Effect of prolonged feeding of broodstock diet with increased inclusion of essential n-3 fatty acids on maturing and spawning performance in 3-year-old Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) broodstock recruits are normally fed a specialized diet with a higher content of essential nutrients for a limited time period prior to fasting and transfer to freshwater. Typically, this period lasts for about six months, but may vary among producers. Reduced use of marine ingredients in commercial salmon diets during the last decades has affected the content of essential nutrients, such as n-3 long chained polyunsaturated fatty acids (LC-PUFA), minerals and vitamins. Furthermore, to minimize the risk of losses and implement new breeding achievements faster, breeding companies have shortened the production cycle of broodstock from 4 to 3 years, which may affect the number of fish that are large enough to mature. In the present study, we have extended the broodstock feeding period from 6 to 15 months prior to the freshwater transfer giving a higher content of n-3 LC-PUFA (higher inclusion of marine oils) from February to December (Phase 1), and thereafter a diet with a higher energy content to ensure growth towards the spring and maturation (Phase 2). Four sea cages with approximately 80.000 salmon postsmolt, two sea cages with males and two with females, were given a control diet and an experimental diet. Samples were taken in Phase 1 at start (1.7 kg), mid (3.4 kg) and end Phase 1/start of Phase 2 (8.3 kg), and end of Phase 2 (13.4 kg). The fish were thereafter fasted, and selected fish transferred to landbased freshwater tanks where light and temperature were used to manipulate the spawning time of the fish in two groups (early or late). Due to disease in the facility, measures of egg quality and hatching were only obtained from the early group. During the trial and spawning period, biometrical measurements were recorded, and samples of liver, gonad, fillet and red blood cells (RBC) were collected for fatty acid composition and blood plasma for analysis of lipid and health-related parameters. Samples were also collected for gonadal transcriptomic analysis by microarray and qPCR (end Phase 2) and plasma steroids (end Phase 2, mid maturation and spawning). Males fed the test diet had a larger body size compared to the control group at the end of Phase 2, while no differences were observed between dietary groups for the females. Total mortality in the trial was lower in the test group compared to the control, losses were caused mainly by sea lice treatments, loser fish or cardiomyopathy syndrome (CMS). The dietary LC-PUFA levels in the test diet were reflected in the tissues particularly during Phase 1, but only different in the fillet samples and eggs at the end of Phase 2 and at spawning. Plasma sex steroids content increased at mid maturation and showed lower levels of androgens and estrogens in females fed the test diet compared to the control. At the end of Phase 2, transcriptional analysis showed upregulation of steroidogenic enzymes, although not reflected in changes in plasma steroids in Phase 2, indicating changes to come during maturation. The differences in LC-PUFA content in tissues and plasma steroids did not appear to affect fecundity, sperm quality, egg survival or hatching rate, but the test group had larger eggs compared to the control in the early spawner-group. Prolonged feeding of n-3 LC-PUFA to pre-puberty Atlantic salmon broodstock appears to be important for higher survival in challenging sea cage environments and has an effect on sex steroid production that, together with high energy diet during early maturation, cause the test group to produce larger eggs.

Calanus finmarchicus hydrolysate improves growth performance in feeding trial with European sea bass juveniles and increases skeletal muscle growth in cell studies.

The world will be dependent on the development of novel feed ingredients from renewable sources to ensure sustainable growth of the aquaculture industry. Zooplankton like Calanus finmarchicus are viable new raw material candidates, as they have optimal nutrient profiles for aquatic animals and may be sustainably harvested in large volumes. In this study, the aim was to investigate if a protein hydrolysate of C. finmarchicus was able to influence the growth performance of fish. The effect of dietary inclusion of hydrolysates was tested in a feeding trial with European sea bass (Dicentrarchus labrax) juveniles, benchmarking calanus hydrolysate (CH) against commercially available hydrolysates. The diet with CH inclusion yielded increased growth, with significantly higher body weight than hydrolysates of sardine and tuna fish at the end of the trial. The observed growth-promoting effects were further examined using an in vitro model with skeletal muscle cells from Atlantic salmon. Through bioactivity experiments with muscle cells grown in media containing CH, low-molecular fractions were found to have the greatest positive effect on proliferation, viability, and expression of muscle-specific genes. Characterization of the most potent fraction revealed an abundance of small peptides, along with amino acids and marine metabolites associated with increased muscle growth.

Differential Expression of miRNAs and Their Predicted Target Genes Indicates That Gene Expression in Atlantic Salmon Gill Is Post-Transcriptionally Regulated by miRNAs in the Parr-Smolt Transformation and Adaptation to Sea Water.

Smoltification (parr-smolt transformation) is a complex developmental process consisting of developmental changes that lead to remodeling of the Atlantic salmon gill. Here, the expression changes of miRNAs and mRNAs were studied by small-RNA sequencing and microarray analysis, respectively, to identify miRNAs and their predicted targets associated with smoltification and subsequent sea water adaptation (SWA). In total, 18 guide miRNAs were identified as differentially expressed (gDE miRNAs). Hierarchical clustering analysis of expression changes divided these into one cluster of 13 gDE miRNAs with decreasing expression during smoltification and SWA that included the miRNA-146, miRNA-30 and miRNA-7132 families. Another smaller cluster that showed increasing expression consisted of miR-101a-3p, miR-193b-5p, miR-499a-5p, miR-727a-3p and miR-8159-5p. The gDE miRNAs were predicted to target 747 of the genes (DE mRNAs), showing expression changes in the microarray analysis. The predicted targets included genes encoding NKA-subunits, aquaporin-subunits, cystic fibrosis transmembrane conductance regulator and the solute carrier family. Furthermore, the predicted target genes were enriched in biological processes associated with smoltification and SWA (e.g., immune system, reactive oxygen species, stress response and extracellular matrix organization). Collectively, the results indicate that remodeling of the gill involves the post-transcriptional regulation of gene expression by the characterized gDE miRNAs.

Expression Analysis in Atlantic Salmon Liver Reveals miRNAs Associated with Smoltification and Seawater Adaptation.

Optimal smoltification is crucial for normal development, growth, and health of farmed Atlantic salmon in seawater. Here, we characterize miRNA expression in liver to reveal whether miRNAs regulate gene expression during this developmental transition. Expression changes of miRNAs and mRNAs was studied by small-RNA sequencing and microarray analysis, respectively. This revealed 62 differentially expressed guide miRNAs (gDE-miRNAs) that could be divided into three groups with characteristic dynamic expression patterns. Three of miRNA families are known as highly expressed in liver. A rare arm shift was observed during smoltification in the Atlantic salmon-specific novel-ssa-miR-16. The gDE-miRNAs were predicted to target 2804 of the genes revealing expression changes in the microarray analysis. Enrichment analysis revealed that targets were significantly enriched in smoltification-associated biological process groups. These included lipid and cholesterol synthesis, carbohydrate metabolism, protein metabolism and protein transport, immune system genes, circadian rhythm and stress response. The results indicate that gDE-miRNAs may regulate many of the changes associated with this developmental transition in liver. The results pave the way for validation of the predicted target genes and further study of gDE-miRNA and their targets by functional assays.

Modulation of hepatic miRNA expression in Atlantic salmon (Salmo salar) by family background and dietary fatty acid composition.

This study finds significant differences in hepatic fatty acid composition between four groups of Atlantic salmon (Salmo salar) consisting of offspring from families selected for high and low capacities to express the delta 6 desaturase isomer b and fed diets with 10% or 75% fish oil. The results demonstrated that hepatic lipid metabolism was affected by experimental conditions (diet/family). The fatty acid composition in the four groups mirrored the differences in dietary composition, but it was also associated with the family groups. Small RNA sequencing followed by RT-qPCR identified 12 differentially expressed microRNAs (DE miRNAs), with expression associated with family groups (miR-146 family members, miR-200b, miR-214, miR-221, miR-125, miR-135, miR-137, miR_nov_1), diets (miR-203, miR-462) or both conditions. All the conserved DE miRNAs have been reported as associated with lipid metabolism in other vertebrates. In silico predictions revealed 37 lipid metabolism pathway genes, including desaturases, transcription factors and key enzymes in the synthesis pathways as putative targets (e.g., srebp-1 and 2, Δ6fad_b and c, hmdh, elovl4 and 5b, cdc42). RT-qPCR analysis of selected target genes showed expression changes that were associated with diet and with family groups (d5fad, d6fad_a, srebp-1). There was a reciprocal difference in the abundance of ssa-miR-203a-3p and srebp-1 in one group comparison, whereas other predicted targets did not reveal any evidence of being negatively regulated by degradation. More experimental studies are needed to validate and fully understand the predicted interactions and how the DE miRNAs may participate in the regulation of hepatic lipid metabolism.

Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2.

Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.

Molecular cloning of the Atlantic salmon activin receptor IIB cDNA - Localization of the receptor and myostatin in vivo and in vitro in muscle cells.

In mammals, the activin receptor type IIB (ActRIIB) binds with high affinity several members of the transforming growth factor-beta (TGF-beta) superfamily, including the negative muscle regulator myostatin (MSTN). In this study, an actRIIB cDNA of 1443 bp was isolated by reverse transcription (RT)-PCR from the liver of Atlantic salmon (Salmo salar) encoding almost the complete receptor. The deduced salmon ActRIIB of 481 amino acids (aa) contained the conserved catalytic domain of serine/threonine protein kinases, and showed the highest sequence identity (83-87%) to the zebrafish, chicken and goldfish ActRIIB. Salmon actRIIB mRNA was identified by RT-PCR in all the examined tissues of juvenile fish that was confirmed by in situ hybridization. In comparison, the salmon MSTN signal was less widespread, and co-expression of the receptor and this putative ligand was only demonstrated in skeletal muscle. Consistently, both ActRIIB and MSTN were immunocytologically identified in salmon myoblasts and differentiated myotubes in culture.