Effects of clove oil and MS-222 on blood hormone profiles in rainbow trout Oncorhynchus mykiss, Walbaum

Clove oil has been demonstrated to be an effective, inexpensive anaesthetic and euthanizing agent for a number of fish species, including rainbow trout, used in aquaculture and fisheries research. However, the potential for clove oil to cause perturbations in important plasma hormone concentrations has not been investigated. The effect of anaesthesia and euthanasia in trout with eugenol (the active ingredient in clove oil) on plasma cortisol, glucose, growth hormone (GH) and two thyroid hormones [tri‐iodothyronine (T₃) and thyroxine (T₄)] was compared with tricaine methanesulfonate (MS‐222) anaesthesia, and stunning by cranial concussion in two experiments. Effects on blood chemistry were different when comparing the particular anaesthetic method being used. Stunning fish significantly increased plasma cortisol and glucose levels (both P<0.05), while euthanizing fish using either clove oil or MS‐222 had no effect on these hormone levels. In contrast, the levels of GH, T₃ and T₄ hormones were unaffected regardless of whether fish were euthanized by stunning, MS‐222 or clove oil. Variation in effects between hormones were observed using clove oil eugenol. In fish sampled 10 min after anaesthetizing with 150 mg L^?1 of eugenol, cortisol levels were significantly decreased (P<0.03), while there were no differences in either glucose or GH levels. Tri‐iodothyronine and T₄ also showed significantly elevated levels (P<0.05) after 10‐min exposure to eugenol. These results highlight the importance of investigating the potential effects of any new anaesthetic or euthanizing compounds on blood plasma parameters, prior to using them in a research setting, or when comparing results to other studies which have utilized alternative anaesthetic compounds.     

The efficacy of clove oil as an anaesthetic for rainbow trout, Oncorhynchus mykiss (Walbaum)

The anaesthetic effects of clove-oil-derived eugenol were studied in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Acute lethality and the effects of multiple exposures to eugenol were measured. The estimated 8-96 h LC₅₀ for eugenol was found to be approximately 9 p.p.m. Times to induction and recovery from anaesthesia were measured and compared with MS-222 under similar conditions. Eugenol generally induced anaesthesia faster and at lower concentrations than MS-222. The recovery times for fish exposed to eugenol were six to 10 times longer than in those exposed to similar concentrations of MS-222. Clove oil eugenol was determined to be an acceptable anaesthetic with potential for use in aquaculture and aquatic research. Doses of 40-60 p.p.m. eugenol were found to induce rapid anaesthesia with a relatively short time for recovery in juvenile trout.     

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum)

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.     

In vitro and in vivo antifungal activity of Satureja cuneifolia ten essential oil on Saprolegnia parasitica strains isolated from rainbow trout (Oncorhynchus mykiss,Walbaum) eggs

In the present study, the chemical composition and the antifungal properties against Saprolegnia parasitica (in vitro and in vivo) of the essential oils of thyme (Satureja cuneifolia) from Mediterranean region of Turkey were evaluated for the first time. The composition of oils was analysed using gas chromatography/mass spectrometry (GC/MS). The major constituents of oil of S. cuneifolia were cavracrol (46,84%) and cymene (16.90%). Antifungal effects of S. cuneifolia essential oil against S. parasitica strains (A1 and E1) were detected by disc diffusion and tube dilution assays. The antifungal effect of S. cuneifolia was determined to be stronger against S. parasitica E1 isolate (MIC 50 μL mL^?1, MLC 250 μL mL^?1) compared with S. parasitica A1 isolate (MIC 50 μL mL^?1, MLC 500 μL mL^?1). Following in vitro assays, effective doses of S. cuneifolia for disease control in rainbow trout eggs experimentally infected with S. parasitica were investigated. For this aim, infected eggs were treated with the essential oil (0, 5, 10, 20 and 50 ppm) during incubation period (21 days) after fertilization. Formalin (5 mL L^?1) was used as positive control. Hatching rate of eggs at the end of incubation period were calculated. The highest hatching rates were recorded in S. parasitica E1 strain at 5 and 10 ppm concentrations of S. cuneifolia and in S. parasitica A1 strain at 10 and 20 ppm (P < 0.05).     

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.     

Indirect estimation of stomach volume of rainbow trout Oncorhynchus mykiss (Walbaum)

The main hypothesis of this study was that if stomach volume is correlated with food intake it can be estimated without laborious and destructive direct measurement. Rainbow trout, Oncorhynchus mykiss (Walbaum), ca. 500–1300 g, were starved for 1, 4, 8 or 16 days at 15°C after which they were fed in excess with dry pellets containing known amounts of X‐ray‐dense markers. Immediately after feeding the fish were killed, X rayed and weighed. Then the stomach was dissected, its contents removed and weighed, and stomach volume was measured. X‐ray plates were developed and feed intake was estimated based on the amount of marker. All measured variables correlated positively with stomach volume. The best fit for linear regression models was obtained for fish starved for 4 days, where stomach content (dry mass) explained 94%, food intake (based on X‐ray measurement) 77% and fish mass 62% of the variation in stomach volume. However, as stomach content measurement can be a lethal, or at least very stressful, event for the fish, the accuracy of food intake measurement (X‐ray) could be increased using multiple regression. In multiple linear regressions, R²‐values varied between 0.79 (16‐day starvation) and 0.91 (1‐day starvation) with food intake and fish mass as explanatory variables for stomach volume. These results indicate that the stomach volume in rainbow trout can be estimated satisfactorily using indirect methods, which are not detrimental to the fish, although feeding history may affect the accuracy of the estimates.     

Olfactory nerve response of masu salmon (Oncorhynchus masou Brevoort) and rainbow trout (O. mykiss Walbaum) to clove oil and MS-222

Two anaesthetics, clove oil and methane sulphonate (MS-222), were examined for their effects on the olfactory nerve response of masu salmon ( Oncorhynchus masou Brevoort) and rainbow trout ( O. mykiss Walbaum). Exposing both species to clove oil for 3 min at concentrations of 50 and 100 mg L⁻¹, or for 10 min at 50 mg L⁻¹, did not significantly reduce their olfactory response. Directly applying clove oil anaesthesia to the olfactory epithelium significantly reduced olfactory response though after 20 min, olfactory response recovered to 70% and 52% of pre-treatment levels in masu salmon and rainbow trout respectively. Compared with the post-anaesthetic recovery of responses after clove oil (50 mg L⁻¹), buffered MS-222 (100 mg L⁻¹) with NaHCO₃ (100 mg L⁻¹), and unbuffered MS-222 (100 mg L⁻¹) treatment for 3 min, the response after MS-222 treatment declined gradually and significantly, but not after clove oil and MS-222+NaHCO₃ treatments. Clove oil appears to be an effective and relatively safe anaesthetic for salmonids with little long-term impact on their olfactory response, which plays a crucial role in their life history.     

Protective effects of a synbiotic against experimental Saprolegnia parasitica infection in rainbow trout (Oncorhynchus mykiss)

A combination of probiotics and prebiotics as synbiotics allows assessing their synergistic effects. This study evaluated the effects of a synbiotic supplement on growth performance, haematological parameters and resistance to Saprolegnia parasitica in rainbow trout, Oncorhynchus mykiss (Walbaum) fingerlings. Fish fed a dietary synbiotic in three levels of 0.5, 1.0 and 1.5 g kg^?1 thrice a day. The fingerlings were challenged with Saprolegnia parasitica after 60 days post feeding and their mortalities recorded up to 15 days. The fingerlings at all three experimental treatments showed significant (P < 0.05) increases in final mean weights and specific growth rates (SGR). The best feed conversion ratio (FCR), feed conversion efficiency (FCE) and maximum survival rate were also obtained by the fish fed 1.0 g synbiotic kg^?1 diet. Furthermore, supplementation with synbiotic significantly increased blood factors at all treatments. After challenges with Saprolegnia parasitica, the synbiotic‐fed groups showed significantly higher survival rates compared with the control group. These results reveal that a dietary synbiotic of 1.0 g kg^?1 fed for 60 days leads to increased growth performance and survival rate as well as improved feeding efficiency in rainbow trout fingerling, rendering them more resistant against infection by Saprolegnia parasitica.     

Effects of ascorbic acid enrichment by immersion of rainbow trout (Oncorhynchus mykiss, Walbaum 1792) eggs and embryos

This study was conducted to examine the effects of different forms and concentrations of ascorbic acid (vitamin C), and different enrichment times (24 and 48 h post ovulation) on egg, embryo and alevin ascorbate concentrations and survival of rainbow trout (enrichment was at the ova stage). In experiments 1 and 2, fertilized eggs were immersed in water containing ascorbate at 0 (control), 100, 1000 mg L^?1 l ‐ascorbic acid (AA) and 2000 mg L^?1 l ‐ascorbyl monophosphate (AP). In experiment 3, 0 (control), 500 and 1000 mg L^?1 AA neutralized (N) with NaOH, 1000 mg L^?1 AA non‐neutralized (NN), 1000 and 2000 mg L^?1 AP immersions were used. The mean total ascorbic acid (TAA) and dehydroascorbic acid (DHA) concentrations were measured before fertilization, at 3 and 24 h after fertilization, at the eyed stage, and in hatched alevins. We observed significant differences in TAA concentration at different immersion levels at 3 and 24 h after fertilization. Survival decreased significantly depending on the level of vitamin C, pH of the solutions and immersion time. We suggest that when broodstock rainbow trout do not have enough vitamin C in their ovaries, immersion of eggs in 1000 mg L^?1 of neutralized AA may be useful.     

Strain differences in feed efficiency measured as residual feed intake in individually reared rainbow trout, Oncorhynchus mykiss (Walbaum)

The efficient use of feed for growth and meat production is important for all animal production industries including aquaculture. Residual feed intake (RFI) is an alternative measure of feed efficiency that has been widely used in livestock production. Residual feed intake was calculated as the difference between intake observed and intake predicted on the basis of a bioenergetics model; a low RFI indicates greater efficiency. Residual feed intake offers some advantages as a selection criterion for improving production efficiency over traditional feed efficiency statistics because it is not a ratio and it typically has a larger coefficient of variation. The RFI of individually reared rainbow trout progeny from six different genetic cross‐types was examined for genetic variation. Proximate analysis and nitrogen retention were also evaluated to determine if differences in RFI correlate to differences in body composition and nutrient retention and varied by cross‐type. Differences between cross‐types indicated a genetic component for RFI, with the most efficient fish of approximately 160 g consuming 0.99 g less and inefficient fish consuming 0.05 g more feed per day than expected. Lower RFI was associated with higher growth rates (r=?0.38, P<0.05) and greater nitrogen retention (r=?0.82 P<0.001).     

Suppression of Saprolegnia infections in rainbow trout (Oncorhynchus mykiss) eggs using protective bacteria and ultraviolet irradiation of the hatchery water

Since formalin is widely used in prevention of Saprolegnia infections in salmonid fish hatcheries, there is a need for more environmentally safe treatment methods. Therefore, we screened 360 bacterial isolates against their ability to antagonize the growth of Saprolegnia parasitica hyphae in vitro, and best strains were selected according to their antagonistic properties and colonization capability on rainbow trout egg surface. Protective bacterial cultures of Pseudomonas sp. M162, Pseudomonas sp. M174 and Janthinobacterium M169 were tested for prevention of Saprolegnia sp. infections during incubation trials of rainbow trout (Oncorhynchus mykiss) eggs with UV irradiated (400 mWs cm^?2) and non‐treated inlet water. UV irradiation of inlet water significantly decreased mortality during the incubation. Lowest mortalities were observed in protective culture treated groups incubated with UV‐irradiated inlet water. UV irradiation increased the dominance of the main bacterial colonizers and variation in the bacterial species diversity between the experimental units.     

Erythromycin and florfenicol treatment of rainbow trout Oncorhynchus mykiss (Walbaum) experimentally infected with Flavobacterium psychrophilum

Flavobacterium psychrophilum is responsible for significant economic losses in rainbow trout aquaculture. Antimicrobial treatment remains the primary means of control; however, there are limited choices available for use. The objectives of the study were therefore to determine the minimum inhibitory concentrations for erythromycin and florfenicol in selected F. psychrophilum isolates and to evaluate their clinical treatment efficacy in experimentally infected rainbow trout. All isolates tested had moderate susceptibility to florfenicol and erythromycin except one isolate, which had low susceptibility to erythromycin. Two isolates (one with moderate and one with low susceptibility to erythromycin) were used in an experimental infection trial. Rainbow trout juveniles were injected intraperitoneally with 10⁸ cfu/fish and after mortality had begun, fish were given erythromycin‐ and florfenicol‐medicated feed at a rate of 75 mg kg^?1 day^?1 and 10 mg kg^?1 day^?1 fish body weight, respectively, for 10 consecutive days. The splenic F. psychrophilum load was determined using an rpoC quantitative PCR throughout the 30‐day trial. Relative to antibiotic‐free controls, erythromycin treatment significantly (p < 0.05) reduced mortality of rainbow trout juveniles infected with FPG101, even when treatment was initiated after clinical signs developed.     

Transfer of toxaphene and chlordane into farmed rainbow trout, Oncorhynchus mykiss (Walbaum) via feed

The study was carried out to quantitate the transfer of toxaphene and chlordane compounds from commercial fish feed into the edible part of rainbow trout, Oncorhynchus mykiss (Walbaum) under normal rearing conditions. Trout were fed with unspiked high energy feed for salmon (fat content 26–30%) over a period of 19 months. The average weight of trout increased from 10 g to more than 2092 g, reaching sizes of 51 cm length. Considerable amounts of toxaphene and chlordane residues were transferred from fish feed into trout muscle. Toxaphene concentrations increased up to 8.6 µg (Σ toxaphene indicator compounds 1–3) kg^?1 wet weight (w.w.) and chlordane reached 5.3 µg Σoxy‐, trans‐, cis‐chlordane + t‐nonachlor kg^?1 w.w. Results are also discussed on the contaminant levels based on the fat content and the effect of sexual maturation is considered in this study. The data allow the establishment of transfer rates for toxaphene and chlordane congeners from high energy diet into the edible part of farmed rainbow trout.     

Changes in smooth muscle contractility of rainbow trout (Oncorhynchus mykiss Walbaum) intestine during acclimation to altered temperature

The effects of altered water temperature in vivo on in vitro smooth muscle contractility of rainbow trout intestine were investigated. Temperature has a significant effect on receptor-mediated intestinal smooth muscle contractility in the rainbow trout. The efficacy of 5-HT, carbachol, and transmural stimulation increased with temperatures above 10°C, with an optimal increase at 15°C. There was also a modest increase in the potency of 5-HT and carbachol within 2 days of establishing trout at 20°C. By day 8, most of these changes had either stabilized or were returning to control values, suggesting that acclimation changes in membranes and enzyme activities were taking effect. However, the contractile responses to carbachol and transmural stimulation were still increasing at this time. This may imply that the muscarinic receptors are more resistant to membrane acclimation changes and may take longer to adapt. Because these experiments were controlled for handling stress and seasonal changes that affect contractility, we have been able to demonstrate some early changes in smooth muscle contractility that occur during acclimation to altered temperature.     

Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout,Oncorhynchus mykiss (Walbaum) and bacterial isolates

The application of natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics. We evaluated the effect of diet supplementation with Thymus vulgaris essential oil (TVEO) on the allochthonous microbial composition of rainbow trout. DNA was extracted directly from the intestinal contents, and the V3‐V4 regions of the 16S rRNA genes were amplified by PCR. The bacterial composition was analysed using temporal temperature gradient electrophoresis (TTGE). No significant changes (P>0.05) were detected in the TTGE profiles of TVEO‐treated trout compared with the controls. The Dice similarity index revealed a high stability (C_s >70%) of the intestinal microbiota in both groups during the 5‐week period. Sequence analyses of the TTGE bands revealed the same bacterial composition in both groups, with most bacteria belonging to the Proteobacteria and Firmicutes phyla. The in vitro antibacterial activity of TVEO was assessed using a range of normal intestinal isolates and fish pathogens. The inhibitory concentrations for all the tested bacteria were higher than the TVEO levels used in trout, which may explain the in vivo results.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum)

From a comparison of 125 bacterial isolates recovered from the digestive tract of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus sp., a culture was obtained which was effective at preventing clinical disease caused by Lactococcus garvieae and Streptococcus iniae when used as a feed additive. The culture, Aeromonas sobria GC2, was incorporated into the feed and fed to rainbow trout (average weight = 20 g) for 14 days at a dose equivalent to 5 x 10(7) cells g(-1) of feed. Whereas the untreated controls experienced losses of 75-100% when challenged intraperitoneally with L. garvieae and S. iniae, the probiotic-treated groups remained healthy with total mortalities of only 0-6%. Formalized and sonicated preparations of GC2 and cell-free supernatant fared less well. The mode of action reflected stimulation of innate immunity, namely an increased number of leucocytes and enhanced phagocytic and respiratory burst activity.