Nutritional regulation of somatostatin expression in rainbow trout, Oncorhynchus mykiss

Previously we characterized three DNAs from the endocrine pancreas (Brockmann body) of rainbow trout (Oncorhynchus mykiss) that encode for distinct preprosomatostatin (PPSS) molecules: one containing somatostatin-14 and its C-terminus (PPSS I) and two containing [Tyr⁷, Gly¹⁰]-somatostatin-14 at their C-termini (PPSS II′ and PPSS II″). In the present study, the regulation of PPSS expression was studied in rainbow trout placed on varying nutritional regimes (fed continuously, fasted, fasted then refed). Fish that were fasted for one week displayed reduced growth compared to their fed counterparts, but no alteration in pancreatic PPSS expression was noted between the two groups. Fish fasted for 4 and 6 weeks also were growth retarded and displayed increased levels of PPSS I mRNA and PPSS II″ mRNA compared to fed animals; PPSS II′ mRNA levels were not affected by food deprivation. Refeeding fish for two weeks following 4 weeks of food deprivation restored growth and reduced PPSS I and PPSS II″ mRNA expression to levels similar to those displayed by continuously fed fish. Changes in PPSS expression were correlated with increases in plasma SS. These results indicate that nutritional state modulates differential expression of PPSSs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Commercial Bacillus probiotic supplementation of rainbow trout (Oncorhynchys mykiss) and brown trout (Salmo trutta): growth,immune responses and intestinal morphology

Probiotic administration is associated with the enhancement of host resistance to environmental and nutritional stressors, improving survival and growth rates. This study was carried out to evaluate the effects of dietary supplementation of Bacillus subtilis and Bacillus cereus toyoi on growth performance, innate immune responses and gut morphology of two trout species feeding a commercial diet high in soybean meal. A commercial probiotic (4.2 × 10⁹ CFU g^?1 of additive) was supplemented to the experimental diets at 0% (control), 0.03% (P₁; 6 × 10³ CFU g^?1 of diet) or 0.06% (P₂; 1.5 × 10⁶ CFU g^?1 of diet) and fed to brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) for 9 and 20 weeks respectively. Rainbow trout showed significantly better growth performance than brown trout, regardless of the dietary treatment. No effect of dietary probiotic supplementation was detected on growth performance, body composition or innate immune parameters (plasma lysozyme, alternative complement and peroxidase activities). In both species, after 9 weeks, intestinal lamina propria and submucosa were widened, with increased presence of inflammatory cells, significantly higher in groups fed probiotics. This inflammatory process, with villi and enterocytes noticeably damaged compared with the control group, was more pronounced in brown trout. Under the current trial conditions, the B. subtilis + B. cereus toyoi had no positive impact in either trout species, on the contrary a harmful effect was observed.     

Growth performance,immune response and disease resistance of rainbow trout (Oncorhynchus mykiss) fed Thymus vulgaris essential oils

Efficacy of Thymus vulgaris essential oils was assessed on growth, immune response and disease resistance of rainbow trout (Oncorhynchus mykiss). Fish weighing 10 g were fed with dietary supplemented of the oils at 0.5, 1.0 and 2.0 ml/kg feed for 2 months. Fish fed with the oils at 0.5 ml/kg feed demonstrated a better weight gain and specific growth rate, compared to other treatments (p < .05). Fish fed with 1.0 ml the oils showed the highest up‐regulation of complement component 3 (C3) and (cluster of differentiation 4) (CD4) genes expression (p < .05), while lysozyme gene expression level significantly increased in fish fed with 2.0 ml of the oils. In addition, at the end of the experiment, the expression of C3 and CD4 genes were significantly up‐regulated in fish fed with 1.0 and 2.0 ml of the oils, while IL‐1ß and lysozyme genes expression levels were significantly decreased in fish fed 2.0 ml oils, towards the end of the trial (p < .05). There was a fluctuation in the levels of Alanine aminotransferase, Aspartate aminotransferase and Alkaline phosphatase in all treatments during the experiment. When treated fish were challenged with Aeromonas hydrophila, the highest survival rate was observed in 0.5 ml/kg treatment, followed by 2 and 1 mg/kg treatments. Overall, these findings demonstrated that dietary administration of T. vulgaris oils especially at 0.5 mg/kg feed can be considered as a potential component for enhancing of the growth, immune responses and disease resistance of trout against motile Aeromonas septicemia caused by A. hydrophila.     

Replacement of fishmeal by canola protein concentrate in diets fed to rainbow trout (Oncorhynchus mykiss)

A dephytinized protein concentrate prepared from canola seed (CPC) was assessed for nutrient digestibility and performance in rainbow trout (Oncorhynchus mykiss). The apparent digestibility coefficients of CPC were: dry matter, 817 g kg^?1; crude protein, 899 g kg^?1; gross energy, 861 g kg^?1; arginine, 945 g kg^?1; lysine, 935 g kg^?1; methionine, 954 g kg^?1; threonine, 893 g kg^?1. A 9‐week performance trial assessed 7 diets. Fishmeal provided 940 g kg^?1 of the protein in the control diet. Test diets consisted of CPC or water‐washed CPC replacing 500 and 750 g kg^?1 of fishmeal protein; and CPC plus an attractant replacing 500 and 750 g kg^?1 of fishmeal protein. No significant differences in performance were observed (P > 0.05). A subsequent 9‐week performance trial evaluated the effect of adding CPC into compound diets containing fishmeal/soybean meal/corn gluten meal. Five diets were prepared: fishmeal provided 670 g kg^?1 of the protein in the control diet, in the remaining diets CPC was incorporated into commercial‐like trout diets at 100, 200 and 300 g kg^?1 replacement of fishmeal protein, the fifth diet included an attractant in the 300 g kg^?1 replacement diet. No significant differences in performance were obtained (P > 0.05). These studies show that dephytinized canola protein concentrate has potential to replace substantial levels of fishmeal in diets for carnivorous fish without compromising performance.     

Optimizing zinc supplementation levels of rainbow trout (Oncorhynchus mykiss) fed practical type fishmeal‐ and plant‐based diets

Rainbow trout (23.1 ± 0.4 g) were fed either a fishmeal‐ or plant‐based diet supplemented with various levels of zinc (0, 15, 30, 60 or 120 mg kg^?1) for 12 weeks. Trout fed the fishmeal diet had significantly higher weight gain than with the plant‐based diet. Zinc supplementation in the fishmeal diet had no effect on growth performance, suggesting that additional dietary supplementation of zinc is not required. However, in trout fed the plant‐based diet, growth increased significantly up to 30 mg kg^?1 zinc after which growth was not affected. Trout fed the plant‐based diet containing no zinc exhibited severe growth retardation, and in fish fed the 0 and 15 mg kg^?1 zinc diets, cataracts were present. Use of broken‐line quadratic modelling suggests that dietary supplementation of zinc needed to prevent deficiency and promote adequate growth in rainbow trout fed the plant‐based diet in this study was 30.1 mg kg^?1 (80 mg kg^?1 total dietary zinc). This is higher than the NRC (2011, Nutrient Requirements of Fish and Shrimp) dietary recommended level of 15 mg kg^?1 for rainbow trout. Following the NRC recommendation could lead to zinc deficiency in rainbow trout fed a plant‐based diet.     

Effects of low‐level dietary microalgae supplementation on the distal intestinal microbiome of farmed rainbow trout Oncorhynchus mykiss (Walbaum)

In this study, high throughput 16S rRNA sequencing was used to investigate the effect of a novel whole‐cell dietary microalgae meal (Schizochytrium limacinum), on the distal intestinal microbiome of farmed rainbow trout, Oncorhynchus mykiss. Heterotrophic microalgae are rich in omega 3 polyunsaturated fatty acids, can be produced sustainably and have been shown to have beneficial effects on host health. After a 15‐week trial period, microbial community profiles were compared between the distal intestinal contents of fish fed either a control diet or a treatment diet that partially replaced fish oil with microalgae meal, at a substitution level of 5%. The results of this research showed that the microbial communities of both fish populations were composed of similar microbial taxa, however, the treatment group fed the microalgae supplement possessed a greater level of microbial diversity than those in the control group. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group, notably operational taxonomic units (OTUs) assigned to the genera Streptococcus, Leuconostoc, Lactobacillus, Lactococcus and Weissella. However, the overall structure of the intestinal microbiome between control and treatment groups was not found to be significantly different. The treatment group displayed a heavier mean weight and condition factor at the end of the trial period. The results of this study suggest that the tested microalgae meal can be used as a replacement for a proportion of fish oil in aquafeeds, with minor changes to the intestinal microbiome of farmed rainbow trout, and positive effects on growth.     

Effects of dietary β-carotene on the immune response of rainbow trout Oncorhynchus mykiss

SUMMARY: We attempt to assess the impact of different levels of dietary β-carotene on immune function in rainbow trout. Semi-purified diets containing 0, 40, 200, and 400 mg β-carotene/kg dry diet were fed for 12 weeks to fish with average weight of 45 g. In addition to the humoral and cellular immune parameters, growth and feed utilization were examined. There were no marked differences in growth and feed utilization showing that β-carotene was not particularly efficient in enhancing growth of rainbow trout. Of the immune parameters measured, total immunoglobulin was significantly highest for the 200 mg β-carotene fed group. Serum complement activity (alternate pathway) at 200 and 400 mg β-carotene supplementation was significantly higher than that of the unsupplemented group. An increasing trend in lysozyme activity was observed, however, the differences among the groups were not significant. Phagocytic activity was similar among diet groups except at the highest level of supplementation where it was the maximum. Oxygen radical production by peripheral blood leukocytes appeared to be lower at higher levels of carotenoid supplementation. Overall, dietary β-carotene clearly enhanced immune response parameters in rainbow trout such as serum complement activity and total plasma immunoglobulin but did not show a definite influence for the other factors examined in the present study.     

Preliminary assessment of in vivo safety of potentially probiotic lactic acid bacteria for American bullfrog culture

The effect of the administration of beneficial lactic acid bacteria (LAB): Lactococcus lactis CRL 1584, L. lactis CRL 1827, Enterococcus gallinarum CRL 1826 and combined CRL 1584+ CRL 1826 on the development of bullfrog embryos from the hatching stage until 31 days (tadpole) was evaluated. In vitro assays indicated that 10³, 10⁶ and 10⁹ CFU mL^?1 single LAB strains remained viable until 24 h in 10% Ringer. Around 10⁹ CFU mL^?1 LAB (individually and combined) were used in an experimental design built to evaluate their effect when administrated at different intervals (three 7‐day cycles with 5‐day rest periods in between) to embryos until day 31. The highest potentially beneficial population (LAB) numbers were detected in the LAB‐treated groups. All the LAB‐treatments increased it and were significantly higher than the controls. Although the highest, potentially pathogenic, population (Red‐Leg Syndrome‐RLS‐related pathogens) numbers were detected in the control and the lowest in the CRL 1584+ CRL 1826‐treated group, they did not differ significantly. Stereoscopic studies showed no malformations in any LAB‐treated group and all the specimens reached the same stage of their biological cycle with a survival >94%. The histological structure of target organs for RLS‐associated pathogens (intestine and skin) and stomach was not affected and the spleen was developed. Only the LAB‐treated groups showed microorganisms associated with the intestinal mucus, without inflammatory reaction in the lamina propria. This is the first report on the evaluation of the absence of adverse effects after LAB administration to bullfrog embryos using indigenous microorganisms.     

Reducing bacterial density in the semen of rainbow trout (Oncorhynchus mykiss) by applying gradient centrifugation and swim‐up washing methods

The objective of this study was to demonstrate the effectiveness of disinfection procedures to reduce bacterial load of rainbow trout, Oncorhynchus mykiss (Walbaum 1792), semen. Fresh semen was obtained from 3–4‐year‐old male species by abdominal sampling of sperm into pre‐cooled test tubes. After sperm cryopreservation and thawing, experiments were accomplished at 4–9°C. ‘Swim‐up’ and gradient centrifugation were used as a sperm washing method with commercial kits. Phosphate buffered saline was also used as washing solution. Bacterial growth tests were employed before and after washing the semen samples. Samples were inoculated on tryptic soy agar (TSA), modified Anacker and Ordal agar (MAOA) as well as brain heart infusion (BHI) agar. After using ‘swim‐up’ method for washing the semen, many bacterial colonies were observed. However, after semen washing with gradient centrifugation, lower bacterial growth was observed on TSA, MAOA and BHI. Some motile (40%) spermatozoa were obtained doing gradient washing procedure. Although sperm motility was not satisfactory, apparently the gradient centrifugation method reduced bacterial contamination as known from the mammalians.     

Non‐invasive recording of brain function in rainbow trout: Evaluations of the effects of MS‐222 anaesthesia induction

Effective methods of producing instantaneous and irreversible unconsciousness at the time of slaughter are crucial for ensuring animal welfare in commercial aquaculture. However, the traditional method of visually evaluating unconsciousness has been shown to be insufficient and may lead to misjudgements of stunning efficiency. In this study, we developed a non‐invasive technique that measures brain activity in fish as an alternative to traditional invasive, brain implants and used it to determine when a change in consciousness occurs in trout during anaesthesia induction. Nine rainbow trout (Oncorhynchus mykiss) were equipped with a custom designed silicone cup fitted with electrodes and submerged in 10°C water with dissolved MS‐222. During anaesthesia, the state of consciousness was assessed by recordings of electroencephalogram (EEG). The EEG recordings were analysed both by visually evoked responses from light stimulation (VERs) and from changes in signal amplitude, median frequency and relative signal power. According to the loss of VERs and decrease in signal amplitude, trout transitioned to surgical depth of anaesthesia within 5 min. Our results show that consciousness, or loss of, can be determined using a non‐invasive system to record EEG in fish.     

Performance and immunological responses of rainbow trout (Oncorhynchus mykiss) fed bioprocessed plant‐based proteins

Marine‐derived fish meal (FM) is a traditional component of commercial aquaculture feeds for many farmed fish species. Modern bioprocessing technologies have been developed to produce high‐protein plant‐based ingredients for aquafeeds to further reduce FM and other conventional animal protein sources. A 90‐day feeding trial using juvenile rainbow trout (Oncorhynchus mykiss) (27.9 ± 1.4 g; mean ± SE) was completed to evaluate growth, feed efficiency, general health and immunological responses to diets containing experimental plant‐based protein sources and reduced FM. Trout were fed one of four dietary treatments: experimental bioprocessed soy protein concentrate (BSPC), commercially available enzymatically hydrolysed soy protein concentrate (CSPC), experimental bioprocessed barley protein concentrate (BBPC) and a FM control (FMC). At trial termination, there were no significant differences in relative growth (RG) or specific growth rate (SGR) between fish fed either FMC or BSPC (p > .05). Fish fed either CSPC or BBPC resulted in significantly lower RG and SGR than the FMC (p < .05). Significant respiratory burst analysis differences were observed among treatments at day 90 (p < .01), for fish fed either FMC or CSPC diets. This assessment of bioprocessed plant‐based protein ingredients facilitates the characterization and incorporation of bioprocessed soy and barley protein as the industry continues to evaluate FM replacements in rainbow trout feeds.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

The effect of total replacement of fish oil with DHA‐Gold® and plant oils on growth and fillet quality of rainbow trout (Oncorhynchus mykiss) fed a plant‐based diet

This study investigated the effect of the replacement of fish oil (FO) with DHA‐Gold (DHA‐G)‐supplemented plant oils (PO) in rainbow trout fed plant‐protein‐based diets. Five diets (450 mg g^?1 digestible protein and 150 mg g^?1 crude lipid) were fed to rainbow trout (initial weight 37 ± 0.5 g) for 12 weeks in a 15 °C recirculating water system. The lipid inclusion types and levels were FO, PO and PO with DHA‐G supplemented at 30 mg g^?1, 60 mg g^?1 or 90 mg g^?1 of the diet replacement for corn oil. Fish fed 90 mg g^?1 DHA‐G were significantly larger and consumed more feed than fish‐fed PO or FO (218 g and 2.6% bwd^?1 versus 181 g and 2.4% and 190 g and 2.3%, respectively). Feed conversion ratio was significantly increased in fish fed 90 mg g^?1 DHA‐G (0.99) as compared to fish‐fed FO (0.90) and 30 mg g^?1 DHA‐G (0.91). Panellists found trout fillets from fish fed the 90 mg g^?1 DHA‐G diet to have significantly fishier aroma and flavour than fish fed the FO diet. Fatty acid analysis demonstrated that 60 mg g^?1 or 90 mg g^?1 DHA‐G supplementation increased PO fed fish fillet DHA to fatty acid levels equivalent or higher than those fish fed a FO diet.     

Comparative effects of four feed types on white spot disease susceptibility and skin immune parameters in rainbow trout, Oncorhynchus mykiss (Walbaum)

The effects on rainbow trout, Oncorhynchus mykiss (Walbaum), immune parameters by differently formulated fish feed types containing immunostimulants have been tested in a double-blind, duplicated and controlled study performed over 50 days. A total of 800 rainbow trout (10-12 g) were kept in eight duplicate fish tanks (each containing 100 fish) and fed at a daily feeding rate of 1.5% of the biomass. The feed types were (1) control feed (C) without additives, (2) feed containing beta-glucan, nucleotides, manno-oligosaccharides (MOS), vitamins C and E (GNMCE), (3) feed containing probiotic bacteria and plant extracts (PP) and (4) feed with nucleotides, manno-oligosaccharides, vitamins C and E (NMCE). Plasma lysozyme activity was increased in fish fed two feed types (GNMCE and NMCE) but slightly depressed in fish fed PP. A non-significant trend for a higher mucous cell density at days 30 and 50 was shown in all fish receiving feeds with additives compared to the control group. All fish became infected with Ichthyophthirius multifiliis when exposed, but fish fed GNMCE showed a significantly lower infection both at days 30 and 50. Expression of genes encoding C3 and MHCII was significantly up-regulated in fish fed GNMCE for 50 days, and the expression of genes coding Hepcidin was significantly down-regulated in fish fed NMCE for 50 days. Beta-glucan was the single component, when used in combination with other feed ingredients, which was found associated with increased parasite resistance, increased lysozyme and immune gene up-regulation.     

Effect of drying on the viability of the probiotic bacterium Lactobacillus plantarum Lab9 (CPQBA 144‐09 DRM 03) impregnated in the feed for tilapia (Oreochromis sp.)

Drying experiments were conducted at different temperatures and air flux velocities to determine the proper drying conditions for reducing moisture in commercial fish feed impregnated with probiotic lactic acid bacteria and to assess the effect on bacteria viability over time. At temperatures of 45°C, the drying time was shorter, without the air flux velocity under study having a relevant influence. The drying conditions influenced the viability of the bacteria in the feed; the least loss of viability was obtained with a velocity of 0.8 m s^?1 and a temperature of 45°C during 15 min. Using these drying conditions, 5 kg of feed was dried and stored for a month at temperature of 26°C and relative humidity of 75%. The viability of the bacteria and the moisture of the feed were measured every 3 days during the storage period. Loss of viability followed first order kinetics, with a constant k of 0.112 days^?1. Thus, the viability of the bacteria in the feed is less than 10⁶ CFU g^?1 after 43 days.     

Citric acid substituted the inclusion of inorganic phosphorus in diet of rainbow trout (Oncorhynchus mykiss)

This study was conducted to evaluate the effects of citric acid (CA) supplementation in diet without inorganic phosphorus (P) on growth, muscle and bone composition, proteolytic activities and serum antioxidant property of rainbow trout. Six diets were designed as the negative diet without monocalcium phosphate (MCP) supplementation, the positive diet containing 10 g kg^?1 MCP and CA supplementation diets with 4, 8, 12, 16 g kg^?1 CA supplementation in negative diet, and then were fed to rainbow trout (113.6 g) for 60 days. Results showed that the fish fed 8 g kg^?1 CA, 12 g kg^?1 CA diet had higher weight gain, higher contents of crude ash and P in bone, and lower feed conversion ratio than those of fish fed negative diet (P < 0.05), and showed the similar levels as those of fish fed positive diet (P > 0.05). The proximate composition and P level of muscle were not affected by dietary CA and MCP. The proteolytic activity in intestine, but not in stomach and gastric digesta, was significantly improved by dietary CA and MCP (P < 0.05), when compared with negative control. The activities of serum superoxide dismutase of 12 g kg^?1 CA and 10 g kg^?1 MCP groups were significantly higher, and the malondialdehyde of 8 g kg^?1 CA and 12 CA g kg^?1 groups were significantly lower than those of negative control (P < 0.05). The above results indicated that the supplementation of CA could substitute the inclusion of MCP in rainbow trout diet and the supplementation level was suggested to be 8–12 g kg^?1.