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.     

Relative physiological effects of laparoscopic surgery and anesthesia with tricaine methanesulfonate (MS‐222) in Atlantic sturgeon Acipenser oxyrinchus oxyrinchus

Laparoscopy is a reliable, minimally‐invasive technique to obtain reproductive information from wild and captive sturgeon. While generally considered safe, the physiological consequences of laparoscopy in sturgeon are unknown. Therefore clinical pathology changes in juvenile, Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) following experimental laparoscopy at 10 and 22°C were described. Control fish were anesthetized with MS‐222 according to the same protocols as surgical fish, but were not incised. Surgical procedures did not affect heart and ventilation rates, signs of stress (skin redness) or time to recover from anesthesia in comparison to control fish. Anesthesia with MS‐222 produced a transient (by 1 h) hemo‐concentration (elevated protein and electrolytes), erythrocyte swelling (increased PCV and MCV) and stress response (elevated cortisol and glucose); and a delayed (by 24 h) increase in RBC, leukopenia and increased N : L ratio. Surgical procedures resulted in a delayed (by 24 h) decrease in plasma proteins, electrolytes, RBC and PCV relative to control fish, which may have resulted from surgically‐induced hemorrhage. Plasma enzyme activities increased in response to anesthesia and surgery and may indicate general stress and tissue damage. Anesthesia had a greater effect on blood value response than surgery, and the proportion of effect increased with temperature as MS‐222 potency and toxicity increases with water temperature. Repeated handling and blood draws within 24 h resulted in a 7% increase in cortisol, 10–14% increase in CK and 9–11% increase in LDH values. Except for plasma enzyme activities, blood values of all fish recovered within 1 week following anesthesia and surgeries. Relative experience of surgeons had no effect on hematology and biochemistry of fish, but healing rates of incisions were improved with better suture technique. Results of this study conclude that the physiological effects of laparoscopy are largely related to the anesthetic, MS‐222, and are generally mild and short‐lived. Improvements in laparoscopic technique might be gained by exploring alternate anesthetic protocols with faster induction and recovery times and reduced physiological effects.     

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.     

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.     

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.     

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 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.     

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.     

Anaesthesia and transport of juvenile tambaqui Colossoma macropomum (Cuvier, 1818) with tricaine methane‐sulphonate: Implications on secondary and oxidative stress responses

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 () in whole blood collected from caudal vasculature. Total antioxidant capacity against peroxyl radicals (ACAP), glutathione‐S‐transferase (GST) activity and lipid peroxidation levels (TBARS) were investigated in gills, brain and liver. All concentrations of TMS^® induced deep anaesthesia in juvenile tambaqui in this study. A concentration of 240 mg/L of TMS^® was sufficient to induce rapid anaesthesia (<3 min) with uneventful recovery (<5 min). In light of the secondary and oxidative stress responses of fish transported using TMS^®, which were generally not significantly different compared to responses of fish transported in anaesthetic‐free water, sedation with 20 mg/L is not advantageous and therefore is dispensable for the transport of this species for up to 10 hr.     

Data‐independent acquisition‐based SWATH‐MS for quantitative proteomics: a tutorial

Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed.     

Addressing the Challenges of High‐Throughput Cancer Tissue Proteomics for Clinical Application: ProCan

The cancer tissue proteome has enormous potential as a source of novel predictive biomarkers in oncology. Progress in the development of mass spectrometry (MS)‐based tissue proteomics now presents an opportunity to exploit this by applying the strategies of comprehensive molecular profiling and big‐data analytics that are refined in other fields of ‘omics research. ProCan (ProCan is a registered trademark) is a program aiming to generate high‐quality tissue proteomic data across a broad spectrum of cancer types. It is based on data‐independent acquisition–MS proteomic analysis of annotated tissue samples sourced through collaboration with expert clinical and cancer research groups. The practical requirements of a high‐throughput translational research program have shaped the approach that ProCan is taking to address challenges in study design, sample preparation, raw data acquisition, and data analysis. The ultimate goal is to establish a large proteomics knowledge‐base that, in combination with other cancer ‘omics data, will accelerate cancer research.     

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

High‐resolution MS/MS spectra of peptides can be deisotoped to identify monoisotopic masses of peptide fragments. The use of such masses should improve protein identification rates. However, deisotoping is not universally used and its benefits have not been fully explored. Here, MS2‐Deisotoper, a tool for use prior to database search, is used to identify monoisotopic peaks in centroided MS/MS spectra. MS2‐Deisotoper works by comparing the mass and relative intensity of each peptide fragment peak to every other peak of greater mass, and by applying a set of rules concerning mass and intensity differences. After comprehensive parameter optimization, it is shown that MS2‐Deisotoper can improve the number of peptide spectrum matches (PSMs) identified by up to 8.2% and proteins by up to 2.8%. It is effective with SILAC and non‐SILAC MS/MS data. The identification of unique peptide sequences is also improved, increasing the number of human proteoforms by 3.7%. Detailed investigation of results shows that deisotoping increases Mascot ion scores, improves FDR estimation for PSMs, and leads to greater protein sequence coverage. At a peptide level, it is found that the efficacy of deisotoping is affected by peptide mass and charge. MS2‐Deisotoper can be used via a user interface or as a command‐line tool.     

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 .     

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.     

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 different stressor agents on gilthead seabream natural cytotoxic activity

Several common situations in gilthead seabream (Sparus aurata L.) farming, such as air exposure, crowding and the use of anaesthetics, have been demonstrated to be stressful. In the present study, these conditions were simulated in the laboratory, after which head-kidney natural cytotoxic cell (NCC) activity was evaluated. For this, several specimens were air exposed for 2 min, returned to the aquarium and sampled from 0 to 4 days after exposure. NCC activity was significantly lower on the day following air-exposure compared with the control (rested fish) but not at any other time studied. Other fish were crowded (100 kg biomass m(-3), 2 h), returned to an aquarium with the same density as the control group (9 kg m(-3)) and sampled from 0 to 4 days after treatment. Head-kidney NCC activity was statistically increased compared with the control (resting) fish, 1 day after crowding. Anaesthesis for 1 h with 60 or 200 microl 2-phenoxyethanol l(-1)had no significant effect on NCC activity, while the use of 50 mg MS222 l(-1)for 1 h reduced such activity (by about 40%) compared with the control. In other experiments, fish were consecutively treated with crowding and anaesthetics. When treated with the lowest 2-phenoxyethanol concentration after crowding, the NCC activity inhibition was abolished compared with the activity in fish treated either with crowding or anaesthetic alone, while the use of the highest concentration increased such inhibition. The use of MS222 after crowding did not produce any differential effect compared with the fish treated with only one of the factors. In conclusion, NCC activity is affected differently according to the stress factor applied (hypoxia, crowding and/or anaesthetics). Differences in the effects provoked by these stressors on other seabream innate immune parameters are discussed.     

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。     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.     

Proteomic analysis of the anti‐inflammatory action of minocycline

Minocycline possesses anti‐inflammatory properties independently of its antibiotic activity although the underlying molecular mechanisms are unclear. Lipopolysaccharide (LPS)‐induced cytokines and pro‐inflammatory protein expression are reduced by minocycline in cultured macrophages. Here, we tested a range of clinically important tetracycline compounds (oxytetracycline, doxycycline, minocycline and tigecycline) and showed that they all inhibited LPS‐induced nitric oxide production. We made the novel finding that tigecycline inhibited LPS‐induced nitric oxide production to a greater extent than the other tetracycline compounds tested. To identify potential targets for minocycline, we assessed alterations in the macrophage proteome induced by LPS in the presence or absence of a minocycline pre‐treatment using 2‐DE and nanoLC‐MS. We found a number of proteins, mainly involved in cellular metabolism (ATP synthase β‐subunit and aldose reductase) or stress response (heat shock proteins), which were altered in expression in response to LPS, some of which were restored, at least in part, by minocycline. This is the first study to document proteomic changes induced by minocycline. The observation that minocycline inhibits some, but not all, of the LPS‐induced proteomic changes shows that minocycline specifically affects some signalling pathways and does not completely inhibit macrophage activation.