Cortisol and testosterone accumulation in a low pH recirculating aquaculture system for rainbow trout (Oncorhynchus mykiss)

Steroids accumulate in recirculating aquaculture system (RAS), although explanatory factors for such accumulation are still poorly explored. This study investigated the effect of water exchange rate and pH in six replicated RAS on the concentration of the stress hormone cortisol in rainbow trout blood plasma and in the holding water and of the sex steroids testosterone, 11‐ketotestosterone (11‐KT) and 17,20β‐dihydroxypregn‐4‐en‐3‐one (17,20β‐P) over a 70‐day experimental period. Three combinations of water exchange rate and pH were used each treatment, with two replications: (i) high water exchange rate (±1700 L kg^?1 feed) and neutral pH (±7.3), (ii) low water exchange rate (±500 L kg^?1 feed) and neutral pH (±7.3) and (iii) low water exchange rate (±500 L kg^?1 feed) and low pH (±5.8). Plasma cortisol concentrations at day 70 were higher (24.4 ± 9.5 ng mL^?1) for fish kept at low pH when compared to fish kept at neutral pH (12.0 ± 0.1 and 8.7 ± 0.2 ng mL^?1). Water cortisol and testosterone concentrations at day 35 were higher at low pH than at neutral pH, whereas water 11‐KT and 17,20β‐P did not differ among treatments. At day 70, there were no significant differences between low and high pH. These results demonstrate that low pH contributes to increased plasma cortisol concentrations and to its accumulation in water, possibly indicating a stress response to low pH. The higher concentration of testosterone but not of the other sex hormones point to unspecified reproductive effects that need further investigation.     

Evaluating the effects of prolonged peracetic acid dosing on water quality and rainbow trout Oncorhynchus mykiss performance in recirculation aquaculture systems

Peracetic acid (PAA) is an effective disinfectant/sanitizer for certain industrial applications. PAA has been described as a powerful oxidant capable of producing water quality benefits comparable to those expected with ozone application; however, the water oxidizing capacity of PAA in aquaculture systems and its effects on fish production require further investigation, particularly within recirculation aquaculture systems (RAS). To this end, a trial was conducted using six replicated RAS; three operated with semi-continuous PAA dosing and three without PAA addition, while culturing rainbow trout Oncorhynchus mykiss. Three target PAA doses (0.05, 0.10, and 0.30 mg/L) were evaluated at approximately monthly intervals. A water recycle rate >99% was maintained and system hydraulic retention time averaged 2.7 days. Rainbow trout performance metrics including growth, survival, and feed conversion ratio were not affected by PAA dosing. Water quality was unaffected by PAA for most tested parameters. Oxidative reduction potential increased directly with PAA dose and was greater (P < 0.05) in RAS where PAA was added, indicating the potential for ORP to monitor PAA residuals. True color was lower (P < 0.05) in RAS with target PAA concentrations of 0.10 and 0.30 mg/L. Off-flavor (geosmin and 2-methylisoborneol) levels in culture water, biofilm, and trout fillets were not affected by PAA dosing under the conditions of this study. Overall, semi-continuous PAA dosing from 0.05-0.30 mg/L was compatible with rainbow trout performance and RAS operation, but did not create water quality improvements like those expected when applying low-dose ozone.     

Micro particles and microbial activity in Danish recirculating rainbow trout (Oncorhynchus mykiss) farms

Increasing intensities of water reuse in recirculating aquaculture systems (RAS) lead to a build-up of micro particles (< 20 μm) in the water. This build-up may have consequences for other water quality parameters and for the fish. This baseline study was carried out to determine the variation in micro particle levels (numbers, volume and surface area) and accompanying bacterial activity in commercially operated outdoor RAS, as well as the effects of different components in the recirculation loop on micro particle dynamics. Water samples were obtained during spring 2017 from 7 Danish Model Trout Farms (MTFs) producing rainbow trout (Oncorhynchus mykiss) in a total of 20 separate RAS units. Micro particle numbers and size distribution, bacterial activity, and inorganic and organic nutrient concentrations were analysed. Micro particle numbers ranged between 6.0·10⁴ – 7.4·10⁵ ml⁻¹ and large variations were found between seemingly similarly operated RAS units within the same farm. There was a strong, positive correlation (p < 0.001) between micro particle levels and bacterial activity in the systems. Although not significant, biofilters generally seemed to trap particles whereas drum filters seemed to reduce particle volume while increasing particle numbers and surface area. The study sustains that bacterial activity in RAS is strongly associated with fine particle loading, and demonstrates for the first time the overall magnitude and level of variation in particle levels and bacterial activity that exists in commercially operated MTFs.     

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.     

Moderate stocking density does not influence the behavioural and physiological responses of rainbow trout (Oncorhynchus mykiss) in organic aquaculture

Welfare in farmed fish got particular attention during the last decades from both governmental and public sides. In aquaculture context, welfare concerns are mainly related to handling procedures, water quality and stoking densities. In Europe, authorities had to clarify the threshold limits of stocking densities to maintain fish good welfare, including for organics aquaculture through the EC regulation 710/2009. However, effects of stocking density on fish welfare are complex and sometimes contradictory. Moreover, there is a lack of knowledge about the impact of density on fish welfare in organic aquaculture. Thus, the aim of the study is to asses welfare state of rainbow trout (Oncorhynchus mykiss) at two initial stocking densities (low density, LD: 12 kg/m³ and high density, HD: 17 kg/m³) fed using organic feed by combining the monitoring of growth performances, behaviour (swimming activity) and physiological indicators (i.e. cortisol, glucose, lactate, hematocrit, red blood cellule count and lysozyme). At the end of experiment, the stocking density reached 21 kg/m³ and 30 kg/m³ for the LD and HD respectively. Overall, growth performances, swimming activity and level of physiological indicators of stress and welfare were similar between HD and LD over the experiment duration. To conclude, we observed no alteration of fish welfare between the two stocking densities monitored. This study suggests that a final stocking density of 30 kg/m³ can be considered for organic aquaculture of rainbow trout respecting welfare.     

A factorial model to predict phosphorus waste output of rainbow trout (Oncorhynchus mykiss)

Minimizing phosphorus (P) wastes is considered to be a key factor for environmental sustainability of freshwater aquaculture operations in many parts of the world. A factorial P model, consisting of digestibility, whole-body P deposition, P waste output and limnological transformation sub-models, was constructed to simulate the effects of different dietary P sources and levels on P utilization in salmonids. This factorial P model was developed based on information from the literature for rainbow trout ( Oncorhynchus mykiss ). This factorial model runs on the platform of a fish bioenergetics model that provides dynamic estimates of feed intake of salmonids based on diet composition and growth rate. Simulations suggest that this model can potentially be a useful tool for waste output management of salmonid culture operations.     

Puffy skin disease (PSD) in rainbow trout,Oncorhynchus mykiss (Walbaum): a case definition

Puffy skin disease (PSD) is a disease that causes skin pathology in rainbow trout, Oncorhynchus mykiss (Walbaum). Incidence of PSD in UK fish farms and fisheries has increased sharply in the last decade, with growing concern from both industry sectors. This paper provides the first comprehensive case definition of PSD, combining clinical and pathological observations of diseased rainbow trout from both fish farms and fisheries. The defining features of PSD, as summarized in the case definition, were focal lateral flank skin lesions that appeared as cutaneous swelling with pigment loss and petechiae. These were associated with lethargy, poor body condition, inappetance and low level mortality. Epidermal hyperplasia and spongiosis, oedema of the dermis stratum spongiosum and a mild diffuse inflammatory cellularity were typical in histopathology of skin. A specific pathogen or aetiology was not identified. Prevalence and severity of skin lesions was greatest during late summer and autumn, with the highest prevalence being 95%. Atypical lesions seen in winter and spring were suggestive of clinical resolution. PSD holds important implications for both trout aquaculture and still water trout fisheries. This case definition will aid future diagnosis, help avoid confusion with other skin conditions and promote prompt and consistent reporting.     

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.     

Metabolism of exogenous histamine in rainbow trout (Oncorhynchus mykiss)

Information on the metabolism of exogenous histamine in fish is of much concern regarding the effect of dietary histamine on fish. Histamine catabolic enzymes, diamine oxidase and histamine N-methyl transferase were measured in the tissues of rainbow trout. Diamine oxidase was detected in the stomach, pylorus caeca and intestine. Histamine N-methyl transferase was detected only in the liver.A change in the contents of histamine and its metabolites was observed in the tissues of rainbow trout after oral administration of histamine. A large amount of imidazole acetic acid was observed in the serum, kidney, liver and muscle. On the other hand, 1-methyl histamine was detected only in the liver. Histamine and its metabolites, imidazole acetic acid and 1-methyl histamine were metabolized and diminished within 48 hr in all tissues.These results showed that histamine was metabolized by two metabolic routes in rainbow trout. One is the main pathway producing imidazole acetic acid by intestinal diamine oxidase and the other is the complementary pathway producing 1-methyl histamine by liver N-methyl-transferase.     

Exploring the microbial diversity of the distal intestinal lumen and mucosa of farmed rainbow trout Oncorhynchus mykiss (Walbaum) using next generation sequencing (NGS)

In this study, next generation sequencing (NGS) was used to survey the 16S rRNA ribotypes of the distal intestinal lumen and mucosal epithelium of farmed rainbow trout. This approach yielded a library consisting of 2 979 715 quality filtered paired sequences, assigned to genus level of taxonomy using the Ribosomal Database Project (RDP). A high level of diversity was observed in both regions. A total of 90 bacterial genera were identified in the lumen of all fish sampled, compared with 159 in the mucosa. The allochthonous microflora was dominated by sequences belonging to the γ Proteobacteria (mean sequence abundance 54.3%), in particular the Enterobacteriaceae, with Yersinia, Serratia, Hafnia and Obesumbacterium the most abundant genera. Fewer γ Proteobacteria (mean sequence abundance 37%) were present in the mucosa, and autochthonous communities consisted of a more even split among the bacterial classes, with increases in sequences assigned to members of the β Proteobacteria (mean sequence abundance 18.4%) and Bacilli (mean sequence abundance 16.8%). The principal bacterial genera recorded in the mucosa were Cetobacterium, Yersinia, Ralstonia, Hafnia and Carnobacterium. The results of this study demonstrate that the luminal and mucosal bacterial communities may be different in their respective structures, and that the mucosal microflora of rainbow trout may be more diverse than previous research has suggested. This research also demonstrated a degree of conservation of bacterial genera between individual fish sampled, and is to the author's knowledge the first time the MiSeq^® NGS platform has been used to explore the rainbow trout intestinal microflora.     

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.     

Effects of traditional rainbow trout (Oncorhynchus mykiss) breeding on dissolved organic nitrogen pools and microbial activity in the water

Seasonal effects of rainbow trout (Oncorhynchus mykiss) breeding in traditional, flow‐through ponds on abundance and activity of bacteria, and on pools and cycling of dissolved free and combined amino acids (DFAA and DCAA), were studied in three aquaculture systems in Denmark. The fish breeding was found to increase both abundance and production of bacteria by about 60% (annual average of all three systems). The bacterial production correlated positively with temperature, and was 10 to 60‐fold higher in summer than winter. Concentrations of DFAA (range: 45–690 nM) increased by 40–350% (mean of 89%) during passage in the systems. The bacterial uptake of DFAA was stimulated by 42% (annual average value of the three systems) between inlets and outlets. Uptake of DFAA was estimated to sustain from 45% (C) and 70% (N) of the bacterial C and N demand. Concentrations of DCAA, e.g., proteins and peptides, varied from 600 to 3900 nM, and increased on average 89% during passage in the three systems. Bacterial capacity for utilization of DCAA was determined as leucine peptidase activity and related to the bacterial density. The cell‐specific peptidase activity was, on average, 103% higher at outlets than inlets. Changes in the peptidase activity did not correlate with variations in DCAA pools or the bacterial production. Concentrations of ammonium, urea and phosphate increased on average by 88%, 103% and 66%, respectively, during passage in the three systems, whereas no changes were observed for nitrate. Our study shows that traditional fish breeding impacts pools and cycling of dissolved organic N, as well as bacterial dynamics. The short residence time of water in the ponds (3–6 h) implies that most of the nutrients produced in the systems were discharged into the downstream water, rather than assimilated by bacteria within the systems.     

Comparison of brown trout (Salmo trutta) reared in fresh water and sea water to freshwater rainbow trout (Oncorhynchus mykiss): II. Phosphorus balance

Brown trout and rainbow trout (average weight 100 g) were reared in fresh water at 12 °C under the same conditions before transferring brown trout to sea water, in order to compare phosphorus utilisation in both species. Apparent phosphorus availability, orthophosphate excretion and phosphorus accretion in the fish were directly determined. Thus, actual phosphorus mass balance was built. Rainbow trout raised in fresh water had a higher phosphorus retention coefficient (maximum 50 %) than brown trout reared in fresh water (maximum 45 %). Transferring brown trout to sea water induced a reduction in phosphorus retention (maximum 39 %). Orthophosphate excretion, ranging 7–20 mg phosphorus per kg wet weight per day, represented 10–20 % of ingested phosphorus. Phosphorus availability was lower in brown trout raised in sea water (65 %) than brown trout raised in fresh water (76 %). Phosphorus balance measurements showed that 90 to 98 % of phosphorus flow could be accounted for.     

Androgen metabolism in the skin of the rainbow trout (Oncorhynchus mykiss)

Pieces of skin of male and female rainbow trout (Oncorhynchus mykiss) were incubated with testosterone and 11-ketotestosterone as substrates. In immature fish the conversion rate was low. In non-spawning adult males 11-ketotestosterone was reduced to 5α-11KDHT (up to 5.2%). In the fish in spawning condition the 5α-reduction rate was only about 1 to 2%. In the same specimens incubated with testosterone a high 11β-hydroxylase activity (23.8-25% in the male and 13% in the female skin) was found. Similar sex specific differences were observed for the occurence of 5α-reduced metabolites (about 20% in the male and 13% in the female tissue).

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Résumé Des fragments de peau de truites arc en ciel (Oncorhynchus mykiss, males ou femelles, ont été incubés avec de la testostérone ou de la 11-cétotestostérone, utilisées comme substrats. Chez les poissons immatures, les taux de conversion restent faibles. Chez les males adultes ne donnant pas de sperme, la 11-cétotestostérone est réduite en 5α-androstane-17β-ol-3,11-dione (jusqu'à 5.2%). Chezles poissons en conditions de fraie, le taux de 5α-réduction est seulement de l'ordre de 1 à 2%. Pour ce derniers individus, les incubations de peau en présence de testostérone montrent l'existence d'une forte activité 11β-hydroxylase (23.8-25% pour le male, et 13% pour la femelle). Des différences liées au sexe sont observées de la même manière dans la production de métabolites 5α-réduits (environ 20% avec le tissu male et 13% avect le tissu femelle).

     

Embryonic expression of UCP2 in rainbow trout (Oncorhynchus mykiss)

Uncoupling proteins are mitochondrial anions transporters that dissociate respiration from ATP synthesis through proton leaks. Uncoupling protein 2 reportedly plays a role in several physiological processes such as energy partitioning, nutrition, and fatty acid metabolism. The mRNA expression of rainbow trout UCP2 genes (UCP2A and UCP2B) was monitored during embryogenesis and early larval development. Both genes were recruited early and displayed similar steadily decreasing patterns from fertilization until hatching. The expression of UCP2A and UCP2B appeared significantly differentiated after hatching and during the yolk sac absorption, with UCP2A displaying higher expression. We suggest that UCP2 expression profiles in the rainbow trout embryo could be associated with the utilization of lipids as a source of energy during development.