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 .     

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.     

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.     

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.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Susceptibility of the Antarctic fish Pagothenia borchgrevinki to MS-222 anaesthesia

Summary Induction of anaesthesia and distribution of the routinely used fish anaesthetic, MS-222 (ethyl m-amino-benzoate methanesulfonate), were monitored in the Antarctic fish Pagothenia borchgrevinki. As expected from the extreme low metabolic rate of Antarctic fish, induction times were significantly longer than those from rainbow trout. Onset of anaesthesia in the Antarctic fish also failed to correlate with either brain or blood MS-222 concentrations, in contrast to the trends observed in freshwater salmonids. High levels of free MS-222 in liver and in the aglomerular kidney, and the presence of acetylated derivatives in the blood, suggest these organs as primary sites of drug metabolism. MS-222 anaesthesia in P. borchgrevinki is also accompanied by dose-dependent changes in haematological parameters.     

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.     

Haematological evaluation of the anaesthetic benzocaine hydrochloride in the freshwater fish Cyprinus carpio L.

The effects of anaesthetization with different concentrations of benzocaine hydrochloride (BH) and neutralized benzocaine hydrochloride (NBH) were studied on the haematology of Cyprinus carpio. Due to its acidic nature and resultant effects on aquarium water, BH produces haemoconcentration effects with disturbances in acid-base function. The use of NBH, whereby water quality effects were drastically reduced, improved the general haematological profile. In contrast, haemodilution resulted when blood was sampled without the use of an anaesthetic agent.     

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.     

Effects of anaesthesia on the chemosensory behaviour of Pacific salmon

As Lewis et al . reported in J. Fish Biol. 26 , 355–358 (1985) that the anaesthetic tricaine methanosulphate (MS 222) damaged the olfactory epithelium of channel catfish, two assays were used to determine whether MS 222 or 2-phenoxyethanol (an alternative anaesthetic) influenced subsequent olfaction-mediated behaviour. Tests revealed that the homing of adult chinook salmon, Oncorhynchus tshawytscha , and the l-serine avoidance response of juvenile coho salmon, O. kisutch , were not altered by anaesthesia.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of four anaesthetics on the innate immune response of gilthead seabream (Sparus aurata L.)

Anaesthesia may depress the immune system in mammals, but there is no available information on this topic in fish. In the present work, four anaesthetics that are used in aquaculture, MS222 (0 19 mM), benzocaine (0.21 mM), 2-phenoxyethanol (16 mM) and quinaldine sulphate (0.083 mM), were tested in order to observe their effects on the gilthead seabream (Sparus aurata L.) innate immune system. The results showed that the four anaesthetics produced increased blood glucose levels after an hour. In addition, benzocaine and 2-phenoxyethanol depressed complement activity and phagocytosis, while MS222 and quinaldine sulphate did not. Some anaesthesia is a common practice in aquaculture, the data obtained should be taken into account to avoid possible immunodepression in farmed fish.     

Evaluation of intramuscular ketoprofen and butorphanol as analgesics in chain dogfish (Scyliorhinus retifer)

The mediation of nociception with analgesic medications has not been well documented in elasmobranchs. The purpose of this study was to determine effective analgesic doses of the opioid agonist‐antagonist, butorphanol, and the non‐steroidal anti‐inflammatory drug, ketoprofen, in an elasmobranch. This was evaluated by repetitively assessing minimum anesthetic concentrations of the immersion anesthetic, tricaine methanesulfonate (MS‐222), required to prevent response to noxious stimuli in chain dogfish (Scyliorhinus retifer) when administered multiple doses of each of the analgesics (0.25, 0.5, 1.0, 2.5, and 5.0 mg/kg butorphanol and 1.0, 1.5, 2.0, and 4.0 mg/kg ketoprofen). Baseline concentrations of MS‐222 required to prevent a response to a noxious stimulus were determined for each animal and served as the controls. Although individual animals displayed a reduction in MS‐222 concentration with various doses of both analgesics, no statistically significant difference was noted between control animals and animals given analgesics. It is plausible that unique elasmobranch anatomy, lack of appropriate medication dose or timing of administration, and other physiological factors not yet identified may have contributed to the lack of apparent efficacy of the analgesics evaluated in this study. Zoo Biol 0:1–10, 2006. © 2006 Wiley‐Liss, Inc.     

Study in vitro of the biosynthesis of Hb in alpha-thalassemic subjects of Puglia

An haematological study and haemoglobin biosynthesis in vitro carried out on the members of family from Ostuni (Apulia), three of which present Hb H in quantities varying from 26 to 28 and 29%, has been reported. On the basis of the haematological and biosynthetical data, the Authors think that the genetics of the alpha-thalassemia in the family under observation is similar to that observed in South East Asia according to the hypothesis of the existence of two alpha loci. The biosynthetical data fall into the values obtained by Kan and Coll. even if with slight differences.