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Abstract: Hydrogen peroxide (H2O2) is currently being considered as a potential disinfectant in aquaculture. Stress impact of H2O2 exposure in sea bass, Dicentrarchus labrax was evaluated by measuring stress indicators; plasma cortisol, lactate, glucose, plasma calcium (Ca), phosphorus (P), sodium (Na), potassium (K), chloride (Cl), magnesium (Mg), hematocrit, hemoglobin, total protein and albumin. The stress indicators were analysed for 30 fish from each of three experimental groups: (i) exposure group: fish that were sampled immediately after a 1h 50ppm H2O2 bath, (ii) recovery group: fish treated as in (i) and sampled after a 24h recovery period without H2O2, and (iii) control group: fish that were sampled at the same time as exposure group fish, but that were not exposed to H2O2. The stress indicators measured were compared against the control. Plasma cortisol and hemoglobin levels were significantly higher than control, after H2O2 exposure and after the 24h recovery. Plasma lactate level was significantly higher after H2O2 exposure, but similar to control levels after the 24h recovery. Plasma glucose level was significantly higher after H2O2 exposure and significantly lower than the control at 24h. Plasma electrolytes showed different responses to H2O2 exposure. Plasma Ca, Na and Mg were significantly higher in the exposure group compared to the control and at 24h, plasma Ca and Mg were similar to the control while plasma Na was significantly lower than control. There were no changes in plasma P, K and Cl as a consequence of H2O2 exposure, but plasma K and Cl were significantly increased at 24h. Hematocrit and albumin were significantly increased after H2O2 exposure and similar to the control at 24h. The results indicate a quick physiological stress response and sensitivity of sea bass to H2O2 exposure, which appears to require more than 24h period for full recovery. [Copyright &y& Elsevier]

In this study, two experiments were performed to investigate the anesthetic efficiency, hematological and biochemical effects of thymol on Nile tilapia, Oreochromis niloticus. In the first experiment, in order to estimate thymol anesthetic efficacy, tilapia juveniles were exposed to anesthetic baths at six different concentrations of thymol (10, 20, 40, 60, 80, and 100 mg L−1) and the induction and recovery times were recorded. The second experiment was performed with five treatments: CON (non-anesthetized fish), TH1 (anesthesia with 80 mg L−1 thymol within 1 min), TH5 (anesthesia with 30 mg L−1 thymol within 5 min), EU1 (anesthesia with 80 mg L−1 eugenol within 1 min), and EU5 (anesthesia with 30 mg L−1 eugenol within 5 min). The results showed that thymol at 10 mg L−1 concentration failed to induce anesthesia; however 20–100 mg L−1 concentrations induced anesthesia within 491–56.3 s. A strong negative relationship was detected between thymol concentrations and induction times (R2 = 0.906). Anesthesia had no significant effects on plasma albumin, triglyceride, cholesterol, alanine aminotransferase, and the blood mean corpuscular hemoglobin levels. The anesthetized fish exhibited significant elevations in plasma cortisol, glucose, lactate, and the blood hematocrit and mean corpuscular volume; these parameters exhibited significant elevations in the fish anesthetized by 30 mg L−1 anesthetics, compared to the 80 mg L−1. Plasma globulin, total protein, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, catalase, superoxide dismutase, and the blood hemoglobin and red blood cell significantly increased, as mean corpuscular hemoglobin concentration decreased, in the fish anesthetized by 30 mg L−1 anesthetics, compared to the CON fish. The anesthetized fish showed similar plasma glutathione levels, all significantly lower than the CON fish. The EU5 fish exhibited significant elevation in the plasma malondialdehyde levels, compared to the CON fish. In conclusion, thymol anesthetic efficacy was similar to eugenol in Nile tilapia weighing ~40 g at water temperature of 26 °C. Considering the physiological responses, slight but significant elevations in plasma proteins, lactate, lactate dehydrogenase, and malondialdehyde levels attest that thymol may induce less stress, hypoxia, and oxidative stress in the fish than eugenol, but further studies are needed to provide robust and clinically applicable data to support this hypothesis. • Thymol at 80 mg L−1 is suitable for Nile tilapia anesthesia. • Thymol effects on plasma enzymes and stress responses are similar to eugenol. • Thymol induces less hypoxia than eugenol after 5-min anesthesia. • Thymol anesthesia leads to lower plasma MDA, compared to eugenol after 5-min anesthesia. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study was to develop, evaluate and set up baseline values for reliable acute and chronic stress indicators in sexually immature red porgy, Pagrus pagrus. The effects of anaesthesia, acute (chasing for 10 min followed by air exposure for 3–4 min) and chronic (different stocking densities: 5 vs. 25 kg m−3 and water temperatures: 15 °C, 19 °C, 25 °C) stressors on stress indicators were studied. Cortisol, lactate, norepinephrine, epinephrine, glucose, electrolytes, and prothrombin time were found to be reliable indicators of acute stress, whereas lactate and catecholamines were found to be reliable indicators of chronic stress. Baseline values were: glucose, 2.05–5.17 mmol l−1; lactate, 0.15–1.5 mmol l−1; cortisol, 0.8–24.9 ng ml−1; potassium, 1.5–4.5 mmol l−1; sodium, 167–206 mmol l−1; chloride, 165–180 mmol l−1; and prothrombin time, 9.26–14.65 s. Plasma epinephrine and norepinephrine concentrations after minimum handling were 2.4–43.2 nM and 9.6–74.5 nM, respectively. The present study also revealed that stocking density expressed in kg m−3 is not an appropriate measure to describe and compare the physiological status of fish under intensive culture conditions. Results suggested that when group and tank sizes are reduced, even at a low stocking density (5 kg m−3), the maintenance cost is higher than the one at high stocking density (25 kg m−3). In addition, low (15 °C) and high (25 °C) water temperatures were important environmental stressors for Pagrus pagrus, as shown by the observed elevated (mean±SEM) norepinephrine (15 °C: 110.8±17.6 nM, 19 °C: 84.0±6.0 nM, 25 °C: 160.8±24.6 nM) plasma concentrations. [Copyright &y& Elsevier]

Abstract: A number of primary and secondary features used as stress indicators in fish were investigated in red sea bream, Pagrus major exposed to different photoperiod regimes. In Experiment 1, fish weighing from 17 to 25 g were subjected to acute stress consisted of netting the fish from the rearing tanks and confinement in a small tank (30 L) half-full of water. The fish were then sampled at 0.5, 1, 4, 24, 48, and 72 h post-stress to investigate the stress-induced levels of different parameters. In Experiment 2, fish were exposed to different photoperiods (6L:6D, 12L:12D, 16L:8D and 24L:0D) for eight weeks. Blood samples were collected from the fish at the 1st, 4th and 8th week after exposure to the photoperiods. Acute stress significantly increased the plasma levels of cortisol (190.1 ng ml−1) and glucose (109.5 mg dl−1) and returned back to the basal levels within 24 h. In contrast, plasma levels of cholesterol (180.5 mg dl−1), total protein (3.0 g dl−1), potassium (4.1 mMol l−1) and Ca++ (11.3 mg dl−1) were decreased significantly and returned to levels indistinguishable from those of control within 72 h except cholesterol, which did not recover within 72 h. In Experiment 2, fish exposed to 6L:6D, 16L:8D and 24L:0D photoperiods had significantly higher cortisol levels than those exposed to the 12L:12D photoperiod during the first week (P <0.05); however, there was no major difference in cortisol levels among the treatments during other sampling times. There were no significant differences in other parameters among the treatments during the course of the study (P >0.05). Fish exposed to the 24L:0D photoperiod showed significantly higher weight gain and SGR followed by 16L:8D, 6L:6D and 12L:12D photoperiods. The plasma levels of different stress indicators in different photoperiods in Experiment 2 were far lower than those of acute stress-induced levels observed in Experiment 1. On the other hand, no apparent signs of chronic stress were observed in fish exposed to different photoperiods. The results demonstrated that photoperiod manipulation caused no significant acute or chronic stress response in red sea bream. [Copyright &y& Elsevier]

Chronic stress is known to impair fish health, performance, and welfare. Although physiological responses to acute stress have been well studied and reliable as well as easily quantified indicators, like circulating cortisol, are available in fish, the same is not true for chronic stress. Specifically, commonly used stress indicators cannot reliably identify the effects of chronic stress in fish. Recently, cortisol concentration in fish scales has been proposed as a novel marker of chronic stress. The purpose of the present study was to verify whether circulating physiological stress indicators and cortisol concentrations in fish scales are reliable markers of chronic stress in European sea bass. For that reason, a predictable, repeated chronic stress protocol was developed and applied. Results showed that, in the absence of an additional acute stressor, physiological indicators like plasma cortisol, glucose, lactate and osmolality were not able to differentiate non-stressed from chronically stressed fish. Only when fish were exposed to an additional stressor, cortisol response of the chronically stressed fish was blunted compared to controls. On the contrary, cortisol concentration in scales differentiate non-stressed from chronically stressed fish, being significantly higher in the later. The same was observed when previously identified as High cortisol Responding (HR) fish, but not Low Responding (LR) fish were tested. Collectively, current results confirm that circulating levels of stress indicators cannot mark chronic stress in E. sea bass, while cortisol concentration in fish scales is able to identify chronic stress situation. • Commonly used plasma stress indicators cannot reliably indicate chronic stress in European sea bass. • Chronic stress can be reflected on the concentration of cortisol in fish scales. • Low (LR) and High (HR) responding individuals show differences in scales cortisol concentration after exposure to chronic stress. [ABSTRACT FROM AUTHOR]

Current European Union regulation explicitly states that farmed fish should be spared any avoidable pain, distress or suffering at the time of slaughter. It has been shown that fish suffer when they are killed in an ice slurry, the most common method of killing farmed fish in the Mediterranean. Thus, it is necessary to find a method of slaughtering Mediterranean fish that is, (1) efficient in inducing unconsciousness with minimal pain and distress, (2) practical to be applied to a large group of animals at the same time, and (3) feasible to be used at sea. The present study assesses the welfare of Gilthead seabream (Sparus aurata) stunned by two different gas mixtures authorised for stunning other farmed species. To achieve this objective, commercial sized seabream were stunned and/or sacrificed under different protocols: a) killed directly in ice slurry, b) exposed to a mixture of 30% CO 2 + 70% N 2 , and then moved to ice slurry and c) exposed to a mixture of 40% CO 2 + 30% N 2 + 30% O 2 and then moved to ice slurry. Electroencephalograms (EEG) were recorded to evaluate the state of consciousness of seabream during stunning, while blood and brains were sampled to obtain acute stress indicators and relative gene expression, respectively. Additionally, dead fish were kept for in situ meat quality evaluation. When exposed to the gas mixtures, fish lost balance at 1 min 23 s ± 31 s with CO 2 + N 2 and 1 min 12 s ± 32 s, with CO 2 + N 2 + O 2 , respectively. Cortisol, lactate and glucose levels were significantly lower in all fish exposed to gas prior to ice slurry than in fish slaughtered directly in ice slurry (p < 0.05). Electroencephalogram records indicated that fish started to lose consciousness when they lost balance and sank to the bottom of the tank. No differences were found in the meat quality (pH and rigor mortis) among the three treatments. Altogether, the study concludes that the use of carbon dioxide together with nitrogen prior to immersion in ice slurry is more humane than ice slurry alone. • A stunning method which can comply with current legislation is proposed for seabream • Seabream stunned by exposure to a mixture of gases is less stressed than when killed directly in ice slurry • The aversion to CO 2 is reduced in the presence of N 2 [ABSTRACT FROM AUTHOR]