Chronic cortisol stimulation enhances hypothalamus-specific enrichment of metabolites in the rainbow trout brain.

The hypothalamus is a key integrating center that is involved in the initiation of the corticosteroid stress response, and in regulating nutrient homeostasis. Although cortisol, the principal glucocorticoid in humans and teleosts, plays a central role in feeding regulation, the mechanisms are far from clear. We tested the hypothesis that the metabolic changes to cortisol exposure signal an energy excess in the hypothalamus, leading to feeding suppression during stress in fish. Rainbow trout (Oncorhynchus mykiss) were administered a slow-release cortisol implant for 3 days, and the metabolite profiles in the plasma, hypothalamus, and the rest of the brain were assessed. Also, U-13C-glucose was injected into the hypothalamus by intracerebroventricular (ICV) route, and the metabolic fate of this energy substrate was followed in the brain regions by metabolomics. Chronic cortisol treatment reduced feed intake, and this corresponded with a downregulation of the orexigenic gene agrp, and an upregulation of the anorexigenic gene cart in the hypothalamus. The U-13C-glucose-mediated metabolite profiling indicated an enhancement of glycolytic flux and tricarboxylic acid intermediates in the rest of the brain compared with the hypothalamus. There was no effect of cortisol treatment on the phosphorylation status of AMPK or mechanistic target of rapamycin in the brain, whereas several endogenous metabolites, including leucine, citrate, and lactate were enriched in the hypothalamus, suggesting a tissue-specific metabolic shift in response to cortisol stimulation. Altogether, our results suggest that the hypothalamus-specific enrichment of leucine and the metabolic fate of this amino acid, including the generation of lipid intermediates, contribute to cortisol-mediated feeding suppression in fish.NEW & NOTEWORTHY Elevated cortisol levels during stress suppress feed intake in animals. We tested whether the feed suppression is associated with cortisol-mediated alteration in hypothalamus metabolism. The brain metabolome revealed a hypothalamus-specific metabolite profile suggesting nutrient excess. Specifically, we noted the enrichment of leucine and citrate in the hypothalamus, and the upregulation of pathways involved in leucine metabolism and fatty acid synthesis. This cortisol-mediated energy substrate repartitioning may modulate the feeding/satiety centers leading to the feeding suppression.

Nano-selenium Antagonizes Heat Stress-Induced Apoptosis of Rainbow Trout (Oncorhynchus mykiss) Hepatocytes by Activating the PI3K/AKT Pathway.

The cold-water fish rainbow trout (Oncorhynchus mykiss) shows poor resistance to heat, which is the main factor restricting their survival and yield. With the advancement of nanotechnology, nano-selenium (nano-Se) has emerged as a key nano-trace element, showing unique advantages, including high biological activity and low toxicity, for studying the response of animals to adverse environmental conditions. However, little is still known regarding the potential protective mechanisms of nano-Se against heat stress-induced cellular damage. Herein, we aimed to investigate the mechanism underlying the antagonistic effects of nano-Se on heat stress. Four groups were assessed: CG18 (0 µg/mL nano-Se, 18 °C), Se18 (5.0 µg/mL nano-Se, 18 °C), CG24 (0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture), and Se24 (5.0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture). We found that after heat treatment (CG24 group), T-AOC, GPx, and CAT activities in rainbow trout hepatocytes showed a decrease of 36%, 33%, and 19%, respectively, while ROS and MDA levels showed an increase of 67% and 93%, respectively (P < 0.05). Meanwhile, the mRNA levels of the apoptosis-related genes caspase3, caspase9, Cyt-c, Bax, and Bax/Bcl-2 in the CG24 group were 41%, 47%, 285%, 65%, and 151% higher than those in the CG18 group, respectively, while those of PI3K and AKT were 31% and 17% lower, respectively (P < 0.05). Besides, flow cytometry analysis showed an increase in the level of apoptotic cells after heat exposure. More importantly, we observed that nano-Se cotreatment (Se24 group) remarkably attenuated heat stress-induced effects (P < 0.05). We conclude that heat stress induces oxidative stress and apoptosis in rainbow trout hepatocytes. Nano-Se ameliorates heat stress-induced apoptosis by activating the PI3K/AKT pathway. Our results provide a new perspective to improve our understanding of the ability of nano-Se to confer heat stress resistance.

miR-330 targeting BCO2 is involved in carotenoid metabolism to regulate skin pigmentation in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: MicroRNAs (miRNAs) play a critical role in regulating skin pigmentation. As a key economic trait, skin color directly affects the market value of rainbow trout (Oncorhynchus mykiss), however, the regulatory mechanism of most miRNAs in fish skin color is still unclear. RESULTS: In this study, the full-length cDNA sequence of ß-carotene oxygenase 2 (BCO2, a key regulator of carotenoid metabolism) from the rainbow trout was obtained using rapid-amplification of cDNA ends (RACE) technology, and qRT-PCR was used to investigate the differential expression of miR-330 and BCO2 in 14 developmental stages and 13 tissues between wild-type rainbow trout (WTrt) and yellow mutant rainbow trout (YMrt). Additionally, the function of miR-330 was verified by overexpression and silencing in vitro and in vivo. The results showed that the complete cDNA sequence of BCO2 was 2057 bp with a 1707 bp ORF, encoding a 568 amino acid protein having a molecular weight of 64.07 kD. Sequence alignment revealed that higher conservation of BCO2 protein amongst fishes than amongst other vertebrates, which was further confirmed by phylogenetic analysis. The analysis of spatial and temporal expression patterns suggested that BCO2 and miR-330 were abundantly expressed from fertilized-stage to multi-cell as well as in the dorsal and ventral skin of WTrt and YMrt, and their expression patterns were opposite in most of the same periods and tissues. In vitro, luciferase reporter assay confirmed that BCO2 was a direct target of miR-330, and transfection of miR-330 mimics into rainbow trout liver cells resulted in a decrease in the expression of BCO2; conversely, miR-330 inhibitor had the opposite effect to the miR-330 mimics. In vivo, miR-330 agomir significantly decreased BCO2 expression in dorsal skin, tail fin, and liver. Furthermore, overexpression of miR-330 could suppress cell proliferation and induce apoptosis. CONCLUSION: Our results showed that miR-330 is involved in the regulation of skin pigmentation in rainbow trout by targeting BCO2 and shows its promise as a potential molecular target to assist the selection of rainbow trout with better skin color patterns.

Effect of 11-Deoxycorticosterone in the Transcriptomic Response to Stress in Rainbow Trout Skeletal Muscle.

In aquaculture, many stressors can negatively affect growth in teleosts. It is believed that cortisol performs glucocorticoid and mineralocorticoid functions because teleosts do not synthesize aldosterone. However, recent data suggest that 11-deoxycorticosterone (DOC) released during stress events may be relevant to modulate the compensatory response. To understand how DOC modifies the skeletal muscle molecular response, we carried out a transcriptomic analysis. Rainbow trout (Oncorhynchus mykiss) were intraperitoneally treated with physiological doses of DOC in individuals pretreated with mifepristone (glucocorticoid receptor antagonist) or eplerenone (mineralocorticoid receptor antagonist). RNA was extracted from the skeletal muscles, and cDNA libraries were constructed from vehicle, DOC, mifepristone, mifepristone plus DOC, eplerenone, and eplerenone plus DOC groups. The RNA-seq analysis revealed 131 differentially expressed transcripts (DETs) induced by DOC with respect to the vehicle group, mainly associated with muscle contraction, sarcomere organization, and cell adhesion. In addition, a DOC versus mifepristone plus DOC analysis revealed 122 DETs related to muscle contraction, sarcomere organization, and skeletal muscle cell differentiation. In a DOC versus eplerenone plus DOC analysis, 133 DETs were associated with autophagosome assembly, circadian regulation of gene expression, and regulation of transcription from RNA pol II promoter. These analyses indicate that DOC has a relevant function in the stress response of skeletal muscles, whose action is differentially modulated by GR and MR and is complementary to cortisol.

Influences of l-ascorbic acid on cytotoxic, biochemical, and genotoxic damages caused by copper II oxide nanoparticles in the rainbow trout gonad cells-2.

In parallel with the raising use of copper oxide nanoparticles (CuO NPs) in various industrial and commercial practices, scientific reports on their release to the environment and toxicity are increasing. The toxicity of CuO NPs is mostly based on their oxidative stress. Therefore, it is necessary to investigate the efficacy of well-known therapeutic agents as antioxidants against CuO NPs damage. This study aimed to investigate the mechanism of this damage and to display whether l-ascorbic acid could preserve against the cell toxicities induced by CuO NPs in the rainbow trout gonad cells-2 (RTG-2). While CuO NPs treatment significantly diminished cell viability, the l-ascorbic acid supplement reversed this. l-ascorbic acid treatment reversed the changes in expressions of sod1, sod2, gpx1a, and gpx4b genes while playing a supportive role in the changes in the expression of the cat gene induced by CuO NPs treatment. Moreover, CuO NPs treatment caused an upregulation in the expressions of growth-related genes (gh1, igf1, and igf2) and l-ascorbic acid treatment further increased these effects. CuO NPs treatment significantly up-regulated the expression of the gapdh gene (glycolytic enzyme gene) compared to the control group, and l-ascorbic acid treatment significantly down-regulated the expression of the gapdh gene compared to CuO NPs treatment. The genotoxicity test demonstrated that l-ascorbic acid treatment increased the genotoxic effect caused by CuO NPs by acting as a co-mutagen. Based on the findings, l-ascorbic acid has the potential to be sometimes inhibitory and sometimes supportive of cellular mechanisms caused by CuO NPs.

Transcriptome sequencing reveals the effect of selenium nanoparticles on primary hepatocytes of rainbow trout.

Heat stress is one of the important threats in rainbow trout culture, and selenium nanoparticles (SeNPs) have an important role in combating heat stress and enhancing immunity. In this study, to enable rainbow trout to survive at higher temperatures, we added 5 µg/mL SeNPs to hepatocytes to study the resistance effect and immune effect of SeNPs against heat stress, thus enabling rainbow trout to adapt to summer temperatures (Average 26 °C) in Lanzhou, Gansu Province, China. Therefore, we investigated the transcriptome expression profile of hepatocytes spiked with SeNPs when exposed to heat stress. A total of 234 differentially expressed genes (DEGs) were firmly established in SeNPs-added group when exposed to heat stress compared to non-SeNPs-added group. Of these DEGs, 156 were up-regulated and 78 were down-regulated. These DEGs were grouped into different Gene Ontology (GO) functional terms and enriched in 75 significantly different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, of which approximately-one-third (19) were associated with immunity. STRING was used to identify 39 key immune DEGs belonging to 5 immune pathways (C-type lectin receptor signaling pathway, FoxO signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Rachidonic acid metabolism). These pathways interact extensively and formed a complex network to regulate heat stress. These results provided an important basis for future elucidation of the role of SeNPs in heat stress resistance and immune enhancement in rainbow trout.

Effect of selenium nanoparticles on alternative splicing in heat-stressed rainbow trout primary hepatocytes.

Alternative splicing (AS) is a ubiquitous post-transcriptional regulatory mechanism in eukaryotes that generates multiple mRNA isoforms from a single gene, increasing diversity of mRNAs and proteins that are essential for eukaryotic developmental processes and responses to environmental stress. Results showed that a total of 37,463 AS events were identified in rainbow trout hepatocytes. In addition, a total of 364 differential alternative splicing (DAS) events were identified in hepatocytes under selenium nanoparticles (SeNPs) and 3632 DAS events were identified under a combination of SeNPs and heat stress (24 °C). Gene Ontology (GO) enrichment showed that some subcategories "immune effector processes", "response to stimuli" and "antioxidant activity" were associated with immunity, abiotic stimuli and antioxidants. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that differentially expressed genes (DEGs) were significantly enriched in spliceosomes by adding SeNPs in heat-stressed hepatocytes. Splicing factor family (SRSF3, SRSF7, SRSF9, U2AF1 and U2AF2) and pre-RNA splicing factors (ACIN1 and PPRF18) were significantly upregulated and promoted AS. Furthermore, addition of SeNPs activated the phosphatidylinositol signaling system and upregulated the related genes PI4KA, DGKH, ITPK1 and Ocrl, and thus attenuated the inflammatory response to heat stress and enhanced resistance to heat stress by activating the adherent plaque kinase-PI3K-Akt signaling pathway and calcium channels. Those findings suggested that AS could be an essential regulatory mechanism in adaptation of rainbow trout to heat-stressed environments.

Molecular characterization and expression analysis of selenoprotein W gene in rainbow trout (Oncorhynchus mykiss) with dietary selenium levels.

Selenium (Se) plays an essential role in the growth of fish and performs its physiological functions mainly through incorporation into selenoproteins. Our previous studies suggested that the selenoprotein W gene (selenow) is sensitive to changes in dietary Se in rainbow trout. However, the molecular characterization and tissue expression pattern of selenow are still unknown. Here, we revealed the molecular characterization, the tissue expression pattern of rainbow trout selenow and analyzed its response to dietary Se. The open reading frame (ORF) of the selenow gene was composed of 393 base pairs (bp) and encodes a 130-amino-acid protein. The 3' untranslated region (UTR) was 372 bp with a selenocysteine insertion sequence (SECIS) element. Remarkably, the rainbow trout selenow gene sequence was longer than those reported for mammals and most other fish. A ß1-α1-ß2-ß3-ß4-α2 pattern made up the secondary structure of SELENOW. Furthermore, multiple sequence alignment revealed that rainbow trout SELENOW showed a high level of identity with SELENOW from Salmo salar. In addition, the selenow gene was ubiquitously distributed in 13 tissues with various abundances and was predominantly expressed in muscle and brain. Interestingly, dietary Se significantly increased selenow mRNA expression in muscle. Our results highlight the vital role of selenow in rainbow trout muscle response to dietary Se levels and provide a theoretical basis for studies of selenow.

Improvement in feed efficiency and reduction in nutrient loading from rainbow trout farms: the role of selective breeding.

Resource efficiency, the ratio of inputs to outputs, is essential for both the economic and environmental performance of any sector of food production. This study quantified the advancement in the feed conversion ratio (FCR) and reduction in nutrient loading from rainbow trout farming in Finland and the degree to which genetic improvements made by a national breeding program have contributed to this advancement. The study combined two datasets. One included annual records on farm-level performance of commercial rainbow trout farms from 1980 onwards, and the other included individuals across eight generations of the national breeding program. The data from the commercial farms showed that from 1980 onwards, the farm-level feed conversion ratio improved by 53.4%, and the specific nitrogen and phosphorus loading from the farms decreased by over 70%. Hence, to produce 1 kg of fish today, only half of the feed is needed compared to the 1980s. The first generation of the breeding program was established in 1992. The FCR was not directly selected for, and hence, the genetic improvement in the FCR is a correlated genetic change in response to the selection for growth and body composition. Since 1992, the estimated genetic improvement in the FCR has been 1.74% per generation, resulting in a cumulative genetic improvement of 11.6% in eight generations. Genetic improvement in the FCR is estimated to be 32.6% of the total improvement in the FCR observed at farms, implying that genetic improvement is a significant contributor to resource efficiency. The use of genetically improved rainbow trout, instead of the base population of fish, reduces feed costs by 18.3% and total production costs by 7.8% at commercial farms (by -0.266€ per kg of ungutted fish). For phosphorus and nitrogen, it can be assumed that the use of fish material with an improved FCR also leads to 18.3% less nitrogen and phosphorus flowing into an aquatic environment. Such improvements in resource efficiency are win-wins for both industry and the environment-the same amount of seafood can be produced with significantly reduced amounts of raw materials and reduced environmental impact.

Resource efficiency, the ratio of inputs to outputs, is essential for both the economic and environmental performance of aquaculture. The data from commercial rainbow trout farms showed that from 1980 onwards, the farm-level feed conversion ratio (FCR) improved by 53.4%, and the specific nitrogen and phosphorus loading from the farms decreased by over 70%. Hence, to produce 1 kg of fish today, only half of the feed is needed compared to the 1980s. Selective breeding is a major contributor to this improvement, and it has resulted in an estimated genetic gain of 1.74% per generation in the FCR. The use of genetically improved rainbow trout, instead of a base population of fish, reduces feed costs and nutrient loading by 18.3% and total production costs by 7.8% at commercial farms. Such improvements in resource efficiency are win­wins for both industry and the environment­the same amount of seafood can be produced with significantly reduced amounts of raw materials and reduced environmental impact.

Regulation of Δ6Fads2 Gene Involved in LC-PUFA Biosynthesis Subjected to Fatty Acid in Large Yellow Croaker (Larimichthys crocea) and Rainbow Trout (Oncorhynchus mykiss).

Δ6 fatty acyl desaturase (Δ6Fads2) is regarded as the first rate-limiting desaturase that catalyzes the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from 18-carbon fatty acid in vertebrates, but the underlying regulatory mechanism of fads2 has not been comprehensively understood. This study aimed to investigate the regulation role of fads2 subjected to fatty acid in large yellow croaker and rainbow trout. In vivo, large yellow croaker and rainbow trout were fed a fish oil (FO) diet, a soybean oil (SO) diet or a linseed oil (LO) diet for 10 weeks. The results show that LO and SO can significantly increase fads2 expression (p < 0.05). In vitro experiments were conducted in HEK293T cells or primary hepatocytes to determine the transcriptional regulation of fads2. The results show that CCAAT/enhancer-binding protein α (C/EBPα) can up-regulate fads2 expression. GATA binding protein 3 (GATA3) can up-regulate fads2 expression in rainbow trout but showed opposite effect in large yellow croaker. Furthermore, C/EBPα protein levels were significantly increased by LO and SO (p < 0.05), gata3 expression was increased in rainbow trout by LO but decreased in large yellow croaker by LO and SO. In conclusion, we revealed that FO replaced by LO and SO increased fads2 expression through a C/EBPα and GATA3 dependent mechanism in large yellow croaker and rainbow trout. This study might provide critical insights into the regulatory mechanisms of fads2 expression and LC-PUFA biosynthesis.

Effects of salinity acclimation on histological characteristics and miRNA expression profiles of scales in juvenile rainbow trout (Oncorhynchus mykiss).

BACKGROUND: The scales serve as an ideal model for studying the regulatory mechanism of bone homeostasis in fish. To explore the effect of salinity acclimation on bone metabolism of juvenile rainbow trout (Oncorhynchus mykiss), three sampling time points during salinity acclimation (7D, 14D and 21D) were selected to detect variations in histological characteristics. In the histological analysis, osteoblast marker enzymes alkaline phosphatase (ALP), osteoclast marker tartrate-resistant acid phosphatase (TRAcP) and calcium salt deposit areas (Von Kossa's) were detected. Changes in calcium (Ca), phosphorus (P) and the molar mass ratio of calcium to phosphorus (Ca/P) in the scales were also detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In addition, the global MicroRNA (miRNA) expression profiles during salinity acclimation were examined using Illumina sequencing platform because of their important regulatory roles in teleost biological processes. RESULTS: Twelve independent miRNA libraries were constructed, a total of 664 known and 92 putative novel miRNAs were identified. A total of 290 differentially expressed (DE) miRNAs were found in clusters with significant trends in the cluster analysis, and five types of clustering patterns were obtained; 22,374 DE predicted target genes of the aforementioned 290 DE miRNAs were obtained, 5957 of which clustered in six types of clustering patterns with a significant trend. To better understand the functions of the DE miRNAs, GO and KEGG analysis was performed on the 5957 target genes, as a result, they were significantly enriched in bone metabolism related signaling pathways such as MAPK signaling pathway, Calcium signaling pathway, Wnt signaling pathway, Mineral absorption and NF-kappa B signaling pathway. Six DE miRNAs were randomly selected and their expression were verified by quantitative real-time PCR (qRT-PCR), the expression trends were consistent with the results of transcriptome sequencing. CONCLUSIONS: The DE miRNAs and DE target genes identified in this study might play an important role in regulation of bone metabolism during salinity acclimation, relative genes or pathways could serve as key candidates for further studies to elucidate molecular mechanism of teleost bone metabolism, and help performing salinity acclimation and developing marine culture of salmonid species.

Periprandial response of central cannabinoid system to different feeding conditions in rainbow trout Oncorhynchus mykiss.

BACKGROUND: The mechanisms that regulate food intake are very complex since they comprise several neuroendocrine and metabolic signals responding to energetic or reward requirements. Previous studies in mammals indicate that cannabinoid system is implicated in homeostatic and hedonic regulation of food intake. In fish, several studies describe the components of this system, but only a little information is available regarding their role in food intake and energy balance regulation. OBJECTIVES: The objective of this study was to evaluate the main components of cannabinoid system related to feeding conditions in fish. METHODS: Samples of blood and different brain areas (telencephalon and hypothalamus) were taken from rainbow trout under different nutritional status (fasted, fed and refed) at different periprandial times (-30, 0, +30 and +180 min). RESULTS: Changes in AEA and 2-AG levels were observed in plasma related to the nutritional status and the sampling times assessed. At central levels, changes in endocannabinoids levels were observed in hypothalamus and in mRNA abundance of cnr1 and tprv1 in telencephalon and faah, gpr55 and fos in both brain areas. DISCUSSION: The results obtained suggest a role of endocannabinoid system in the regulation of food intake in fish at central level but further studies are required to fully elucidate the mechanisms involved.

Effects of replacing fishmeal with cottonseed protein concentrate on growth performance, blood metabolites, and the intestinal health of juvenile rainbow trout (Oncorhynchus mykiss).

Cottonseed protein concentrate (CPC) is a potential non-food protein source for fishmeal replacement in fish feed. However, a high inclusion level of CPC in diets may have adverse effects on the metabolism and health of carnivorous fish. This study aimed to investigate CPC as a fishmeal alternative in the diet of rainbow trout Oncorhynchus mykiss based on growth performance, blood metabolites, and intestinal health. Five isonitrogenous (46% crude protein) and isolipidic (16% crude lipid) diets were formulated: a control diet (30% fishmeal) and four experimental diets with substitution of fishmeal by CPC at 25%, 50%, 75%, and 100%. A total of 600 fish (mean body weight 11.24g) were hand-fed the five formulated diets to apparent satiation for eight weeks. The results showed no adverse effects on growth performance when 75% dietary fishmeal was replaced by CPC. However, reduced growth and feed intake were observed in rainbow trout fed a fishmeal-free diet based on CPC (CPC100%). Changes in serum metabolites were also observed in CPC100% compared with the control group, including an increase in alanine aminotransferase (ALT), a decrease in alkaline phosphatase (ALP), alterations in free amino acids, and reductions in cholesterol metabolism. In addition, the CPC-based diet resulted in reduced intestinal trypsin, decreased villus height and width in the distal intestine, upregulated mRNA expression levels of inflammatory cytokines in the intestine, and impaired gut microbiota with reduced bacterial diversity and decreased abundance of Bacillaceae compared with the control group. The findings suggest that the optimum substitution rate of dietary fishmeal by CPC for rainbow trout should be less than 75%.

Effect of an Established Nutritional Level of Selenium on Energy Metabolism and Gene Expression in the Liver of Rainbow Trout.

The nutritional selenium (Se) has been demonstrated to have health-boosting effects on fish. However, its effect on fish energy metabolism remains unclear. This study explores the effect and underlying mechanism of the action of nutritional Se on energy metabolism in fish. Rainbow trout (Oncorhynchus mykiss) were fed a basal diet (0 mg Se/kg diet) and a diet containing an already established nutritional Se level (2 mg Se/kg diet, based on Se-yeast) for 30 days. After the feeding experiment, the plasma and liver biochemical profiles and liver transcriptome were analyzed. The results showed that the present nutritional level of Se significantly increased liver triglyceride, total cholesterol, and plasma total cholesterol contents (P < 0.05) compared with the control. Transcriptome analysis showed that 336 and 219 genes were significantly upregulated and downregulated, respectively. Gene enrichment analysis showed that many differentially expressed genes (DEGs) were associated with lipid metabolism pathways (fatty acid biosynthesis, fatty acid elongation, and unsaturated fatty acid biosynthesis), carbohydrate metabolism pathways (glycolysis, the pentose phosphate pathway, and the citrate cycle), and the oxidative phosphorylation pathway. Real-time quantitative PCR (Q-PCR) validation results showed that the expression profiles of 15 genes exhibited similar trends both in RNA sequencing (RNA-seq) and Q-PCR analysis. These results reveal that optimum dietary Se activates glucose catabolic processes, fatty acid biosynthetic processes, and energy production and hence produces higher liver lipid content. This study concludes that the previously established level of nutritional Se (Se-yeast) (2 mg/kg diet, fed basis) for rainbow trout promotes energy storage in the liver, which may benefit fish growth to some extent.

Dietary effects of a low-molecular weight fraction (<10 kDa) from shrimp waste hydrolysate on growth performance and immunity of rainbow trout (Oncorhynchus mykiss): Employing nanodelivery systems.

Aquaculture by-products have been of great interest for producing protein hydrolysates with multiple biological activities. The present experiment was carried out to evaluate dietary effects of a low-molecular fraction (<10 kDa) from shrimp waste hydrolysate in forms of unprotected and nanocapsulated on growth and immunity of rainbow trout. Therefore, six diets were designed including a control diet (no supplementation), D1 (1 g kg-1 of unprotected fraction), D2 (1 g kg-1 chitosan nanocapsules), D3 (1 g kg-1 liposome nanocapsules), D4 (1 g kg-1 of fraction-loaded chitosan nanocapsules), D5 (1 g kg-1 of fraction-loaded liposome nanocapsules). Fish (0.91 ± 0.15 g) were fed with experimental diets until apparent satiation for six weeks followed by a 5-day experimental challenge with Streptococcus iniae. Results revealed that growth is strongly affected in fish receiving the fraction with D4 treatment showing the highest weight gain, SGR, final weight and the lowest FCR (p < 0.05). Nanocapsules without fraction did not show remarkable effects when compared to control group. In terms of serum and mucus immune parameters of lysozyme, complement activity, myeloperoxidase activity, and total protease, fish from D4 group showed the highest measured values followed by D5 (p < 0.05). Key immune related genes of IL-6 and TNF-α were noticeably up-regulated in fish from D1, D4, and D5 groups, which were consistent with survival rate after 5 days challenge with Streptococcus iniae. All together, the present findings highlighted the application of chitosan and liposome nanocarriers in aquaculture and potential of low-molecular weight fraction (<10 kDa) from shrimp wastes hydrolysate to improve growth performance and immune status of rainbow trout.

Cutaneous mucosal immune-parameters and intestinal immune-relevant genes expression in streptococcal-infected rainbow trout (Oncorhynchus mykiss): A comparative study with the administration of florfenicol and olive leaf extract.

This study evaluated changes in cutaneous mucosal immunity (total protein (TP) and immunoglobulin (TIg), lysozyme, protease, esterase, and alkaline phosphatase (ALP)) and some immune-related genes expression (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-8, hepcidin-like antimicrobial peptides (HAMP), and immunoglobulin M (IgM)) in the intestine of rainbow trout (Oncorhynchus mykiss) orally-administrated florfenicol (FFC) and/or olive leaf extract (OLE), experimentally infected with Streptococcus iniae. The juvenile fish (55 ± 7.6 g) were divided into different groups according to the use of added OLE (80 g kg-1 food), the presence/absence of FFC (15 mg kg-1 body weight for 10 consecutive days), and the streptococcal infectivity (2.87 × 107 CFU mL-1 as 30% of LD50-96h). The extract's chemical composition was analyzed using the high-performance liquid chromatography (HPLC) system. The skin mucus and intestine of fish were sampled after a 10-day therapeutic period for all groups, and their noted indices were measured. Our results signified that the oleuropein, quercetin, and trans-ferulic acid were the most obvious active components of OLE which were found by HPLC analysis. The combined use of OLE and FFC could lowered some skin mucus immunological indices (e.g., TP, TIg, and ALP), and the gene expression of inflammatory cytokines (e.g., TNF-α and IL-1ß) of rainbow trout. Moreover, lysozyme and protease activities respectively were invigorated by the FFC and OLE treatment. Also, the use of OLE as a potential medicine induced the gene expression of HAMP. As the prevention approach, it would be recommended to find the best dose of OLE alone or in combination with the drug through therapeutics period before the farm involved in the streptococcal infection.

Administration of grape (Vitis vinifera) seed extract to rainbow trout (Oncorhynchus mykiss) modulates growth performance, some biochemical parameters, and antioxidant-relevant gene expression.

Grape seed, as a main source of polyphenols, has many nutritional and medicinal properties in humans. In the current study, the effects of dietary ethanolic grape seed extract (GSE) on the growth performance, antioxidant activity, and some biochemical parameters in rainbow trout were investigated. Ninety fish (initial weight 78.47 g) were randomly distributed among nine cement tanks (1.8 m × 0.22 m × 0.35 m) with 10 fish per tank. Three experimental diets containing either 0, 10, or 50 g kg-1 GSE were prepared and each diet was randomly assigned to three tanks of fish for 60 days. Results showed that feeding GSE enhanced some growth parameters including the specific growth rate and condition factor in comparison with the control group. Among different serum metabolites, the glucose levels in treatment groups significantly decreased compared to the control group. The total product of lipid peroxidation indicated as malondialdehyde significantly decreased in both the GSE-added treatment groups. The gene expression related to the antioxidant enzymes, catalase, glutathione peroxidase 1, and glutathione S-transferase A, were upregulated in the intestine of fish that received a low dose of GSE. The results of the current study suggest that GSE, especially at 10 g kg-1, diet had the potential to improve (1) specific growth rate and condition factor, (2) biochemical parameters including glucose and lipid peroxidation product, and (3) upregulated the expression of antioxidant genes including catalase, glutathione peroxidase 1, and glutathione S-transferase A in rainbow trout.

Humoral and skin mucosal immune parameters, intestinal immune related genes expression and antioxidant defense in rainbow trout (Oncorhynchus mykiss) fed olive (Olea europea L.) waste.

A six-week feeding trial was carried out to evaluate the effects of inclusion of dietary olive waste cake (OWC, 0, 0.5, 2.5 and 5 g kg-1 diet) on performance, antioxidant condition and immune responses of rainbow trout (Oncorhynchus mykiss) (2.5 ± 0.1 g). Supplementing diet with 2.5 and 5 g OWC kg-1 diet significantly enhanced serum and mucosal lysozyme activity in fish. Regarding mucosal immunity, fish fed 2.5 and 5 g OWC kg-1 diets had higher skin mucus total Ig concentrations than other groups. In relation to antioxidant status, those in 2.5 g OWC kg-1 and the control groups exhibited the highest and the least liver superoxide dismutase and glutathione peroxidase activities, respectively. Furthermore, the activity of liver glutathione S transferase in fish fed 2.5 and 5 g OWC kg-1 diets was higher than the other treatments. In respect to gut cytokines gene expression, our findings demonstrated dietary OWC did not influence interlukines-1ß and 10 genes expression, but relative expression of IL8 gene gradually up-regulated with increasing dietary OWC level. Moreover, fish fed 0.5 g OWC kg-1 and the control diets had the highest and the lowest gut tumor necrosis factor-α gene expression values, respectively. The relative expression of transforming growth factor-ß significantly down-regulated in gut of fish fed 2.5 and 5 g OWC kg-1 diets compared to other groups. Supplementing diet with OWC pronouncedly improved growth and feed conversion ratio in fish compared to the control. Overall, the findings of this study suggested that inclusion of 2.5 g OWC kg-1 diet can improve growth rate, oxidative stress status, humoral and skin mucosal immune responses in O. mykiss fingerlings and it can be considered as a functional feed additive for this species.

Supplementation of arginine, ornithine and citrulline in rainbow trout (Oncorhynchus mykiss): Effects on growth, amino acid levels in plasma and gene expression responses in liver tissue.

Functional amino acids (FAA) regulate metabolic pathways directly linked to health, survival, growth and development. Arginine is a FAA with crucial roles in protein deposition and the immune response. In mammals, supplementation of arginine's precursor amino acid, citrulline, is known to increase circulating arginine to levels beyond direct arginine supplementation, however, citrulline supplementation is poorly studied in fish. To address this knowledge gap, we supplemented the diet of rainbow trout with arginine and its precursor amino acids, ornithine and citrulline, at 3 levels (0.5%, 1% and 2% of the total diet) during a 14-week experiment. We sampled fish at 3 h and 24 h post-feeding to investigate immediate and steady-state effects, respectively. There were no differences in fish growth for any of the diets across a range of indicators. In blood plasma, out of 26 amino acids detected, 11 and 6 displayed significant changes 24 h and 3 h post-prandial, respectively. Arginine, ornithine and citrulline levels were all significantly increased by the citrulline supplemented diets. In muscle, 8 amino acids were significantly altered by supplemented diets, while there were no significant changes in liver. Arginine was increased by 2% citrulline supplementation in muscle tissue. We also investigated the transcriptional responses of urea cycle, nitric oxide cycle and rate-limiting polyamine synthesis enzymes, related to arginine's metabolism, in liver. At both time points, only 2 enzymes were significantly altered by the supplemented diets, however several significant changes were observed comparing 3 h and 24 h post-prandial expression levels. Of these, the paralogous polyamine synthesis enzyme encoding genes ODC1 and ODC2 displayed the largest increases in 3 h post-prandial fish. These findings demonstrate that endogenous synthesis of arginine is possible from a citrulline supplemented diet and improve our understanding of arginine metabolism in fish.

SIRT1 mediates the effect of stress on hypothalamic clock genes and food intake regulators in rainbow trout, Oncorhynchus mykiss.

Stress negatively affects a wide range of physiological and behavioural functions (circadian physiology and food intake, among others), thus compromising animal welfare. Cortisol mediates the effect of stress on food intake, but other mediators (such as sirtuins) may participate in that related to circadian physiology. We evaluated 1) the effect of stress on the day-night variation of hypothalamic clock genes and food intake regulators, 2) changes of mRNA abundance in cortisol biosynthesis at the head kidney, and 3) changes of glucocorticoid receptors in both tissues of rainbow trout, together with the involvement of SIRT1 in such effect. Trout receiving or not SIRT1 inhibitor (EX527) and subjected or not to stress by high stocking density (72 h), were sampled at day- (ZT10) and night-time (ZT18). Our results indicate that SIRT1 mediates the effect of stress on mRNA abundance of clock genes in trout hypothalamus, but it also influences those changes occurring on food intake-related peptides. High stocking density inhibits clock genes expression, but enhances that of food intake-related peptides. EX527 treatment prevents stress-related changes observed in clock genes, thus evidencing a key role played by SIRT1 in mediating this effect on trout circadian oscillators. On the other hand, EX527 treatment partially prevents changes of food intake-related peptides, indicating that an interaction between SIRT1 and other mediators (such as cortisol) exists during response to stress. In support of that, our results reveal that SIRT1 influences cortisol biosynthesis during stress. Whatever the case is, further research will help understanding the underlying mechanisms involved.