Apparent amino acid availability from feedstuffs in extruded diets for rainbow trout Oncorhynchus mykiss

Apparent amino acid availability coefficients of several typical and novel feed ingredients were determined in rainbow trout using extruded diets and the faecal stripping technique. The ingredients were tested included five fish meals, three terrestrial animal by‐products, five plant protein concentrates, four plant meals, and seven low‐protein plant ingredients. Amino acid availability from the fish meals was relatively high ranging from 90 to 101%. Lower coefficients overall were observed for Menhaden fish meal FAQ when compared to the other fish meals. No differences in apparent amino acid availability were detected among the animal by‐products. Within the plant concentrate group, rice protein concentrate and barley protein concentrate exhibited generally lower amino acid availabilities compared to other concentrates tested. Among the plant meals, only the availabilities of histidine, valine, isoleucine and lysine in flaxseed meal were lower than those of soybean meal. Apparent amino acid availabilities among the low‐protein plant products were variable and significantly different.     

Digestibility in selected rainbow trout families and relation to growth and feed utilisation

Experiments have been carried out aimed at clarifying variations in the digestibility of dietary nutrients in rainbow trout families and studying how differences in digestibility may be related to growth and feed utilisation at various growth rates. The digestibility of protein, lipid, carbohydrates (nitrogen-free extracts, NFE) and dry matter was analysed in two experiments involving eight rainbow trout families [Ab, Ba, Cd, Dc (first study); V, X, Y, Z (second study)]. In the first experiment rainbow trout were reared for 128 days at 13.0°C, and in the second experiment, they were reared for 84 days at 16.8°C. In both experiments, the fish were fed ad libitum and reared from an initial weight of 70–100 g to a final weight of 500–700 g. When the fish reached a weight of approximately 200 g, some individuals were moved to another experimental system in which the digestibility of protein, lipid, nitrogen-free extracts and dry matter was measured. Taken as a whole, our results indicate that selective breeding still offers a large potential for improved growth and feed utilisation in rainbow trout strains. In the first study, family Dc showed a higher specific growth rate (SGR) than the other three families (P < 0.05), and family Ba showed a lower feed conversion ratio (FCR) than family Ab (P < 0.05); there were no observed differences in digestibility despite some differences in growth. In the second study, family Y grew faster than all of the other families (P < 0.05), and family Z grew faster than families V and X (P < 0.01). A comparable pattern was seen for FCR, with family Y utilising feed better than family V (P < 0.05), and families V, Y and Z performing better than family X (P < 0.001). Protein digestibility was higher in the two fastest growing families (Y and Z) than in the slower growing family X (P < 0.05), while lipid digestion was higher in family Y than in family V (P < 0.05). A comparison of the results from both experiments revealed that protein digestibility in particular was closely related to the SGR and the FCR at high growth rates. However, despite the advantageous protein digestibility on fish growth, analysis of the protein retention efficiency (PRE) showed that when protein was ingested in relatively large amounts, as in the fastest growing families, the “excess” nitrogen was excreted and therefore did not contribute to protein deposition in the fish body. Hence, the potential weight gain offered by improved protein digestibility does not materialise when the protein intake is above a certain level. Other factors must therefore explain the positive relation between fast growth and high protein digestibility.     

The uptake and esterification of radiolabelled fatty acids by enterocytes isolated from rainbow trout (Oncorhynchus mykiss)

To study the intestinal fatty acid absorption in fish in vitro, enterocytes were isolated from the intestine of rainbow trout and incubated with an equimolar mixture of seven fatty acids [16:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3] in which the component carrying a radioactive label was varied. The fatty acid mixture was presented in the form of micelles formed by sonication with sodium taurocholate. Control studies showed that the presence of sodium taurocholate in the incubation medium caused an immediate 23% increase in the mortality of the cells but the remaining cells were viable. The effect of the bile salt on cellular permeability was evident at longer exposure periods. The proportions of 14C-labelled 20:5n-3 and 22:6n-3 present as monomeric fatty acids in the micellar solutions were higher than those of 16:0, 18:1n-9, 18:2n-6, 18:3n-3 and 20:4n-6. The rates of uptake of 14C-labelled 20:4n-6, 20:5n-3 and 22:6n-3 by enterocytes were significantly lower than those of the other fatty acid substrates over the first minute of incubation but no significant differences in uptake rate between fatty acids were obvious over a 15 min incubation period. Notable differences were observed between substrates in their distribution pattern in enterocyte lipid classes. Although most of the radioactivity from all radiolabelled substrates was recovered in triacylglycerols, the amounts of 14C- labelled 16:0, 20:4n-6, 20:5n-3 and 22:6n-3 recovered in the polar lipid fraction were higher than those of 14C-labelled C18 substrates, particularly after 15 min. Conversely, the initial esterification rates of 18:1n-9, 18:2n-6 and 18:3n-3 into triacylglycerols were significantly higher than those of 20:4n-6, 20:5n-3 and 22:6n-3. It is concluded that isolated enterocytes can be used for the study of the mechanism of intestinal fatty acid absorption in fish.     

Incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids in phospholipid classes in cultured rainbow trout (Salmo gairdneri) cells

The incorporation and metabolism of various (n-3) and (n-6) polyunsaturated fatty acids supplemented to the culture medium was investigated in the rainbow trout cell line, RTG-2. The distribution, and the occurrence and relative extent of further desaturation and elongation of the incorporated acids was determined in individual phospholipid classes by analysis of the fatty acid compositions. RTG-2 cells exhibited Δ6 and Δ5 desaturase activities whereas Δ4 desaturase activity was almost totally absent. The percentage of precursor acids was greatest in the phosphatidic acid/cardiolipin fraction (PA/CL), suggesting a role for possibly PA in the initial incorporation of these acids into the phospholipid pool. The compositional data indicated that individual intermediates and products of the desaturation pathways were associated with specific phospholipid classes probably via mechanisms depending upon the specificities of the acylating enzymes. The composition of phosphatidylinositol (PI) and the tightly controlled mechanisms for generating/maintaining it are consistent with a role for this phospholipid in providing precursor fatty acid for eicosanoid synthesis.     

Effects of dichlorvos and formalin on fatty acid metabolism of rainbow trout (Oncorhynchus mykiss) skin cells in primary culture

The effects of sub-lethal doses of dichlorvos and formalin, antimicrobial/parasitic agents used in aquaculture, on lipid composition and metabolism of rainbow trout skin cells in primary culture were investigated. [1-¹⁴C]Stearic (18:0), [1-¹⁴C]lin 18:2n-6) and [1-¹⁴C]linolenic (18:3n-3) acids were used as tracers to determine effects on fatty acid incorporation and metabolism. Formalin increased cell numbers and reduced the lipid content of the cells and the incorporation of radioactive fatty acids. The effects of dichlorvos were qualitatively similar but quantitatively less. Formalin induced relatively small but significant changes in lipid class composition including a decreased proportion of phosphatidycholine with increased proportions of sphatidylethanolamine and phosphatidylserine. Dichlorvos had no significant effect on lipid class compositions. The trout primary skin cells expressed substantial 9, 6 and 5 fatty acyl desaturase activities. Although, as expected, the cells were m active towards [1-¹⁴C]18:3n-3, the cells were unusually active towards [1-¹⁴C]18:2n-6. Both dichlorvos and, especially, formalin appeared to significantly inhibit 9 and 6 desaturation. Changes in the distribution of radioactivity between individual spholipid classes was also influenced by formalin and dichlorvos, and this may be related to changes in desaturase activity. This study has shown that topically active agents used in aquaculture, formalin and dichlorvos, had a range of effects on the rainbow trout skin cell cultures that may affect cell proliferation and lipid and fatty acid metabolism. Both agents significantly inhibited desaturation of fatty acids, particularly of 18:2n-6 to 20:4n-6 and, as 20:4n-6 is a major eicosanoid precursor ish and considering the importance of eicosanoids in the biochemistry of skin, it is suggested that these agents may have direct effects on fish skin that could have important consequences for fish health in general.     

虹鳟免疫诱导型基因Cathelicidin2启动子功能分析

本研究通过实时荧光定量PCR实验对虹鳟(Oncorhynchus mykiss)Cathelicidin2(Cath2)基因的转录模式进行了分析。结果显示,该基因在鳃、头肾等与机体免疫防御功能密切相关的组织内转录,在细菌和病毒感染后,转录水平均显著升高。对基因上游调控序列进行启动子和转录因子结合位点预测,发现该启动子具有真核生物典型的TATA盒和CAAT盒结构,基因上游直至第一内含子区域内,密集存在多个免疫相关转录因子结合位点,其中,2个核因子κB(Nuclear factor kappa B,NFκB)预测结合位点均位于核心启动子正链区域。在草鱼(Ctenopharyngodon idellus)肾组织细胞系内,绿色荧光蛋白和萤火虫荧光素酶基因都能够在该启动子驱动下表达,表明其具有启动子活性,且启动子活性在受到免疫诱导后增强,包括细菌脂多糖(Lipopolysaccharides,LPS)模拟的细菌感染和聚肌胞苷酸(Polyinosinic polycytidylic acid,Poly I:C)模拟的病毒感染。双荧光素酶报告基因检测显示,启动子活性在与NFκB转录因子表达时,增强至4.39倍,证明Cath2基因受NFκB通路调控。研究表明,虹鳟Cath2基因能够在多种免疫刺激诱导下表达,其启动子可以应用为免疫诱导型的基因工程元件,驱动外源免疫基因在鱼体内适时表达,抵御外界病原感染,同时,避免非必要条件下的过度表达形成生长负担。     

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.     

Partial replacement of soybean meal with Methylobacterium extorquens single‐cell protein in feeds for rainbow trout (Oncorhynchus mykiss Walbaum)

A feeding trial was conducted with juvenile rainbow trout (15–16 g initial weight) to assess the effects of including single‐cell protein (SCP) produced from Methylobacterium extorquens in trout feeds. Three isonitrogenous and isoenergetic diets were produced: a control diet and two experimental diets containing 5% or 10% bacterial protein meal replacing soybean meal. Triplicate tanks, each containing 35 fish, were fed each diet to apparent satiation in a constant‐temperature (15°C), flow‐through tank system for 12 weeks. No statistically significant differences in final fish weight or other fish growth parameters were observed. Similarly, feed efficiency parameters showed no significant differences among groups. Nutrient retention indices (protein, fat, energy) were relatively high and similar among fish in each dietary treatment group, as were whole body proximate compositions. Fish survival was high, with a small but statistically significant increase for the 10% SCP diet. Overall, results demonstrate that SCP from M. extorquens is a safe and effective alternative protein for rainbow trout diets at the low inclusion levels tested. Slightly lower weight gain in fish fed the 10% SCP diet was largely due to lower feed intake, suggesting that adding palatability‐enhancing ingredients to feeds may allow higher levels of M. extorquens SCP to be used without compromising fish growth.     

Oral pharmacological treatments for ichthyophthiriosis of rainbow trout (Oncorhynchus mykiss)

The aim of this work was to explore the efficacy of a veterinary drug, Triclabendazole (5-chloro-6-(2, 3-dichlorophenoxy)-2-methylthio-1H-benzimidazole), in an inclusion complex with β-cyclodextrin as a suitable treatment for parasitic diseases caused by Ichthyophthirius multifiliis in rainbow trout. The efficacy was determined by the reduction in the infection intensity. The complexes were prepared by the kneading method and were characterized by DSC and X-ray diffractometry. The selected stoichoimetry was 1:3 because of the higher percentage of Triclabendazole complexed with cyclodextrin. Administration of Triclabendazole and complex was carried out by including appropriate doses in animal feed. Our studies suggest that the Triclabendazole–cyclodextrin complex results in a reduction in the infection degree and trophont size is decreased in the animals treated. The oral treatment of Triclabendazole in inclusion complexes may be an alternative to bath treatments in trout farming.     

Effects of diets containing a bacterial protein meal on growth and feed utilisation in rainbow trout (Oncorhynchus mykiss)

Triplicate groups of rainbow trout with initial weight 361 g were fed either a fish meal based control diet or diets containing 9, 18 or 27% bacterial protein meal (BPM) or 9% of an autolysate (AU) of the BPM. No significant treatment effects were found on specific growth rates (SGR), feed intake, feed efficiency ratio (FER), or retention of nitrogen, amino acids or energy. The apparent digestibility coefficients (ADC) of nitrogen, energy and most indispensable amino acids decreased when BPM was included in the diet. The ADC of lipid, sum of amino acids, arginine, lysine, threonine and most of the dispensable amino acids were reduced at 27% BPM inclusion compared to the control, 9% and 18% BPM diets. None of the ADC estimated was different in the 9% AU diet compared to the 9% BPM diet. The loss of nitrogen and energy in faeces per kg gain increased as the dietary BPM or AU levels increased, and the energy used for activity and maintenance was higher in fish fed the 27% BPM diet than in fish fed the other diets (P < 0.05). There were no significant differences in the urea concentrations in plasma, liver and muscle, whereas the uric acid level in plasma was elevated in trout fed the 27% BPM diet. Histological evaluation of tissue from the stomach, pyloric caeca, mid-intestine and distal intestine did not reveal any diet-related morphological changes.In conclusion, no significant differences in growth and feed efficiency were found in the rainbow trout fed diets containing up to 27% BPM, and the AU did not increase fish performance compared to the BPM. Based on the data from this study, at levels up to 27% dietary inclusion, BPM is a good replacement for fish meal in diets for rainbow trout.     

Evaluation of the influence of Lupinus angustifolius kernel meal on dietary nutrient and energy utilization efficiency by rainbow trout (Oncorhynchus mykiss)

This study examined the utilization efficiencies of three diets when fed to rainbow trout in a 28‐day growth study. Each of the diets was fed at one of three ration levels, and an additional starved treatment was also included. The diets differed by an increasing concentration of lupin (Lupinus angustifolius cv. Myallie) kernel meal (MKM) inclusion. Two lupin kernel meal inclusion levels of 15% (MKM15%) and 30% (MKM30%) were studied. The diets were formulated to equivalent digestible protein and energy specifications based upon the predetermined digestibility values for each of the ingredients used. There were no significant differences in digestible protein level among the diets. However, a significantly higher level of digestible energy of both MKM diets was measured, as well as a significantly higher level of digestible phosphorus in the MKM30% diet. No significant differences between the diets were observed with respect the utilization of dietary digestible energy. Over the full data range, the energy utilization efficiency was described by the linear equation of; y = 0.747x ? 26.174, R² = 0.985. Efficiency of protein utilization over lower digestible protein intake levels was also linear (y = 0.599x ? 0.142, R² = 0.905), but over the full range was better described by a nonlinear function. The comparison of the three diets in this study shows that the dietary inclusion of lupin kernel had no significant effect on the gain of either protein or lipid energy relative to protein or lipid energy intake, respectively. Protein energy use efficiency constants varied depending on the feed intake level, but were not significantly affected by diet type. The efficiency of use of lipid energy for lipid energy retention was also not affected by diet type. The findings of this study demonstrate that the inclusion of lupin kernel meal, at up to 30% of the diet, does not affect the ability of rainbow trout to utilize the dietary digestible protein and energy of diet in which it is included.     

Evaluating dietary supply of microminerals as a premix in a complete plant ingredient‐based diet to juvenile rainbow trout (Oncorhynchus mykiss)

Two basal diets M0 and V0 were formulated with marine and plant based ingredient composition. Seven experimental diets were prepared from the two basal diets namely M0, M100, V0, V30, V60, V100 and V150 by incorporating different levels of a micromineral premix (Cu, Fe, Mn, Se and Zn). Triplicate groups of rainbow trout (initial weight: 20 g) reared at 17°C were fed one of each diet to apparent visual satiation over 12 weeks. Among the V diet fed fish, growth and feed intake exhibited maximal response at V60 level of premix inclusion; Apparent availability coefficient of Fe, Cu and Zn decreased linearly with increasing level of premix whereas apparent availability coefficient of Mn and Se was unaffected. The available dietary concentration in basal V0 diet was for Fe, 20.6; Cu, 2.8; Mn, 6.5; Zn, 17.3 and Se, 0.195 (in mg/kg DM) and in the M0 diet for Fe, 63.3; Cu, 5.2; Mn, 2.9; Zn, 35.2 and Se, 0.87 (in mg/kg DM). In reference to NRC (Nutrient requirements of fish and shrimp. Washington, DC: National Research Council, The National Academies Press, 2011) recommendations, the V0 basal diet accounted for 34.3%, 92.9%, 53.9%, 115% and 130.2% and the contribution from M0 diet for 105.5%, 173.3%, 24.2%, 234.7% and 580% of the minimal dietary inclusion levels of Fe, Cu, Mn, Zn and Se to rainbow trout, respectively. However, data on whole body mineral contents showed that normal levels were maintained only for Cu and Mn through supply from basal V0 diet. For Zn and Se, available supply even from the highest supplemented diet (V150) was not sufficient to maintain normal body mineral levels of rainbow trout in the present study. On the whole, optimal dietary inclusion levels of microminerals are altered while using fishmeal‐free diets for rainbow trout.     

Growth and feed utilisation of rainbow trout subjected to changes in feed lipid concentrations

Rainbow trout Oncorhynchus mykiss grew from 44 to 326 g in 96days when held at 12 °C. Fish were fed to satiation twice dailywith either high (L₁: 30.8%, L₂:31.4%) or lower-lipid feeds (C₁: 18.8%,C₂: 21.8%). Four feeding treatments were studied.Group C₁C₂ received feed C₁ for 43 days(days 0–43) and C₂ thereafter (days 44–96).Groups L₁L₂, L₁C₂ andC₁L₂ were subjected to dietary changes asindicated by the feed designations. After a short period of feedadaptation, fish ingested similar amounts of feed energy i.e., they ateless by weight of the lipid-rich (L) feeds. Feed lipid content did notaffect growth but fish fed L-feed had reduced feed conversion ratio(FCR) compared to fish fed C-feed (0.731 vs. 0.773) during days0–43 (P < 0.01). After 96 days,L₁L₂-fish were lower in body protein(15.8%) than the C₁C₂-fish (16.8%)(P < 0.01). L-feeds also tended to increase percentage lipidand reduce percentage whole body moisture and ash. A higher net proteinutilisation (NPU) was recorded in fish fed L-feeds (43.6%)compared to fish fed C-feeds (38.8%) in days 0–43(P < 0.05). This seemed to be the result of a lower proteinintake rather than a protein-sparing effect of feed lipid. Above athreshold value of approximately 6.5 mg protein eaten·g bodywt_{minus 1}·day_–1, NPU decreased.     

Growth, digestibility and nutrient utilization of rainbow trout (Oncorhynchus mykiss) and European sea bass (Dicentrarchus labrax) juveniles fed different dietary soybean oil levels

A shortage of marine raw ingredients, such as fish oil, is predicted in the near future. The use of suitable alternative lipid sources, such as vegetable oils, is crucial for sustainable growth of the aquaculture sector. Three isonitrogenous (50% Crude Protein) and isoenergetic (19 kJ g⁻¹) diets, in which fish oil was gradually replaced by soybean oil (0, 25 and 50% of the oil fraction) were tested for 12 weeks, using European sea bass (Dicentrarchus labrax) and rainbow trout (Oncorhynchus mykiss) juveniles. No dietary effects (p>0.05) on growth performance, feed conversion, digestibility or body composition were observed with either species. The results obtained suggest the inclusion of soybean oil up to 50% of the dietary lipid is possible, in diets for sea bass and/or rainbow trout juveniles.     

Measurement of plasma leucine flux in rainbow trout (Salmo gairneri R.) using osmotic pump. Preliminary investigations on influence of diet

A method for the direct measurement of plasma amino acid flux, in rainbow trout, using the continuous infusion of L-[U-¹⁴C]-leucine with ALZET mini-osmotic pumps implanted into the peritoneal cavity, was developed. The fish were fed successively on three different diets (a commercial control diet, a semi-purified diet and a purified diet) during the 4 weeks of experiment. The amounts of radioactivity in the free pool and the protein of both the plasma and skin mucus were measured in these fish. The total flux of leucine was calculated either from the specific activity of leucine in the plasma (61.8 mg leucine. 100 g^-1.d^-1) or from the amounts of labelled and unlabelled leucine flowing into the skin mucus protein (61.5 mg leucine. 100g^-1.day^-1). The total plasma flux was not affected by changes in the diet. The contributions of total leucine oxidation and whole body protein turnover to plasma leucine flux (80% and 20% respectively) were estimated in fish fed the semi-purified diet.     

Effects of intracerebroventricular treatment with oleate or octanoate on fatty acid metabolism in Brockmann bodies and liver of rainbow trout

Intracerebroventricular (ICV) treatment with oleate or octanoate induced in Brockmann bodies (BB) and liver of rainbow trout changes in several parameters related to fatty acid (FA) sensing through FA metabolism, FA transport through FA translocase (FAT/CD36) and mitochondrial activity. Changes observed in BB were completely different to those observed in previous studies after treatment with the same FA either intraperitoneal (IP) or in vitro. Therefore, FA sensing in BB is apparently direct and not related to previous FA sensing in hypothalamus, but it could be influenced by changes in the levels of peripheral hormones like insulin. In contrast, results obtained after ICV treatment with FA in liver were comparable to those observed after IP treatment but different to those observed after in vitro treatment. Therefore, FA sensing in liver is apparently indirect and be the consequence of previous hypothalamic FA sensing followed by vagal and/or sympathetic outflow.     

Inclusion of macroalgae meal (Macrocystis pyrifera) as feed ingredient for rainbow trout (Oncorhynchus mykiss): effect on flesh fatty acid composition

The use of macroalgae as an additional component in animal feeding has been studied. However, information on how it could influence muscle composition of fish body is scarce. This study evaluates four diets with different macroalgae inclusion levels (0%, 1.5%, 3% and 6%) to test the effect on body fatty acid composition of rainbow trout. Tanks with a volume of 600 L were stocked with 60.6 ± 7.9 g fish at a density of 45 individuals tank^?1 and fed for 124 days. At the end of the experiment there were not significant differences (P<0.05) in muscle proximate composition among fish fed the different treatments. However, it was determined that inclusion of 3% and 6% of macroalgae meal resulted in a significant increase (P<0.05) of polyunsaturated fatty acids (PUFAs) in muscle. In summary, macroalgae meal in rainbow trout diets do not enhance the quantity of protein and lipid contents at muscle level but an addition of 3–6% might contribute to increase the level of PUFAs, specially EPA, DHA and LIN. Thus, use of macroalgae meal might help to increase lipid quality content in the final product due the beneficial effects of PUFAs for human health.     

Partial to total replacement of fishmeal by poultry by‐product meal in diets for juvenile rainbow trout (Oncorhynchus mykiss) and their effect on fatty acids from muscle tissue and the time required to retrieve the effect

Triplicate groups of rainbow trout (Oncorhynchus mykiss) (1.4 ± 0.1 g) were fed experimental diets containing four levels of protein substitution to totally replace fishmeal with poultry by‐product meal (0, 33, 67 and 100%). Fishmeal (FM) from tuna fish by‐products and poultry by‐product meal ‘pet food grade' (PBM) were used. Diets were formulated to be isonitrogenous (43.5% crude protein) and isolipidic (12.5% crude fat). After 80 days of feeding, no significant differences were found in terms of thermal unit growth coefficient (TGC). However, the EPA levels in muscle tissue among treatments 0PBM, 24PBM and 44PBM were found to be significantly higher than those for the 59PBM diet. Nevertheless, a re‐feeding period with the control diet showed that 28 days was long enough to regain the FA profile needed. We concluded that PBM used up to 44% in diets for juvenile rainbow trout can be used without a significantly decrease in EPA and DHA under the present conditions. The use of a whole fishmeal diet could be an important strategy to recuperate the fatty acid profile obtained when trout is feed on PBM basis. It will be important to perform longer experiments with larger fish to confirm these results.     

Designing an in‐house ELISA to detect antibody against viral haemorrhagic septicaemia virus using recombinant N protein in Iranian farmed rainbow trout (Oncorhynchus mykiss)

Viral haemorrhagic septicaemia (VHS) is one of the most important viral diseases in rainbow trout that has caused great losses to Iranian rainbow trout aquaculture industry in the last 3 years. Therefore, rapid and reliable diagnosis of VHS virus infections is of great importance. An enzyme linked immunosorbent assay (ELISA) method was performed to study serum antibodies against viral haemorrhagic septicaemia virus (VHSV) using recombinant fragments of their N protein. For this purpose, the virus was first isolated from an infected farm. A part of the nucleocapsid (1–505 bp) gene was amplified by RT‐PCR using specific primers. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of Escherichia coli. Then, recombinant plasmids were tested for protein expression in E. coli Rosetta strain. SDS‐PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 61 KDa. Analysis of trout serum samples from seven previously infected farms and two VHS free farms showed that the designed ELISA method was effective in diagnosing the infected fish. The results revealed that the developed serological assay using designed ELISA based on recombinant protein (N) has the potential to be used in monitoring studies and to determine the prevalence of VHS in rainbow trout farms. The present data allow evaluating the levels of nonneutralizing antibodies without crude virus preparations.