experiments concerning peroxide metabolism enzymes the use of anaesthetic MS 222 is not advisable.     

Haematological assessment of the effects of the anaesthetic MS 222 in natural and neutralized form in three fresh water fish species: haemoglobin electrophoresis, ATP levels and corpuscular fragility curves

The effects of MS 222 and neutralized MS 222 anaesthesia on haemoglobin electrophoresis, erythrocyte adenosine triphosphate (ATP) levels and corpuscular fragility curves were studied in Cyprinus carpio, Sarotherodon mossambicus and Salmo gairdneri . Haemoglobin electrophoresis showed no significant intra-species differences in the percentage composition of the various fractions for any concentration of MS 222 and neutralized MS 222 used. Significant interspecies differences were, however, still observed. ATP levels showed intra and interspecies differences ascribed to the response of the fish species to MS 222-induced stress and not to actual changes in erythrocyte ATP concentrations. Differences were also observed in corpuscular fragility curves for all three species when using MS 222 or neutralized MS 222 compared to curves obtained without the use of the anaesthetic, but the mechanisms involved are not clear.     

Some observations on the use of MS 222 Sandoz with grey mullet (Mugil chelo Cuvier)

Two trials, each using grey mullet, were carried out to investigate the use of the anaesthetic, MS 222 Sandoz, in the handling and transportation of fish. The first trial examined the effect of using various concentrations of MS 222 Sandoz and it was concluded that concentrations of between 1: 75 000 and 1: 100000 were sufficient to tranquillize the fish for pre-transport handling. A further trial using the anaesthetic at a concentration of 1: 100 000 and with a fish biomass of 50 g/1 demonstrated that tranquillized fish could safely be held at this density for a 48 h period. It was concluded that the use of anaesthetic such as MS 222 Sandoz could aid handling and loading operations and induce quiescence for a transportation period of up to 48 h duration.     

Tricaine methane‐sulfonate (MS‐222) application in fish anaesthesia

Tricaine methane‐sulfonate (MS‐222) is one of the most widely used anaesthetics for poikilotherms worldwide. This paper outlines its anaesthetic efficacy and dosage in fish and legislation for its use, fish stress responses to MS‐222 anaesthesia and its effect on fish physiology and blood properties, pharmacokinetics, genotoxicity, immune response, potential interference with fish hepatic cytochrome P450 spectra, and its impact on nerve sensitivity. Key questions arising from the available data are analysed, such as regulatory constraints on its use, the need for the standardization of buffering protocols, and interdependencies of the factors impacting the specific applicative efficacy of MS‐222. Current research has provided an abundance of data on MS‐222 use in fish, although the applications within these studies are often impractical at the farming level. Specific emphasis is therefore placed on highlighting application strategies on a practical basis, presenting potential future research on topics that require in‐depth analysis (preparation and storage of anaesthetic solutions, pre‐anaesthetic sedation and stress reduction, cortisol response in aquarium fish, toxicity of MS‐222 metabolites, and possible immunodepressive properties). Additionally, both from a scientific and practical perspective, it is necessary to have a better understanding of safety margins, induction, immersion and recovery times for many (marine and freshwater, farmed and ornamental) fish species in order to achieve optimal utilization.     

The efficacy of eugenol and tricaine methanesulphonate as anaesthetics for juvenile Chinese sea bass (Lateolabrax maculatus) during simulated transport

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.     

Haematological assessment of the effects of the anaesthetic MS 222 in natural and neutralized form in three freshwater fish species: interspecies differences

Interspecies haematological differences to MS 222 and neutralized MS 222 anaesthesia were investigated in Sarotherodon mossambicus, Cyprinus carpio and Salmo gairdneri acclimated under identical laboratory conditions. Anaesthesia with MS 222 resulted in a 'chemical stress' in all fish, as was evident from changes in the haematological profiles of the animals. Such species specific variations in the haematology persisted throughout the whole experiment protocol which employed different concentrations of the anaesthetic. The use of neutralized MS 222, whereby aquarium water quality remained unchanged, improved the haematological profile. Possible reasons for the interspecies differences observed are discussed.     

Immersion anaesthesia with tricaine methanesulphonate or propofol on different sizes and strains of silver catfish Rhamdia quelen

The efficacy of immersion anaesthesia with tricaine methanesulphonate (MS222) or propofol on albino and grey silver catfish Rhamdia quelen was assessed through induction and recovery times and observation of mortality. Besides reporting a novel, efficient and practical use of propofol as an immersion anaesthetic, the study shows that it is essential to consider size and strain when anaesthetizing R. quelen with MS222 or propofol bath solution in order to minimize physiological impact.     

Effects of anaesthesia with MS 222, neutralized MS 222 and benzocaine on the blood constituents of rainbow trout,Salmo gairdneri†

Rainbow trout (Salmo gairdneri Richardson) were subjected to 15 min anaesthesia with unbuffered MS 222, neutralized MS 222 and benzocaine with and without physical stress. Blood samples were taken through cannulae inserted into the dorsal aorta. The Hct values and Hb concentrations increased with all the anaesthetics, which also caused swelling of erythrocytes. The initial values were restored within 4–12 h of recovery. Each anaesthetic elevated the blood lactate concentration, but the initial level was regained within 12 h. The blood glucose level decreased the most during anaesthesia with unbuffered MS 222, but the initial level was rapidly restored. Benzocaine caused the least hypoglycaemia during anaesthesia, but the subsequent hyperglycaemia, as in the fish anaesthetized with neutralized MS 222, lasted 24 h. Neutralized MS 222 and benzocaine elevated the plasma K + concentration more rapidly than unbuffered MS 222. The initial levels were regained in 4 days. All anaesthetics raised the Mg ⁺⁺ concentration. The Po₂ in the dorsal aorta decreased during anaesthesia with unbuffered MS 222 by about 85 mmHg, while the Pco₂ increased by about 1.5 mmHg. Their initial levels were regained within 20 min. During anaesthesia the pH value decreased by 0.3 units and returned to the initial value within 2–4 h of recovery. MS 222 seemed to be an asphyxiant.     

A New Anaesthetic Protocol for Adult Zebrafish (Danio rerio): Propofol Combined with Lidocaine

Background

The increasing use of zebrafish model has not been accompanied by the evolution of proper anaesthesia for this species in research. The most used anaesthetic in fishes, MS222, may induce aversion, reduction of heart rate, and consequently high mortality, especially during long exposures. Therefore, we aim to explore new anaesthetic protocols to be used in zebrafish by studying the quality of anaesthesia and recovery induced by different concentrations of propofol alone and in combination with different concentrations of lidocaine.

Material and Methods

In experiment A, eighty-three AB zebrafish were randomly assigned to 7 different groups: control, 2.5 (2.5P), 5 (5P) or 7.5 μg/ml (7.5P) of propofol; and 2.5 μg/ml of propofol combined with 50, (P/50L), 100 (P/100L) or 150 μg/ml (P/150L) of lidocaine. Zebrafish were placed in an anaesthetic water bath and time to lose the equilibrium, reflex to touch, reflex to a tail pinch, and respiratory rate were measured. Time to gain equilibrium was also assessed in a clean tank. Five and 24 hours after anaesthesia recovery, zebrafish were evaluated concerning activity and reactivity. Afterwards, in a second phase of experiments (experiment B), the best protocol of the experiment A was compared with a new group of 8 fishes treated with 100 mg/L of MS222 (100M).

Results

In experiment A, only different concentrations of propofol/lidocaine combination induced full anaesthesia in all animals. Thus only these groups were compared with a standard dose of MS222 in experiment B. Propofol/lidocaine induced a quicker loss of equilibrium, and loss of response to light and painful stimuli compared with MS222. However zebrafish treated with MS222 recovered quickly than the ones treated with propofol/lidocaine.

Conclusion

In conclusion, propofol/lidocaine combination and MS222 have advantages in different situations. MS222 is ideal for minor procedures when a quick recovery is important, while propofol/lidocaine is best to induce a quick and complete anaesthesia.     

Serum immunoreactive insulin levels in intact and regenerating postmetamorphic Xenopus laevis

In order to confirm the presence of immunoreactive insulin (IRI) in the serum of postmetamorphic Xenopus laevis, radioimmunoassay (RIA) methods were used. The concentration of hormone found in samples of blood serum taken from nonanaesthetized intact male and female animals by the guillotine method was 10.46 +/- 0.76 microU/ml. Significantly higher IRI concentrations were found in our intact animals anaesthetized in MS 222 at pH 3.5 (21.9 microU/ml) compared with intact controls anaesthetized in MS 222 adjusted to pH 7.0 (14.4 microU/ml). During the wound-healing stage subsequent to forelimb amputation in the experimental cases (0 hours to 3 days) anaesthetized in MS 222 pH 7.0, there were intervals of significantly elevated serum IRI followed by a period of decreased IRI concentration compared with the levels in anaesthetized (MS 222 pH 7.0) and nonanaesthetized intact controls. These fluctuations were due, presumably, to stress caused by amputational injury and/or anaesthetic. Serum IRI increased steadily from 3 to 14 days postamputation then remained stable for the balance of the regeneration period (28 days) compared with nonanesthetized intact controls. A positive correlation was found between immunoreactive insulin and glucose levels in the serum of our animals. However, no correlation exists between serum IRI levels and serum osmolality in the data.     

Effects of anaesthetic MS 222 on the orientation of juvenile salmon, Oncorhynchus tschawytscha Walbaum

Juvenile salmon, Oncorhynchus tschawytscha Walbaum, were trained to orientate in a direction (270°) and then anaesthetized with ethyl m–aminobenzoate methane sulphonate (MS 222) in a test to determine whether anaesthesia affected the learnt orientation.
Before anaesthesia the control group of twelve fish showed a mean unimodal orientation of 264° and a bimodal axis of orientation of 258°/78° with a confidence limit (second order analysis) of 218°←→285°. After administration of MS 222 nine out of ten fish showed marked changes in orientation and random behaviour, persisting in two fish for more than 14 days. The mean of means unimodal orientation, after anaesthesia, was 337° (random) and the bimodal axis of orientation was 337°/157° (random).
Eight weeks after completion of the anaesthesia trial the fish were retested. Each fish, except one, showed a mean bimodal axis of orientation that fell within the confidence limit of the control. The mean axis of orientation for ten fish over a 3–day period was 270°/90°, (r) = 0.28.
The results support the view that the reference orientation in this study (270°) was a learnt, not an innate directional preference. The effects anaesthesia may have on salmon behaviour during migration are discussed.     

The effect of tricaine methanesulphonate (MS-222) on the microhaematocrit of fish blood

The effects of the anaesthetic, MS-222, on the microhaematocrit value of freshwater fish have been examined. Blood containing MS-222 showed a higher haematocrit value than blood without the anaesthetic and haemolysis occurred in the former after a variable time depending on the concentration. The results are discussed in relation to previous findings.     

Inhibitory effect of anaesthesia with 2-phenoxyethanol as compared to MS222 on glucose release in isolated hepatocytes from rainbow trout (Salmo gairdneri)

1. Glucose production by freshly isolated hepatocytes from rainbow trout was studied after anaesthesia of the animals with 2-phenoxy ethanol (2PE) or tricaine methanesulphonate (MS222). 2. At the end of the procedure, hepatic contents of glycogen, glucose, lactate, ATP, ADP, AMP, were not significantly different between the two treatments. 3. Glucose production was considerably lower for 2PE than for MS222 anaesthetized trouts. This discrepancy results probably from an inhibition of glycogenolysis, suggesting that 2PE anaesthetized animals were less stressed than MS222 anaesthetized ones.     

麻醉剂MS-222对斑马鱼行为的影响

实验室条件下观察并测定了斑马鱼(Danio rerio)在不同MS-222浓度处理下的麻醉行为、斑马鱼在高剂量MS-222致死过程中的行为变化、MS-222对斑马鱼摄食条件反射的影响。试验表明:(1)斑马鱼的麻醉行为是一个渐变的过程,可分为不被麻醉(Ⅰ)、轻度麻醉(Ⅱ)、中度麻醉(Ⅲ)深度麻醉(Ⅳ);(2)不同MS-222浓度下斑马鱼进入不同麻醉程度的时间有差异;(3)经MS-222处理900s过程中,80-90mg·L^-1浓度组进入Ⅲ级麻醉程度并有100%的存活率,而麻醉浓度在100mg·L^-1以上时可以迅速在49s内使鱼进入Ⅲ级麻醉程度以及在178s后即可进入Ⅳ级麻醉程度,并在中度麻醉和深度麻醉的过渡中死亡,存活率不超过10%;(4)MS-222对摄食条件反射影响的试验中,光刺激-不麻醉、无光刺激-不麻醉、光刺激-麻醉、无光刺激-麻醉四组班马鱼从投喂到摄食所用时间分别为12.06±1.34s、13.20±1.13s、56.56±56.48s、36.20±25.74s,麻醉组的摄食速度慢于对照组,说明MS-222影响了斑马鱼的摄食条件反射。     

Haematological assessment of the effects of the anaesthetic MS 222 in natural and neutralized form in three freshwater fish species: intraspecies differences

The effects of different concentrations of natural MS 222 and neutralized MS 222 were studied on the haematology of Cyprinus carpio, Sarotherodon mossambicus and Salmo gairdneri . As judged from the results, MS 222, which is acidic in nature, produced haemo-concentration effects in all species studied, being least in the trout followed by carp and Sarotherodon mossambicus . These differences are ascribed to acid-base regulatory functions and metabolic activities of the fish species investigated. The use of neutralized MS 222 improved the haematological profiles markedly and resulted in stabilization of acid-base parameters and red blood cells sizes and numbers. Haemoconcentration effects, however, still persisted. Trout were found to be more susceptible to the stress of MS 222 anaesthesia than carp and Sarotherodon mossambicus .     

Antibacterial peptides in stimulated human granulocytes: characterization of ubiquitinated histone H1A.

Antibacterial peptides were isolated from human peripheral granulocytes of a healthy donor who had been treated with granulocyte-colony stimulating factor (G-CSF) and cortisol. Peptides were solubilized in acidified chloroform/methanol, and partitioned in chloroform/methanol/water. Water- soluble polypeptides were separated by cation-exchange and reversed-phase chromatography. Several previously characterized antibacterial polypeptides were identified; defensins 1-3, defensin 4, lysozyme, eosinophil cationic protein, and calgranulin A. In addition, several histone fragments were isolated and exhibited activity against the Gram- positive bacterium Bacillus megaterium strain Bm11. These fragments included two C-terminal fragments of histone H1A, three C-terminal fragments of histone H1D, one fragment of histone H1B, and two fragments of histone H4. The molecular masses of both histone H1A fragments, as determined by electrospray (ES) MS, were 270 Da higher than those calculated from their amino acid sequences. The two histone H1A fragments corresponded to Lys152-Lys222 (7527 +/- 1 Da) and Lys167-Lys222 (6023 +/- 1 Da). Tandem MS (MS/MS) of the 7.5 kDa and 6.0 kDa fragments indicated that the post-translational modification is on Lys222, the epsilon-amino group of which was conjugated with the alpha-carboxyl group of the tripeptide Arg-Gly-Gly. This finding was substantiated by digestion of the 7.5-kDa polypeptide with trypsin and analysis of the resulting peptides by ES MS and MS/MS. The tripeptide Arg-Gly-Gly corresponded uniquely to the three C-terminal residues of ubiquitin, demonstrating the presence of ubiquitinated histone H1A.     

Effect of tricainemethanesulfonate (MS222), clove oil and electro-anaesthesia on respiratory burst activity in whole blood and serum alternative complement response in rainbow trout (Oncorhynchus mykiss), during the narcosis stage

There is a little available information on the suppressive effect of anaesthesia on immune response in fish, especially electro-anaesthesia. In the present study, two anaesthetics, MS222 (50 ppm), clove oil (25 ppm), and electro-anaesthesia were tested in rainbow trout (Oncorhynchus mykiss) during the narcosis stage in order to observe their effects on the innate immune system. The results showed that electro-anaesthesia reduces light emission in chemiluminescence assay both 1 and 24 h post anaesthesia. Clove oil and MS222 decreased light emission 24 h post anaesthesia. In addition, clove oil, MS222 and electro-anaesthesia had no effect on alternative complement (ACH50) response. From the perspective of aquaculture practice, these data show that the type of anaesthesia should be taken into account to avoid possible immunosuppression in rainbow trout.     

Effects of a high concentration of CO2 on electrocardiograms in the carp, Cyprinus carpio

1. The effects of a high concentration of CO2 (PCO2 = 250 mmHg and PO2 = 360 mmHg in water) and MS222 (tricaine methanesulfonate, 1/8000 or 1/5000) on the electrocardiogram (ECG) in carp were examined using five kinds of bipolar leads from the body surface. 2. In the carp anesthetized with the high concentration of CO2 for 30 min, the QRS duration, PQ interval and and direction of the QRS axis on the frontal plane significantly changed. Even after recovery from anesthesia, delay in the QRS duration was still recognized. 3. The concentration of CO2 used in this study had an anesthetic action to the same degree as 1/8000 of MS222 and had a much more severe effect on the ECG of the carp than 1/5000 of MS222.     

Temperature effects on anaesthesia with clove oil in six temperate‐zone fishes

The potential use of clove oil (eugenol) as an anaesthetic for Atlantic salmon Salmo salar , brown trout Salmo trutta , rainbow trout Oncorhynchus mykiss , whitefish Coregonus lavaretus , perch Perca fluviatilis and roach Rutilus rutilus was examined at 5, 10, 15 and 20° C using three anaesthetic concentrations (varying from 20 to 200 mg l⁻¹) at each temperature. Substantial species differences in sensitivity to clove oil were observed, even amongst congeners, and there may be some disadvantages (slow recovery and possibly mortality) with using clove oil for 0+ year whitefish and at low temperatures for perch and roach.