Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

The impact of 17-estradiol (E₂) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E₂ and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E₂-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E₂- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na⁺,K⁺-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E₂ and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na⁺,K⁺-ATPase activity, relative -subunit Na⁺,K⁺-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E₂- and 4-NP-treated fish was strongly correlated with a decreased gill Na⁺,K⁺-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E₂ activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.     

The effect of allostatic load on hypothalamic–pituitary–interrenal (HPI) axis before and after secondary vaccination in Atlantic salmon postsmolts (Salmo salar L.)

The experiment consisted of three experimental groups: (1) “vaccine and stress”, (2) “stress and vaccine” and (3) control. All groups have previously been vaccinated 6 months prior to the start of the experiment. At the start of the experiment, the “vaccine and stress” group was vaccinated with Pentium Forte Plus for the second time (25.02.2008) and then given a daily stressor (confinement stressor 267 kg m-3 in 15 min) for a period of 4 weeks. The “stress and vaccine” group was given a similar daily stressor for 4 weeks and then vaccinated for the second time. The control group was neither stressed nor vaccinated a second time. The results indicates that fish in the “stress and vaccine” group may have entered an allostatic overload type 2 due to oversensitivity to ACTH, a reduced efficient negative feedback system with elevated baseline levels of plasma cortisol and reduced immune response with pronounced effects on the well-being of the animal. The “vaccine and stress” group may likewise have entered an allostatic overload type 1 response, with oversensitivity to ACTH and transient reduced efficient negative feedback system. This study shows that if plasma cortisol becomes elevated prior to vaccination, it could perhaps instigate an allostatic overload type 2 with dire consequences on animal welfare. To reduce the risk of compromising the animal welfare during commercial vaccination of salmon, one propose to grade the fish minimum a week prior to vaccination or grade simultaneously with vaccination. This could reduce the overall allostatic load during handling and vaccination and secure a healthy fish with intact immune response and improved animal welfare.     

Spatial and temporal distribution of Lepeophtheirus salmonis (Kr?yer) larvae in a sea loch containing Atlantic salmon, Salmo salar L., farms on the north-west coast of Scotland.

A plankton survey investigated spatial and temporal distributions of larval sea lice in a sea loch on the north-west coast of Scotland. Plankton tows were collected approximately weekly at two depths (0 and 5 m) at five sample stations over a 2-year period. The survey began in March 2002 when Atlantic salmon, Salmo salar L., farms in the study area stocked with fish and finished in March 2004, after farms completed harvesting. Over 90% of lice recovered were Lepeophtheirus salmonis (Kr?yer); therefore, the results of this paper refer largely to that species. The data were analysed using generalized additive models with explanatory variables for time, depth and station. Greatest densities of nauplii were recovered at stations adjacent to farms indicating that the local salmon farms were a likely source of larvae at times during the production cycle. There were significant temporal trends in larval densities and the stocking and harvesting of farms can possibly account for these increases and decreases, particularly at the start and at the end of the production cycle. The distribution of copepodids was more widespread than that of nauplii, indicating that the larvae can be transported several kilometres from the point of release.     

Effects of acclimation temperatures of 7 and 14 °C on the contractility of Atlantic salmon (Salmo salar) intestinal smooth muscle in vitro

The contractility of intestinal smooth muscle of Atlantic salmon (Salmo salar) acclimatized to temperatures of 7 and 14 °C was compared over a prolonged period of time (9 months) in freshwater. Initial studies were carried out on intestine isolated from Atlantic salmon to estimate optimal conditions for contractility. Subsequent studies were carried out with the neurotransmitter/agonist 5-HT and TS, which activates neuronal elements in the intestine, in a Krebs-Henseleit solution containing HEPES buffer maintained at pH 7.85 and at the temperature of the experimental group (i.e., 7 or 14 °C). There was a significant interaction with time and temperature for the maximal response of 5-HT (p=0.005), effective concentration producing 50% of the maximal response (EC₅₀) to 5-HT (p=0.026) and maximal response to TS (p=0.002), demonstrating that gastrointestinal contractility of Atlantic salmon is altered by both time (month) and temperature. No significant changes were found with the effective frequency producing 50% of the maximal response (EF₅₀) to TS. The potency (EC₅₀ of 5-HT) increase between 7 and 14 °C was statistically significant (p=0.026) but the small differences found with potency were most likely not physiologically significant. A time shift was recognized in the maximal responses to 5-HT, by applying a time variable to equations for the curves of the responses. This demonstrated that the curves are similar for both temperatures but alterations in the time of the maximal response changes was different. Thus, there is a delay in the time (month) changes of gastrointestinal contractility related to acclimation temperature and would be an area for future study.     

Can conservation‐oriented,captive breeding limit behavioural and growth divergence between offspring of wild and captive origin Atlantic salmon (Salmo salar)?

Captive rearing is being used increasingly to maintain demographics and genetic diversity of threatened fish populations and species, but its effectiveness can be hindered by domestication, that is, inadvertent selection for performance in captivity at the cost of that in the wild. Some captive rearing programmes have begun to take steps to limit such domestication, but the results are ambiguous, as the degree of generational exposure to captivity is often difficult to control. Using an endangered population of Atlantic salmon (Salmo salar) currently undergoing conservation‐oriented captive rearing, we tested for domestication effects on dominance (dyadic trials) and growth (seminatural stream channels with differing densities and group proportions) of juvenile offspring of wild and captive origin parents. Pedigree data afforded the ability to compare these effects among three specific study groups: wild, single‐generation captives and two to three generation captives. Our results indicate that, despite conservation breeding practices, a divergence in growth can occur in as little as one generation without divergence in dominance behaviour. Further, evidence suggests that trait divergence did not increase with generations in captivity. Given the experimental design, results and supporting literature, we conclude that this contemporary divergence is likely genetic and driven by a combination of factors, including variation in selective histories influencing behaviour.     

Vitamin K-dependent γ-glutamylcarboxylase in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Due to problems with bone deformities in farmed Atlantic salmon, there is a growing interest in the possible involvement of vitamin K in normal bone development, and sensitive biomarkers for evaluating vitamin K status are therefore needed. The vitamin K-dependent (VKD) enzyme γ-glutamylcarboxylase (GGCX, EC 6.4.x.x) requires vitamin K as a cofactor for its post-translational modification of glutamic acid (Glu) residues to γ-carboxyglutamic acid (Gla) residues in VKD proteins, and is required for their function in haemostasis and bone metabolism. The present study was designed to evaluate the enzyme assay for GGCX activity in isolated liver microsomes and its distribution in the tissues of Atlantic salmon. The effect of KH₂ and menadione on the GGCX activity in salmon liver was also compared. Results from the present study show a widespread tissue distribution and expression of GGCX in Atlantic salmon. The GGCX activity and ggcx expression in all bony tissues examined imply the presence of vitamin K, and suggest the involvement of vitamin K in bone metabolism of Atlantic salmon. We propose the GGCX assay as a sensitive marker for vitamin K status, and confirm that menadione does not work as a cofactor for GGCX in Atlantic salmon liver.     

Does the AquasmartTM feeding system reduce sea louse [Lepeophtheirus salmonis (Krøyer)] infestation on farmed Atlantic salmon (Salmo salar L.) in winter?

Infections of sea lice [Lepeophtheirus salmonis (Krøyer)] were studied over winter on a working, commercial salmon farm in relation to two types of feeding system: a regular, pneumatic spray feeder and an irregular, ‘demand feed’ sensor system (Aquasmart^TM). Contrary to prediction, the Aquasmart^TM system alone had no significant effect on chalimus infestation compared with fish fed by pneumatic spray. However, it was associated with significantly reduced numbers of mobile stages, both at the beginning of the study (P < 0.05) and, more particularly, in conjunction with treatment of all pens using hydrogen peroxide (P < 0.001). It is suggested that feeding regime may represent a useful tool in reducing lice infestation when co‐ordinated with other methods of control, such as peroxide treatment, and that further work in this field is merited.     

The effects of long-term 20 mg/L carbon dioxide exposure on the health and performance of Atlantic salmon Salmo salar post-smolts in water recirculation aquaculture systems

Previous research and experience has linked elevated dissolved carbon dioxide (CO₂) to reduced growth performance, poor feed conversion, and a variety of health issues in farm-raised fish, including Atlantic salmon Salmo salar. Supplemental control measures in water recirculation aquaculture systems (RAS) to reduce CO₂ accumulation, however, such as increased water pumping to decrease tank hydraulic retention time, can represent significant costs for operators. We exposed post-smolt S₀ Atlantic salmon (197 ± 2 g, 423 days post-hatch) to either high (20 ± 1 mg/L) or low (8 ± <1 mg/L) dissolved CO₂ in six replicated freshwater RAS for 384 days to investigate differences in performance and health as the salmon were grown to harvest size. All RAS were operated at moderate water exchange rates (1.0% of the total recirculating flow), a 24-h photoperiod was provided, fish were fed to satiation, and densities were maintained between 40 and 80 kg/m³. Over the study period, dissolved oxygen was kept at saturation, mean water temperature was 14.1 ± 0.1 °C, and alkalinity averaged 237 mg/L as CaCO₃. At study’s end, no significant differences in fish weight (high CO₂ mean weight = 2879 ± 35 g; low CO₂ mean weight = 2896 ± 12 g), feed conversion ratio (1.14 ± 0.12 vs. 1.22 ± 0.13, respectively), or thermal growth coefficient (1.45 ± 0.01 vs. 1.46 ± 0.01, respectively), were observed. No significant differences in survival (high CO₂ mean survival = 99.1 ± 0.4%; low CO₂ mean survival = 98.9 ± 0.3%) or culls due to saprolegniasis (3.5 ± 1% vs. 3.0 ± 1%, respectively) were determined, and no nephrocalcinosis was observed through histopathological evaluation. Blood gas and chemistry evaluation revealed higher pCO₂, bicarbonate, and total CO₂, and lower chloride and glucose, in the high CO₂ cohort. Molecular analyses of gill enzyme regulation showed significantly higher expression of Na⁺/K⁺ ATPase α1a in high CO₂ fish at 3-weeks post-challenge, indicating physiological adaptation to the higher CO₂ environment without any noticeable long-term impacts on health or performance. Overall, the results of this study suggest that, at 237 mg/L as CaCO₃ mean alkalinity, post-smolt Atlantic salmon can be raised in freshwater RAS to harvest size with up to 20 mg/L CO₂ without significantly impacting fish health and performance.     

Dynamics of Caligus rogercresseyi (Boxshall & Bravo, 2000) in farmed Atlantic salmon (Salmo salar) in southern Chile: Are we controlling sea lice?

Caligus rogercresseyi generates the greatest losses in the salmon industry in Chile. The relationship between salmon farming and sea lice is made up of various components: the parasite, host, environment and farming practices, which make it difficult to identify patterns in parasite population dynamics to define prevention and control strategies. The objectives of this study were to analyse and compare the effect of farming, sanitary practices and environmental variables on the abundance of gravid females (GF) and juveniles (JUV) of C. rogercresseyi on Salmo salar in three Salmon Neighborhood Areas (SNAs) in Region 10, south of Chile. Linear mixed‐effects models of the negative binomial distribution were used to evaluate the effect of the different explanatory variables on GF and JUV. Productive variables were the key drivers explaining the abundance of GF and JUV. Results suggest that C. rogercresseyi is not controlled and JUV are persistent in the three SNAs, and sanitary practices do not control the dissemination of the parasite among sites. Environmental variables had a low impact on sea lice abundance. There is a need to perform analysis for modelling of parasite population dynamics to improve Integrated Pest Management, including changes in the governance to achieve an effective prevention and control.     

Does the capacity for energy utilization affect the survival of post‐smolt Atlantic salmon,Salmo salar L., during natural outbreaks of infectious pancreatic necrosis?

If osmotic stress and reduced seawater tolerance are predisposing factors for infectious pancreatic necrosis (IPN) outbreaks in farmed Atlantic salmon, increased survival by enhancing access to energy would be expected. The aim of the present study was, therefore, to increase energy access in 1-year old Atlantic salmon after sea transfer by increasing the level of dietary fat, by exchanging some of the dietary oil with more easily oxidized medium chain triacylglycerols, or by dietary supplementation of potentially energy enhancing additives such as clofibrate and tetradecylthioacetic acid (TTA). A natural outbreak of IPN occurred 8 weeks after sea transfer, and a significant dietary effect explaining 76% of the variation in mortality was observed. Relative percentage survival for the fish fed TTA in sea water was 70% when compared with the unsupplemented control, reducing mortality from 7.8 to 2.3%. Muscle fat content and plasma chloride were related to IPN mortality, suggesting that reduced hypoosmoregulatory capacity might be a predisposing factor to the onset of an IPN outbreak. Based on the observation of a threefold increase in white muscle mitochondrial fatty acid oxidizing activity by TTA, it is suggested that TTA has resulted in a re-allocation of dietary fatty acids from storage to energy producing oxidation.     

The influence of temperature, dietary polyunsaturated fatty acids, α-tocopherol and spermine on fatty acid composition and indices of oxidative stress in juvenile Arctic char, Salvelinus alpinus (L.)

Juvenile Arctic char, Salvelinus alpinus (L.), were fed two levels of polyunsaturated fatty acids (PUFA) (5.5 or 2.5% of diet (37 or 16% of lipid respectively)), -tocopheryl acetate (70 or 300 mg kg–1 diet) and the antioxidant spermine (0 or 250 mg kg–1 diet) in a 3×2 factorial design and analysed for tissue fatty acid composition and indices of oxidative stress. The effect of temperature was evaluated by first maintaining the fish at 12 °C for 70 days and then 0.6 °C for 71 days. Liver and muscle fatty acid compositions were significantly influenced by the fatty acid compositions of the feed, although extensive modification of some fatty acids took place prior to deposition in the tissues. Maintaining char at low temperature increased liver PUFA content particularly in fish fed the low PUFA diets while no major changes were seen in muscle. Tissue - tocopherol deposition was mainly influenced by dietary - tocopheryl acetate. Dietary spermine had no influence on tissue levels of spermine. Dietary manipulation had no major influence on haematology and blood chemistry. High dietary PUFA increased the content of thiobarbituric acid reactive substances (TBARS) in both liver and muscle. Neither -tocopheryl acetate nor spermine influenced tissue TBARS content. Lowering the environmental temperature influenced several parameters including haematology, blood chemistry and muscle spermine content, but none were related to diet. It is concluded that Arctic char is tolerant to a wide range of dietary content of PUFA (16 to 37% of lipid) and - tocopherol (70 to 300 mg kg–1), and that spermine offers no further protection against oxidative stress. It is suggested that toxic levels of PUFA is related to the amount in dietary lipid where more than 50% of lipid may be harmful to the fish.     

Essential fatty acid deficiency in freshwater fish: the effects of linoleic, α-linolenic, γ-linolenic and stearidonic acids on the metabolism of [1-14C]18:3n-3 in a carp cell culture model

The desaturation of [1-¹⁴C]18:3n-3 to 20:5n-3 and 22:6n-3 is enhanced in an essential fatty acid deficient cell line (EPC-EFAD) in comparison with the parent cell line (EPC) from carp. In the present study, the effects of competing, unlabeled C₁₈ polyunsaturated fatty acids (PUFA), linoleic (18:2n-6), -linolenic (18:3n-3), -linolenic (18:3n-6) and stearidonic (18:4n-3) acids, on the metabolism of [1-¹⁴C]18:3n-3 were investigated in EPC-EFAD cells in comparison with EPC cells. The incorporation of [1-¹⁴C]18:3n-3 in both cell lines was significantly reduced by competing C₁₈ PUFA, with the rank order being 18:4n-3>18:3n-3 = 18:2n-6>18:3n-6. In the absence of competing PUFA, radioactivity from [1-¹⁴C]18:3n-3 in EPC cells was predominantly recovered in phosphatidylethanolamine followed by phosphatidylcholine. This pattern was unaffected by competing n-6PUFA, but n-3PUFA reversed this pattern as did essential fatty acid deficiency in the presence of all competing PUFA. The altered lipid class distribution was most pronounced in cells supplemented with 18:4n-3. Competing C₁₈ PUFA significantly decreased the proportions of radioactivity recovered in 22:6n-3, pentaene and tetraene products, with the proportions of radioactivity recovered in 18:3n-3 and 20:3n-3 increased, in both cell lines. However, the inhibitory effect of competing C₁₈ PUFA on the desaturation of [1-¹⁴C]18:3n-3 was significantly greater in EPC-EFAD cells. The magnitude of the inhibitory effects of C₁₈ PUFA on [1-¹⁴C]18:3n-3 desaturation was dependent upon the specific fatty acid with the rank order being 18:4n-3>18:3n-3>18:2n-6, with 18:3n-6 having little inhibitory effect on the metabolism of [1-¹⁴C]18:3n-3 in EPC cells. The differential effects of the C₁₈ PUFA on [1-¹⁴C]18:3n-3 metabolism were consistent with mass competition in combination with increased desaturation activity in EPC-EFAD cells and the known substrate fatty acid specificities of desaturase enzymes. However, the mechanism underpinning the greater efficacy with which the unlabeled C₁₈PUFA competed with [1-¹⁴C]18:3n-3 in the desaturation pathway in EPC-EFAD cells was unclear.     

Modulation of key metabolic enzyme of <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton) juvenile: effect of dietary starch type,protein level and exogenous α-amylase in the diet

A 60-day feeding trial was conducted to delineate the effect of both gelatinized (G) and non-gelatinized (NG) corn with or without supplementation of exogenous α-amylase, either at optimum (35%) or sub-optimum (27%) protein levels, on blood glucose, and the key metabolic enzymes of glycolysis (hexokinase, HK), gluconeogenesis (glucose-6 phosphatase, G6Pase and fructose-1,6 bisphosphatase, FBPase), lipogenesis (glucose-6 phaosphate dehydrogenase, G6PD) and amino acid metabolism (alanine amino transfersae, ALT and aspartate amino transferase, AST) in Labeo rohita. Three hundred and sixty juveniles (average weight 10 ± 0.15 g) were randomly distributed into 12 treatment groups with each of two replicates. Twelve semi-purified diets containing either 35 or 27% crude protein were prepared by including G or NG corn as carbohydrate source with different levels of microbial α-amylase (0, 50, 100 and 150 mg kg⁻¹). The G corn fed groups showed significantly higher (P < 0.05) blood glucose and G6PD activity, whereas G6Pase, FBPase, ALT and AST activity in liver was higher in the NG corn fed group. Dietary corn type, α-amylase level in diet or their interaction had no significant effect (P > 0.05) on liver HK activity, but the optimum crude protein (35%) fed group showed higher HK activity than their low protein counterparts. The sub-optimum crude protein (27%) fed group showed significantly higher (P < 0.05) G6PD activity than the optimum protein fed group, whereas the reverse trend was observed for HK, G6Pase, FBPase, ALT and AST activity. Addition of 50 mg α-amylase kg⁻¹ feed showed increased blood glucose and G6PD activity of the NG corn fed group, whereas the reverse trend was found for G6Pase, FBPase, ALT and AST activity in liver, which was similar to that of the G or NG corn supplemented with 100/150 mg α-amylase kg⁻¹ feed. Data on enzyme activities suggest that NG corn in the diet significantly induced more gluconeogenic and amino acid metabolic enzyme activity, whereas G corn induced increased lipogenic enzyme activity. Increased amino acid catabolic enzyme (ALT and AST) activity was observed either at optimum protein (35%) irrespective of corn type or NG corn without supplementation of α-amylase irrespective of protein level in the diet.