Influences of Aeromonas salmonicida lipopolysaccharide, prednisolone and water temperature on plasma protein composition in salmonids

To further characterize the putative role of constitutive and inducible plasma proteins in innate resistance to furunculosis, the present authors compared the alterations in profiles of plasma proteins in resistant and susceptible salmonids, i.e. rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), respectively. Rainbow trout were injected with prednisolone acetate and exposed to higher water temperature (18 °C versus 10 °C), or injected with purified lipopolysaccharide (LPS) from a virulent strain of Aeromonas salmonicida , and plasma components were examined by two-dimensional polyacrylamide electrophoresis . Two days after A. salmonicida LPS exposure, rainbow trout had a four- to five-fold increase in concentrations of plasma proteins composed of p48, p19 and p16 subunits, and a significant decrease in a 100-kDa protein group. Consistent elevation or depletion of proteins corresponding to previously reported rainbow trout A. salmonicida LPS-binding pentraxins and lectins in plasma were not observed. Brook trout exposed to A. salmonicida LPS did not have any consistent plasma protein changes. There were no significant alterations in major plasma proteins following temperature shock and prednisolone acetate administration in rainbow trout plasma. These studies demonstrate that rainbow trout with LPS-induced sterile inflammation have few alterations in major plasma proteins or LPS-binding proteins, and do not exhibit the spectrum of acute phase changes induced by inflammation in mammals.     

Stress response of juvenile rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)to chemical cues released from stressed conspecifics

The objective of this study was to determine whether exposure of rainbow trout (Oncorhynchus mykiss) to water containing a stressed trout or skin extract from stressed and non-stressed trout would elicit a stress response in conspecifics. Juvenile rainbow trout were exposed for 1 hour to water containing a stressed fish, homogenized skin extracts from a non-stressed fish, skin extract from a stressed fish and water with none of these factors. The stress response was measured over a 24-h period (1, 6, 12, 24 h after exposure). Plasma cortisol levels increased at 12 h in fish exposed to water from a stressed fish and skin extract from a stressed fish. Plasma glucose and hepatic hsp70 levels were not affected by treatments. The results suggest that rainbow trout elicit a stress response when exposed to stress-related alarm cues released from conspecifics.     

光照颜色对虹鳟行为反应、血浆皮质醇和生化指标的影响

光照是影响鱼类行为、生理以及生长的主要环境因子之一.为研究光照颜色对虹鳟行为反应、血浆皮质醇、生化指标的影响,实验根据鱼类行为学研究方法,分别将白、红、黄、蓝和绿5种光照颜色灯带铺设在室内试验水槽中央底部,将虹鳟放置在水槽同一侧,观察虹鳟在水槽中穿越灯带尾次数及行为反应;根据行为生理学研究方法,分析比较在不同光照颜色下...     

The ability of clove oil and MS-222 to minimize handling stress in rainbow trout (Oncorhynchus mykiss Walbaum)

The effects of 60‐mg L^?1 clove oil and 60‐mg L^?1 tricaine methanesulphonate (MS‐222) on the blood chemistry of rainbow trout were compared after exposure to handling stress via caudal puncture blood sampling. Fish sampled by caudal puncture and subsequently exposed to anaesthetics showed a typical handling stress response over a 48‐h period. There were no significant differences between the responses of fish exposed to equal concentrations of clove oil and MS‐222, with the following exceptions: the blood glucose at full anaesthesia, and lactate at full recovery increased significantly in the clove oil‐exposed fish. In a subsequent experiment, the stress response observed in fish sampled by caudal puncture and exposed to clove oil and MS‐222 was compared with a non‐anaesthetized control group. The increases in plasma cortisol levels were significantly lower at recovery in fish treated with either anaesthetic compared with the control fish. Fish exposed to MS‐222 had significantly higher cortisol levels at 1 h. These findings show that few differences exist between the anaesthetic effects of clove oil and MS‐222 on the physiological response of fish to stress. However, clove oil is more effective at reducing the short‐term stress response induced by handling and blood sampling, and is recommended as an alternative fish anaesthetic.     

Changes of Non-Specific Immune Parameters in Rainbow Trout,Oncorhynchus mykiss,After Exposure to Antimicrobial Agents Used in Aquaculture

Changes of plasma lysozyme and serum bactericidal activity, ceruloplasmin, total plasma protein, serum glucose, hematocrit and leucocrit levels in rainbow trout (Oncorhynchus mykiss) were studied after exposure to formalin and chloramine-T, which are used in aquaculture as anti-ectoparasitic and antibacterial agents. Fish were exposed to formalin at a concentration of 250 ppm for 60 min and exposed to chloramine-T for 5 ppm for 3 h. The parameters were analyzed for rainbow trout exposed to formalin and chloramine-T soon after treatments and rainbow trout exposed to freshwater after 24 h (return) following the treatments. Plasma lysozyme activity decreased after exposure to formalin; however, it was not affected by chloramine-T exposure. Serum bactericidal activity reduced after both formalin and chloramine-T exposure and did not change after returning to freshwater. Ceruloplasmin and total plasma protein were not affected by both treatments. Serum glucose levels of exposed fish elevated and remained at higher levels during recovery period when compared to control values. Hematocrit values of fish exposed to formalin increased; however, no significant change was observed in fish exposed to chloramine-T. Leucocrit levels of fish treated with both formalin and chloramine-T were higher than that of controls.     

Measurement of Resting and Stress-elevated Serum Cortisol in Rainbow Trout Oncorhynchus mykiss in Experimental Net-pens

A commercially available heterogeneous, solid-phase tube enzyme-linked immunoassay (ELISA) was modified and validated for the measurement of serum cortisol in rainbow trout Oncorhynchus mykiss . The assay is accurate and precise. Resting and stress-elevated serum cortisol concentrations were measured in rainbow trout with a sensitivity of 1.5 ng/ml. Fish held in net-pens at a density of 0.4 kg/m³/cm had a resting cortisol level of 16.5 ± 3.8 ng/ml (mean ± SE). At 3 h postdisturbance, serum cortisol levels were not affected by the removal of fish from adjacent net-pens with dip nets or by the use of 200 mg/L tricaine methanesulfonate (MS-222) as an anesthetic for obtaining samples. However, an acute stress (60 s removal from water) elevated serum cortisol levels to 73.7 ± 9.4 ng/ml.     

Stocking density and cohort sampling effects on endocrine interactions in rainbow trout

Studies were conducted to explore the effect of cohort sampling and stocking density on the interactions between plasma growth hormone (GH), thyroid hormone and cortisol concentrations in rainbow trout. Depending on the experimental design, plasma GH concentrations were either suppressed, or elevated by sequential removal of cohorts from the holding aquarium. Since plasma cortisol concentrations consistently increased during cohort sampling, regardless of experimental design, it would appear that the apparent correlation (direct or inverse) is the result of concomitant changes, i.e. not necessarily a cause-effect relationship. Plasma GH concentrations of rainbow trout were not correlated with eviscerated body weight.Trout stocked at 150 kg m^–3 exhibited a significantly lower mean growth rate, hepatosomatic index, hepatic lipid reserve, plasma triiodo-l-thyronine (T₃) concentration andin vitro hepatic T₃ production than trout stocked at 60 kg m^–3. These observations are consistent with the former group being food deprived or ration restricted. Stocking density appeared to have no effect on plasma GH or cortisol concentrations, or on the pituitary-interrenal axis response to stressor challenge, or the thyroid tissue response to exogenous TSH challenge.     

Physiological Responses of Rainbow Trout Oncorhynchus mykiss to Crowding and Anesthesia with AQUI-S™

Abstract.— Following exposure to the anesthetic AQUI-S™, plasma cortisol concentration in immature rainbow trout was measured as (mean) 293 ± 48 ng/ mL, which was significantly ( P > 0.05) higher than the mean concentration in resting fish. Cortisol concentrations remained significantly ( P > 0.05) elevated for at least 24 h after treatment. This was accompanied by a significant increase and decrease in hematocrit and plasma potassium, respectively. These perturbations continued for at least 48 h following recovery from anesthesia. Plasma concentrations of total protein and sodium remained unchanged following anesthesia with AQUI-S™. Crowding stress is commonly encountered by fish during manipulation in aquaculture situations. Anesthetising fish prior to, and during, manipulation may reduce the associated stress. Changes in cortisol values resulting from crowding (30 min; 0.1 kg/L) during anesthesia with AQUI-S™ were not appreciably different from those in fish crowded without anesthesia. Thus, anesthesia with AQUI-S™ at the recommended dose of 17 mg/L did not appear to be effective for alleviating the stress of crowding under the conditions of our experiments.     

Plasma growth hormone levels in fed and fasted rainbow trout (Oncorhynchus mykiss) are decreased following handling stress

Plasma growth hormone concentrations of rainbow trout,Oncorhynchus mykiss, fasted for six weeks, were significantly (p < 0.01) higher than in comparable fed animals; in the fasted fish, the levels fell progressively following acute stress (by displacing the fish within their home aquarium), with significant differences from pre-stressed fish evident between one and thirty-two hours after application of the stressor. Plasma growth hormone concentrations also fell significantly in the fed group, but differences were evident only between two and eight hours after stressor application.Plasma cortisol concentrations in pre- and post-stressed fed and fasted fish were similar. There was a bimodal response to stressor application in both groups, with significantly higher values relative to the pre-stressed sample evident one and eight hours after disturbance, but not after two, four or thirty-two hours. The changes in plasma cortisol levels between the initial (09:00h) sample and the sample taken eight hours later resembles the diet pattern seen in trout given access to self-demand feeders.Plasma glucose concentrations in pre-stressed fed animals were higher than in pre-stressed fasted fish. This relationship was also evident between one and four hours and thirty-two hours after stressor application. The post-stress rise in plasma glucose concentration was evident between one and four hours in the fed group, and between four and eight hours in fasted fish.The diel changes in plasma growth hormone and glucose concentrations could not be attributed to normal circadian patterns, and there was no apparent correlation between changes in plasma growth hormone and cortisol concentrations. There was a significant inverse correlation between plasma glucose and growth hormone concentrations when the total data set were analyzed, but these correlations were not apparent when the treatment groups were analyzed separately.     

The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products

Abstract The possible mechanism of inactivation of the toxicity of Aeromonas salmonicida extracellular products (ECP) by normal rainbow trout serum was investigated using juvenile rainbow trout. ECP was prepared from culture supernatant by an acetone precipitation method. The ECP was incubated with normal rainbow trout serum at 20°C for 2 h, and the interrelationship between ECP proteolytic activity and immune complex-initiating, haemolytic complement activity (CH₅₀) of normal serum against antibody-sensitized goldfish red blood cells was evaluated. When normal serum was incubated with increasing concentrations of ECP, the CH₅₀ activity of serum decreased. The CH₅₀ activity was completely abolished in serum treated with undiluted ECP. ECP treated with serum was administered to trout intraperitoneally to determine mortality. All the fish receiving untreated ECP (0.05 ml = 0.5 mg protein) alone died within 24 h. When ECP was treated with serum at 1:1 to 4:1 (serum: ECP) in volume a similar high mortality was produced. These inocula possessed high protease activity and no or low CH₅₀ activity. However, mortality decreased and finally no mortality was recorded as ECP was treated with large volumes of serum (9:1 to 19:1). These inocula had lower protease activity and considerably higher CH₅₀ activity. Fish receiving ECP treated with heat-inactivated serum at 19:1 showed 100% mortality. A serum: ECP inoculum derived from fish which had been administered lipopolysaccharide from Salmonella enteritidis and which possessed a low CH₅₀ activity also gave a high mortality when used at 19:1. These results suggest that rainbow trout complement is implicated in the inactivation of toxicity of A. salmonicida ECP.     

Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma cortisol

Basal levels of plasma cortisol in unstressed salmonid fish are normally in the range 0–5 ng ml⁻¹. An acute stress such as handling or 1 h confinement caused a temporary elevation of the plasma cortisol levels of both brown trout,Salmo trutta L., and rainbow trout,Salmo gairdneri Richardson, in the range 40–200 ng ml⁻¹ with a return to basal levels within 24–48 h. The extent of the cortisol elevation in response to an acute stress was dependent upon both the species and strain of trout. Chronic stresses, such as prolonged confinement or crowding, resulted in an elevation of plasma cortisol levels to approximately 10 ng ml⁻¹. Under these circumstances, blood cortisol levels remained elevated for periods of up to 4 weeks before acclimation finally occurred. It is shown, by means of intraperitoneal implantation of cortisol, that chronic elevation of plasma cortisol levels in the brown trout results in a dose-dependent increase in mortality due to common bacterial and fungal diseases. This effect is apparent at plasma cortisol levels as low as 10 ng ml⁻¹, levels below those often reported as being representative of ‘unstressed’ fish. These findings are discussed in relation to the known immunosuppressive effects of corticosteroids in teleost fish.     

The effect of some steroid hormones on the growth and protein metabolism of rainbow trout

Two synthetic androgens, dimethazine and norethandrolone, and an oestrogenic compound diethylstilboestrol were administered orally at different concentrations in the diet to juvenile rainbow trout and their effect on protein metabolism, growth and food conversion efficiency examined. Both dimethazine and norethandrolone, when given at low concentrations (2.5 and 5.0 mg/kg food) for 60 days significantly increased weight gain. These increases in weight gain were due to both the increased rate of protein synthesis and improved food conversion efficiency. High concentrations of dimethazine (10 and 20 mg/kg food) produced an initial increase in weight gain but it was not significantly different from that of untreated fish after 48 days. There was no change in the hepatosomatic indices of the fish treated with dimethazine or norethandrolone. However, the renosomatic indices of rainbow trout fed dimethazine were significantly greater than those of the controls. Diethylstilboestrol (1.2 mg/kg food) produced an adverse effect on growth.     

Experimental validation of geosmin uptake in rainbow trout,Oncorhynchus mykiss (Waldbaum) suggests biotransformation

The bioconcentration of waterborne geosmin in rainbow trout, Oncorhynchus mykiss (Waldbaum) was assessed. Fifty rainbow trout with a mean (SD) weight of 226.6 (29.0) g and lipid content of 6.2 (0.6) % (w/w) were exposed to geosmin in static water for 0, 2, 4, 6, 8, 12, 24, 36, 48 and 120 hr, with one tank containing five fish for each exposure period. Geosmin concentrations were measured in fish tissue and water samples collected over time. With time the geosmin concentration in the fish increased and decreased in the water. However, the total absolute amount of geosmin in the system declined over time which could be explained by induction of biotransformation. This is in accordance with the decreasing lipid normalized geosmin levels in the liver compared with the liver‐free carcass. Geosmin distribution within rainbow trout clearly is not exclusively governed by the lipid content of tissues. In vivo geosmin bioconcentration in rainbow trout is slower and the body burden reached is lower than the generally accepted theoretical model predicts.     

Plasma ladderlectin concentration following sterile inflammation and Aeromonas salmonicida subsp. salmonicida infection

Plasma samples obtained from rainbow trout either experimentally infected with Aeromonas salmonicida or injected with either A. salmonicida lipopolysaccharide (LPS) or a commercial A. salmonicida vaccine (Lipogen) were analysed by enzyme immunoassay to evaluate changes in rainbow trout ladderlectin (RTLL) concentrations during the acute phase response (APR). Plasma RTLL concentrations in fish injected with A. salmonicida LPS, vaccine or live A. salmonicida varied over a 10 day period, but did not significantly increase. In contrast, fish experimentally infected with A. salmonicida exhibited a modest, but statistically significant ( P  <   0.05), decrease in RTLL concentration. These studies demonstrate that RTLL is not detectably induced during the trout APR to sterile inflammation or A. salmonicida infection, but plasma concentration of this protein may be reduced during bacterial infection.     

Does oral 3,5,3 ′-triiodo-l-thyronine affect dietary glucose utilization and plasma insulin levels in rainbow trout (Salmo gairdneri)?

A factorial experiment was conducted to determine the effect and interaction of dietary carbohydrate level and triiodo-L-thyronine (T₃) supplementation on the growth, physiological response and plasma insulin and cortisol levels of rainbow trout. The oral administration of T₃ significantly increased the growth, protein efficiency ratio and feed efficiency of trout, indicating an increased protein and perhaps energy utilization in these fish. However, T, administration did not significantly increase the utilization of dietary glucose as an energy source by the trout. Similarly, the administration of T₃ did not significantly affect plasma insulin levels in either the fed or the fasted trout. Plasma insulin levels were significantly higher in fed trout reared on the non-T₃ supplemented high carbohydrate diet in comparison to trout reared on the low carbohydrate diets. This indicates that increased dietary carbohydrate stimulates increased insulin secretion in the trout. Therefore, although rainbow trout are not insulin-deficient, they can still be considered a diabetic-like animal due to their poor glucose tolerance. Plasma cortisol levels were not affected by diet composition and altered plasma glucose levels.     

Altered stress responses in rainbow trout following a dietary administration of cortisol and β-napthoflavone

Previous studies have demonstrated that fish inhabiting polluted waterways often have an impaired stress response at the organismal level. Given the possible link between the organismal (i.e. cortisol) and cellular (i.e. heat shock proteins; hsp) stress responses, we conducted this study to examine the ability of rainbow trout to respond to a 2 h, +14 °C heat stress (HS) challenge following a 28 d, sub-chronic exposure to increased concentrations of cortisol (5 mg kg⁻¹ b.w.), β-napthoflavone (bnf; 50 mg kg⁻¹ b.w.), and a combination of both (mixture), through the diet (1.5% b.w. every 48 h). While control fish responded to the HS by significantly increasing components of their organismal (cortisol, glucose, and lactate) and cellular (hepatic hsp70 protein) stress responses 6 and 24 h post HS, cortisol-, bnf-, and mixture-fed fish had impaired stress responses at both levels of organization. Additionally, hepatic hsp70 levels were significantly reduced 6 h post HS in cortisol-fed fish. While bnf-fed fish had significantly higher EROD activity, cortisol potentiated EROD activity in the mixture-fed fish. Similarly, plasma cortisol concentrations in the mixture-fed fish were significantly lower relative to cortisol-fed fish. These data are the first to indicate that sub-chronically stressed fish can have impaired stress responses at both the organismal and cellular levels. These findings raise questions regarding: (a) the universal and simple applicability of biological indicators of stress in fish; (b) the possible functional relationship between these two levels of stress responses; and (c) the importance of hsps in the generalized stress response of the whole organism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stress-induced changes in the affinity and abundance of cytosolic cortisol-binding sites in the liver of rainbow trout,Oncorhynchus mykiss (Walbaum), are not accompanied by changes in measurable nuclear binding

Plasma cortisol levels and the number (N_max) and affinity (K_d) of specific hepatic cortisol-binding sites were determined in rainbow trout subjected to chronic confinement stress for 14 days. Confinement significantly elevated plasma cortisol levels to 47.3 ± 13.5 ng ml^–1 within 24h and although levels declined to 8.0 ± 3.0 ng ml^–1 after 14 days, they were significantly higher throughout than levels in unstressed control fish (< 2.0 ng ml^–1). There was a 60% reduction in cytosolic N_max in stressed fish during the first 24h of confinement (35.8 ± 7.9 cf. 129.0 ± 15.2 fmol mg^–1 protein), a decline which was sustained at 7 days after the onset of stress but, although numbers of binding sites in the liver of stressed fish were still lower than in unstressed fish, the difference was no longer significant after 14 days of confinement. There was an accompanying significant rise in the K_d of cortisol binding in stressed fish during confinement, from 4.0 ± 0.6 nM at time 0 to 8.4 ± 0.8 nM after 24 h confinement. This increment in K_d was sustained at a level significantly higher than in control fish throughout the 14 day confinement period, despite marked reductions in cortisol levels and increases in N_max in stressed fish. Throughout the study, specific binding of cortisol could not be consistently detected in high-salt nuclear extracts from stressed or unstressed fish, suggesting either that high-affinity binding sites for cortisol were absent from these preparations, that receptors were present but unable to interact with ligand because they were occupied, or that receptors were present but not being extracted. These possibilities were investigated using a range of extraction procedures, by varying the temperature of incubation, by employing dexamethasone as ligand and by examining binding in purified, intact, nuclei. Estradiol was employed as a methodological control throughout and substantial amounts of specific estradiol binding were detected in all compartments and preparations. Specific cortisol-binding sites were detected in intact nuclei of both stressed and unstressed fish, at levels an order of magnitude lower than estradiol binding in the same preparations. These data demonstrate that activation of the pituitary-interrenal axis leads to significant changes in the nature of target-tissue binding of cortisol in rainbow trout, and reveal a clear difference in the subcellular distribution of binding-sites for estradiol and cortisol, which reflects the situation in mammalian tissues.     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Effects of dietary inclusions of red beet and betaine on the acute stress response and muscle lipid peroxidation in rainbow trout

This study evaluates the effects of red beet (RB) and betaine on rainbow trout submitted to an acute stress challenge. A control diet was compared with four experimental diets in which red beet (14 and 28%) and betaine (0.9 and 1.63%) were incorporated in different concentrations according to a factorial design. Cortisol in plasma and fin, glucose and lactate plasma levels, and malondialdehide (MDA) in muscle were all measured before the stress challenge and 30 min and 6 and 12 h after the stress challenge as parameters to determine the diet effects. RB and betaine had no effect on cortisol, glucose, and MDA basal levels. However, lactate basal levels were significantly lower on fish fed with RB and betaine. Thirty minutes after the stress challenge, there was a significant increase in plasma and fin cortisol, glucose and lactate concentrations, although fish fed with diets containing RB and betaine showed significantly higher plasma cortisol values. MDA values of fish fed with 14% RB and 0.9% betaine were significantly higher than MDA values from fish fed with 28% RB and 1.63% betaine. After 6 and 12 h, plasma and fin cortisol and lactate levels recovered in a similar trend. Glucose plasma levels recovered in almost all groups 12 h after the stress. Also, MDA values recovered basal levels after 6 and 12 h. RB and betaine did not enhance the tolerance to the stress challenge compared to the control group, although the presence of these ingredients had no negative effect on any of the stress indicators.     

Anaesthetic efficacy and biochemical effects of 1,8‐cineole in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792)

The aim of the present study was to investigate the anaesthetic efficacy and biochemical effects of 1,8‐cineole (cineole) in rainbow trout (Oncorhynchus mykiss). The fish were exposed to 200, 300, 400, 500, 600 and 800 μl/L cineole and time of induction of anaesthesia and recovery from anaesthesia were recorded. Thereafter, the fish haematological and biochemical responses to anaesthesia with different concentrations of cineole were studies. Moreover, the haematological and biochemical response of fish anaesthetized for 300 s with either cineole (283 μl/L) or eugenol (25 μl/L) were compared. Cineole at the concentrations of 200–800 μl/L induced stages 2, 3 and 4 anaesthesia within 109‐29.3, 226‐59 and 418‐117 s respectively. Increase in anaesthesia induction time led to higher stress responses and enzymes’ activity characterized by elevation in red blood cell (RBC), white blood cell (WBC), blood haematocrit and haemoglobin, and plasma cortisol, glucose and lactate levels, and aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase and creatine phosphokinase (CPK) activities. However, cineole concentrations had no significant effects on plasma alanine aminotransferase (ALT) activity and ions levels. Comparison of cineole with eugenol showed that cineole showed less change in blood RBC and plasma AST, ALP, CPK, cortisol and glucose levels compared to eugenol. In conclusion, cineole is efficient to anaesthetize trout at concentrations of 200–800 μl/L. Increase in cineole concentration shortens anaesthesia induction time, stress response and probably tissue damage. The concentrations of 600–800 μl/L cineol is recommended for rapid sampling as it causes the least stress and enzymatic responses. The present results suggest that cineole causes slightly lower side effects in trout compared to eugenol.