Direct actions of growth hormone in rainbow trout, Oncorhynchus mykiss, skeletal muscle cells in vitro.

The growth hormone (GH)-insulin-like growth factor-1 (IGF-1) system regulates skeletal muscle growth and function. GH has a major function of targeting the liver to regulate IGF-1 production and release, and IGF-1 mediates the primary anabolic action of GH on growth. However, skeletal muscle is a target tissue of GH as evidenced by dynamic GH receptor expression, but it is unclear if GH elicits any direct actions on extrahepatic tissues as it is difficult to distinguish the effects of IGF-1 from GH. Fish growth regulation is complex compared to mammals, as genome duplication events have resulted in multiple isoforms of GHs, GHRs, IGFs, and IGFRs expressed in most fish tissues. This study investigated the potential for GH direct actions on fish skeletal muscle using an in vitro system, where rainbow trout myogenic precursor cells (MPCs) were cultured in normal and serum-deprived media, to mimic in vivo fasting conditions. Fasting reduces IGF-1 signaling in the muscle, which is critical for disentangling the roles of GH from IGF-1. The direct effects of GH were analyzed by measuring changes in myogenic proliferation and differentiation genes, as well as genes regulating muscle growth and proteolysis. This study provides the first in-depth analysis of the direct actions of GH on serum-deprived fish muscle cells in vitro. Data suggest that GH induces the expression of markers for proliferation and muscle growth in the presence of serum, but all observed GH action was blocked in serum-deprived conditions. Additionally, serum deprivation alone reduced the expression of several proliferation and differentiation markers, while increasing growth and proteolysis markers. Results also demonstrate dynamic gene expression response in the presence of GH and a JAK inhibitor in serum-provided but not serum-deprived conditions. These data provide a better understanding of GH signaling in relation to serum in trout muscle cells in vitro.

Characterization of a Leptin Receptor Paralog and Its Response to Fasting in Rainbow Trout (Oncorhynchus mykiss).

Leptin is a cytokine that regulates appetite and energy expenditure, where in fishes it is primarily produced in the liver and acts to mobilize carbohydrates. Most fishes have only one leptin receptor (LepR/LepRA1), however, paralogs have recently been documented in a few species. Here we reveal a second leptin receptor (LepRA2) in rainbow trout that is 77% similar to trout LepRA1. Phylogenetic analyses show a salmonid specific genome duplication event as the probable origin of the second LepR in trout. Tissues distributions showed tissue specific expression of these receptors, with lepra1 highest in the ovaries, nearly 50-fold higher than lepra2. Interestingly, lepra2 was most highly expressed in the liver while hepatic lepra1 levels were low. Feed deprivation elicited a decline in plasma leptin, an increase in hepatic lepra2 by one week and remained elevated at two weeks, while liver expression of lepra1 remained low. By contrast, muscle lepra1 mRNA increased at one and two weeks of fasting, while adipose lepra1 was concordantly lower in fasted fish. lepra2 transcript levels were not affected in muscle and fat. These data show lepra1 and lepra2 are differentially expressed across tissues and during feed deprivation, suggesting paralog- and tissue-specific functions for these leptin receptors.

Distinct microbial assemblages associated with genetic selection for high- and low- muscle yield in rainbow trout.

BACKGROUND: Fish gut microbial assemblages play a crucial role in the growth rate, metabolism, and immunity of the host. We hypothesized that the gut microbiota of rainbow trout was correlated with breeding program based genetic selection for muscle yield. To test this hypothesis, fecal samples from 19 fish representing an F2 high-muscle genetic line (ARS-FY-H) and 20 fish representing an F1 low-muscle yield genetic line (ARS-FY-L) were chosen for microbiota profiling using the 16S rRNA gene. Significant differences in microbial assemblages between these two genetic lines might represent the effect of host genetic selection in structuring the gut microbiota of the host. RESULTS: Tukey's transformed inverse Simpson indices indicated that high muscle yield genetic line (ARS-FY-H) samples have higher microbial diversity compared to those of the low muscle yield genetic line (ARS-FY-L) (LMM, χ2(1) =14.11, p < 0.05). The fecal samples showed statistically distinct structure in microbial assemblages between the genetic lines (F1,36 = 4.7, p < 0.05, R2 = 11.9%). Functional profiling of bacterial operational taxonomic units predicted characteristic functional capabilities of the microbial communities in the high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic line samples. CONCLUSION: The significant differences of the microbial assemblages between high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic lines indicate a possible effect of genetic selection on the microbial diversity of the host. The functional composition of taxa demonstrates a correlation between bacteria and improving the muscle accretion in the host, probably, by producing various metabolites and enzymes that might aid in digestion. Further research is required to elucidate the mechanisms involved in shaping the microbial community through host genetic selection.

Analysis of the fecal microbiota of fast- and slow-growing rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that fecal samples representing the gut microbiota of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiota can improve the profitability of aquaculture. The first objective of this study was to test this hypothesis if gut microbiota is associated with fish growth rate (body weight). Four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family for 16S rRNA microbiota profiling. Microbiota diversity varies with different DNA extraction methods. The second objective of this study was to compare the effects of five commonly used DNA extraction methods on the microbiota profiling and to determine the most appropriate extraction method for this study. These methods were Promega-Maxwell, Phenol-chloroform, MO-BIO, Qiagen-Blood/Tissue, and Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. RESULTS: Differences in DNA extraction methods resulted in significant variation in the identification of bacteria that compose the gut microbiota. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. However, an indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia, and Peptostreptococcus) are indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as indicator taxa for the slow-growing fish. CONCLUSION: DNA extraction methodology should be carefully considered for accurate profiling of the gut microbiota. Although the microbiota was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

Essential amino acids exhibit variable effects on protein degradation in rainbow trout (Oncorhynchus mykiss) primary myocytes.

The functional role of amino acids as regulators of protein degradation was investigated using primary myogenic precursor cell culture as in vitro model of rainbow trout white muscle. Seven-day old myocytes were starved of amino acids for two hours then exposed to media that contained amino acid treatments, during which protein degradation rates were analyzed over five hours by measuring cellular release of 3H-tyrosine. Increasing concentrations of essential amino acids (EAA) reduced protein degradation rates; this effect was dose-dependent within the physiological range found in plasma. Addition of leucine or phenylalanine at 5 mM and 2.5 mM, respectively, decreased rates of protein degradation compared to media without amino acid supplementation, suggesting that these amino acids directly regulate muscle proteolysis. Protein degradation rates were similar in cells exposed to media without EAA and media lacking only leucine, further supporting a role for leucine as a central regulator of protein turnover. Addition of 5 mM lysine or valine to media without amino acids increased protein degradation; this response was attenuated as EAA were added back into media, supporting that a lysine or valine imbalance is costly for muscle protein retention. In summary, there is evidence for amino acids as both positive and negative regulators of protein turnover in rainbow trout muscle. These findings suggest that there may be an optimal plasma amino acid profile that minimizes protein turnover and that this could be achieved through diet formulation.

Effects of sex steroids on expression of genes regulating growth-related mechanisms in rainbow trout (Oncorhynchus mykiss).

Effects of a single injection of 17ß-estradiol (E2), testosterone (T), or 5ß-dihydrotestosterone (DHT) on expression of genes central to the growth hormone (GH)/insulin-like growth factor (IGF) axis, muscle-regulatory factors, transforming growth factor-beta (TGFß) superfamily signaling cascade, and estrogen receptors were determined in rainbow trout (Oncorhynchus mykiss) liver and white muscle tissue. In liver in addition to regulating GH sensitivity and IGF production, sex steroids also affected expression of IGF binding proteins, as E2, T, and DHT increased expression of igfbp2b and E2 also increased expression of igfbp2 and igfbp4. Regulation of this system also occurred in white muscle in which E2 increased expression of igf1, igf2, and igfbp5b1, suggesting anabolic capacity may be maintained in white muscle in the presence of E2. In contrast, DHT decreased expression of igfbp5b1. DHT and T decreased expression of myogenin, while other muscle regulatory factors were either not affected or responded similarly for all steroid treatments. Genes within the TGFß superfamily signaling cascade responded to steroid treatment in both liver and muscle, suggesting a regulatory role for sex steroids in the ability to transmit signals initiated by TGFß superfamily ligands, with a greater number of genes responding in liver than in muscle. Estrogen receptors were also regulated by sex steroids, with era1 expression increasing for all treatments in muscle, but only E2- and T-treatment in liver. E2 reduced expression of erb2 in liver. Collectively, these data identify how physiological mechanisms are regulated by sex steroids in a manner that promotes the disparate effects of androgens and estrogens on growth in salmonids.

Differences in growth, fillet quality, and fatty acid metabolism-related gene expression between juvenile male and female rainbow trout.

Sexual maturation occurs at the expense of stored energy and nutrients, including lipids; however, little is known regarding sex effects on nutrient regulatory mechanisms in rainbow trout prior to maturity. Thirty-two, 14-month-old, male and female rainbow trout were sampled for growth, carcass yield, fillet composition, and gene expression of liver, white muscle, and visceral adipose tissue. Growth parameters, including gonadosomatic index, were not affected by sex. Females had higher percent separable muscle yield, but there were no sex effects on fillet proximate composition. Fillet shear force indicated females produce firmer fillets than males. Male livers had greater expression of three cofactors within the mTOR signaling pathway that act to inhibit TORC1 assembly; mo25, rictor, and pras40. Male liver also exhibited increased expression of ß-oxidation genes cpt1b and ehhadh. These findings are indicative of increased mitochondrial ß-oxidation in male liver. Females exhibited increased expression of the mTOR cofactor raptor in white muscle and had higher expression levels of several genes within the fatty acid synthesis pathway, including gpat, srebp1, scd1, and cd36. Female muscle also had increased expression of ß-oxidation genes cpt1d and cpt2. Increased expression of both fatty acid synthesis and ß-oxidation genes suggests female muscle may have greater fatty acid turnover. Differences between sexes were primarily associated with variation of gene expression within the mTOR signaling pathway. Overall, data suggest there is differential regulation of gene expression in male and female rainbow trout tissues prior to the onset of sexual maturity that may lead to nutrient repartitioning during maturation.

Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes.

The ubiquitin-proteasome system (UPS) is recognized as the major contributor to total proteolysis in mammalian skeletal muscle, responsible for 50% or more of total protein degradation in skeletal muscle, whereas the autophagic-lysosome system (ALS) plays a more minor role. While the relative contribution of these systems to muscle loss is well documented in mammals, little is known in fish species. The current study uses myotubes derived from rainbow trout myogenic precursor cells as an in vitro model of white muscle tissue. Cells were incubated in complete or serum-deprived media or media supplemented with insulin-like growth factor-1 (IGF-1) and exposed to selective proteolytic inhibitors to determine the relative contribution of the ALS and UPS to total protein degradation in myotubes in different culture conditions. Results indicate that the ALS is responsible for 30-34% and 50% of total protein degradation in myotubes in complete and serum-deprived media, respectively. The UPS appears to contribute much less to total protein degradation at almost 4% in cells in complete media to nearly 17% in serum-deprived cells. IGF-1 decreases activity of both systems, as it inhibited the upregulation of both proteolytic systems induced by serum deprivation. The combined inhibition of both the ALS and UPS reduced degradation by a maximum of 55% in serum-deprived cells, suggesting an important contribution of other proteolytic systems to total protein degradation. Collectively, these data identify the ALS as a potential target for strategies aimed at improving muscle protein retention and fillet yield through reductions in protein degradation.

Effects of triploidy on growth and protein degradation in skeletal muscle during recovery from feed deprivation in juvenile rainbow trout (Oncorhynchus mykiss).

Identifying physiological differences between diploid and triploid rainbow trout will help define how ploidy affects mechanisms that impact growth and nutrient utilization. Juvenile diploid and triploid female rainbow trout (Oncorhynchus mykiss) were either continually fed or fasted for one week, followed by four weeks of refeeding, and indices of growth and proteolysis-related gene expression in skeletal muscle were measured. Fasting reduced growth, and based on gene expression analysis, increased capacity for protein degradation. Regardless of feeding treatment, triploids displayed slightly greater feed intake and specific growth rates than diploids. Continually fed triploids displayed lower expression of several autophagy-related genes than diploids, suggesting that reduced rates of protein degradation contributed to their faster growth. Reduced expression of ubiquitin ligases fbxo32 and fbxo25 and autophagy-related genes during refeeding implicates reduced proteolysis in recovery growth. At one week of refeeding triploids exhibited greater gains in eviscerated body weight and length, whereas diploids exhibited greater gains in gastrointestinal tract weights. During refeeding two autophagy-related genes, atg4b and lc3b, decreased within one week to continually fed levels in the triploids, but in diploids overshot in expression at one and two weeks of refeeding then rebounding above continually fed levels by week four, suggesting a delayed return to basal levels of proteolysis.

Leptin Receptor Deficiency Results in Hyperphagia and Increased Fatty Acid Mobilization during Fasting in Rainbow Trout (Oncorhynchus mykiss).

Leptin is a pleiotropic hormone known for regulating appetite and metabolism. To characterize the role of leptin signaling in rainbow trout, we used CRISPR/Cas9 genome editing to disrupt the leptin receptor (LepR) genes, lepra1 and lepra2. We compared wildtype (WT) and mutant fish that were either fed to satiation or feed deprived for six weeks. The LepR mutants exhibited a hyperphagic phenotype, which led to heavier body weight, faster specific growth rate, increased viscero- and hepatosomatic indices, and greater condition factor. Muscle glycogen, plasma leptin, and leptin transcripts (lepa1) were also elevated in fed LepR mutant fish. Expression levels of several hypothalamic genes involved in feed regulation were analyzed (agrp, npy, orexin, cart-1, cart-2, pomc-a1, pomc-b). No differences were detected between fed WT and mutants except for pomc-b (proopiomelanocortin-b), where levels were 7.5-fold higher in LepR fed mutants, suggesting that pomc-b expression is regulated by leptin signaling. Fatty acid (FA) content did not statistically differ in muscle of fed mutant fish compared to WT. However, fasted mutants exhibited significantly lower muscle FA concentrations, suggesting that LepR mutants exhibit increased FA mobilization during fasting. These data demonstrate a key role for leptin signaling in lipid and energy mobilization in a teleost fish.

Generation of a reference transcriptome for evaluating rainbow trout responses to various stressors.

BACKGROUND: Fish under intensive culture conditions are exposed to a variety of acute and chronic stressors, including high rearing densities, sub-optimal water quality, and severe thermal fluctuations. Such stressors are inherent in aquaculture production and can induce physiological responses with adverse effects on traits important to producers and consumers, including those associated with growth, nutrition, reproduction, immune response, and fillet quality. Understanding and monitoring the biological mechanisms underlying stress responses will facilitate alleviating their negative effects through selective breeding and changes in management practices, resulting in improved animal welfare and production efficiency. RESULTS: Physiological responses to five treatments associated with stress were characterized by measuring plasma lysozyme activity, glucose, lactate, chloride, and cortisol concentrations, in addition to stress-associated transcripts by quantitative PCR. Results indicate that the fish had significant stressor-specific changes in their physiological conditions. Sequencing of a pooled normalized transcriptome library created from gill, brain, liver, spleen, kidney and muscle RNA of control and stressed fish produced 3,160,306 expressed sequence tags which were assembled and annotated. SNP discovery resulted in identification of ~58,000 putative single nucleotide polymorphisms including 24,479 which were predicted to fall within exons. Of these, 4907 were predicted to occupy the first position of a codon and 4110 the second, increasing the probability to impact amino acid sequence variation and potentially gene function. CONCLUSION: We have generated and characterized a reference transcriptome for rainbow trout that represents multiple tissues responding to multiple stressors common to aquaculture production environments. This resource compliments existing public transcriptome data and will facilitate approaches aiming to evaluate gene expression associated with stress in this species.

Effects of sex steroids on indices of protein turnover in rainbow trout (Oncorhynchusmykiss) white muscle.

Effects of 17ß-estradiol (E2), testosterone, and 5α-dihydrotestosterone (DHT) on protein turnover and proteolytic gene expression were determined in rainbow trout (Oncorhynchus mykiss) primary myocytes and white muscle tissue. E2 reduced rates of protein synthesis and increased rates of protein degradation in primary myocytes by 45% and 27%, respectively. DHT reduced rates of protein synthesis by 27%. Testosterone did not affect protein synthesis and neither testosterone nor DHT affected rates of protein degradation. Single injections of E2 increased expression of ubiquitin ligase genes fbxo32, fbxo25, and murf1, and the proteasome subunit psmd6 by 24h after injection. Within the cathepsin-lysosome pathway, E2 increased expression of cathepsins ctsd and ctsl, as well as autophagy-related genes atg4b and lc3b. Additionally, E2 injection up-regulated the expression of casp3 and casp9 caspase genes. Incubation of primary myocytes with E2 also increased expression of ubiquitin ligase genes. Therefore, catabolic effects of E2 on protein turnover result in part from E2-induced increases in proteolytic gene expression directly in muscle. Injection of testosterone increased milli-calpain (capn2) and casp3 expression, and DHT increased ctsd expression in vivo, whereas both androgens up-regulated fbxo32 expression in primary myocytes. These results suggest that effects of androgens on protein turnover in muscle are not driven primarily by direct effects of these hormones in this tissue.

Hepatic Fatty Acid and Transcriptome Profiles during the Transition from Vegetable- to Fish Oil-Based Diets in Rainbow Trout (Oncorhynchus mykiss).

A finishing diet strategy is effective at increasing fillet long-chain n-3 fatty acid content in fish consuming sustainable plant oil-based diets. This study investigates the outcomes of a fish oil finishing diet upon the hepatic fatty acid and transcriptome profile in rainbow trout (Oncorhynchus mykiss). Fish were placed on one of three feeding treatments: (1) FO: a fish oil (FO) diet for 20 weeks, (2) VO/FO: a vegetable oil (VO) diet during weeks 1-12 then the FO diet for 8 weeks, or (3) VO/fd/FO: the VO diet between weeks 1-12, 2 weeks of feed deprivation, then the FO diet for 6 weeks. Hepatic fatty acid and transcriptome profiles were analyzed at week 12, 14, and 20. Hepatic fatty acid profiles at week 12 were similar to dietary profiles; transcriptomic analyses indicated 131 differentially regulated genes (DEG) between VO- and FO-fed fish, characterized by VO-induced up-regulation of cholesterol and long-chain fatty acyl-CoA synthesis and oxidation-reduction processes. At week 14, the hepatic fatty acid profile was similar between VO/FO and FO, although concentrations of 18:3n-3 remained higher in the VO/FO group. Thirty-three DEG were detected at week 14 with enrichment of genes associated with extracellular matrix assembly, supporting liver remodeling during the early finishing diet period. Only five DEG were detected at week 20 between VO/FO and FO. Collectively, these findings suggest that it takes several weeks for liver to reach a homeostatic state, even after the hepatic fatty acid equilibration following a finishing diet.

Replacing fish oil and astaxanthin by microalgal sources produced different metabolic responses in juvenile rainbow trout fed 2 types of practical diets.

Dietary fish oil supplementation provides n-3 long-chained polyunsaturated fatty acids for supporting fish growth and metabolism and enriching fillet with eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; c22:6n-3). Two experiments were performed as a 3 × 2 factorial arrangement of dietary treatments for 16 wk to determine effects and mechanisms of replacing 0%, 50%, and 100% fish oil with DHA-rich microalgae in combination with synthetic vs. microalgal source of astaxanthin in plant protein meal (PM)- or fishmeal (FM)- based diets for juvenile rainbow trout (Oncorhynchus mykiss). Fish (22 ± 0.26 g) were stocked at 17/tank and 3 tanks/diet. The 100% fish oil replacement impaired (P < 0.0001) growth performance, dietary protein and energy utilization, body indices, and tissue accumulation of DHA and EPA in both diet series. The impairments were associated (P < 0.05) with upregulation of hepatic gene expression related to growth (ghr1and igf1) and biosynthesis of DHA and EPA (fads6 and evol5) that was more dramatic in the FM than PM diet-fed fish, and more pronounced on tissue EPA than DHA concentrations. The source of astaxanthin exerted interaction effects with the fish oil replacement on several measures including muscle total cholesterol concentrations. In conclusion, replacing fish oil by the DHA-rich microalgae produced more negative metabolic responses than the substitution of synthetic astaxanthin by the microalgal source in juvenile rainbow trout fed 2 types of practical diets.

Effects of insulin-like growth factor-I, insulin, and leucine on protein turnover and ubiquitin ligase expression in rainbow trout primary myocytes.

The effects of insulin-like growth factor-I (IGF-I), insulin, and leucine on protein turnover and pathways that regulate proteolytic gene expression and protein polyubiquitination were investigated in primary cultures of 4-day-old rainbow trout myocytes. Supplementing media with 100 nM IGF-I increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 14% (P < 0.05). Treatment with 1 microM insulin increased protein synthesis by 13% (P < 0.05) and decreased protein degradation by 17% (P < 0.05). Supplementing media containing 0.6 mM leucine with an additional 2.5 mM leucine did not increase protein synthesis rates but reduced rates of protein degradation by 8% (P < 0.05). IGF-I (1 nM-100 nM) and insulin (1 nM-1 microM) independently reduced the abundance of ubiquitin ligase mRNA in a dose-dependent manner, with maximal reductions of approximately 70% for muscle atrophy F-box (Fbx) 32, 40% for Fbx25, and 25% for muscle RING finger-1 (MuRF1, P < 0.05). IGF-I and insulin stimulated phosphorylation of FOXO1 and FOXO4 (P < 0.05), which was inhibited by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, and decreased the abundance of polyubiquitinated proteins by 10-20% (P < 0.05). Supplementing media with leucine reduced Fbx32 expression by 25% (P < 0.05) but did not affect Fbx25 nor MuRF1 transcript abundance. Serum deprivation decreased rates of protein synthesis by 60% (P < 0.05), increased protein degradation by 40% (P < 0.05), and increased expression of all ubiquitin ligases. These data suggest that, similar to mammals, the inhibitory effects of IGF-I and insulin on proteolysis occur via P I3-kinase/protein kinase B signaling and are partially responsible for the ability of these compounds to promote protein accretion.

Molecular Mechanisms Regulating Muscle Plasticity in Fish.

Growth rates in fish are largely dependent on genetic and environmental factors, of which the latter can be highly variable throughout development. For this reason, muscle growth in fish is particularly dynamic as muscle structure and function can be altered by environmental conditions, a concept referred to as muscle plasticity. Myogenic regulatory factors (MRFs) like Myogenin, MyoD, and Pax7 control the myogenic mechanisms regulating quiescent muscle cell maintenance, proliferation, and differentiation, critical processes central for muscle plasticity. This review focuses on recent advancements in molecular mechanisms involving microRNAs (miRNAs) and DNA methylation that regulate the expression and activity of MRFs in fish. Findings provide overwhelming support that these mechanisms are significant regulators of muscle plasticity, particularly in response to environmental factors like temperature and nutritional challenges. Genetic variation in DNA methylation and miRNA expression also correlate with variation in body weight and growth, suggesting that genetic markers related to these mechanisms may be useful for genomic selection strategies. Collectively, this knowledge improves the understanding of mechanisms regulating muscle plasticity and can contribute to the development of husbandry and breeding strategies that improve growth performance and the ability of the fish to respond to environmental challenges.

Transcriptomic Response to Selective Breeding for Fast Growth in Rainbow Trout (Oncorhynchus mykiss).

Genetic improvement for faster growth is a conventional approach to increase growth rates in aquaculture species; however, the genetic and physiological factors regulating growth performance in fish are not fully characterized. The objective of this study was to identify physiological mechanisms associated with faster growth rates by comparing the liver and muscle transcriptome of a rainbow trout line selectively bred for fast growth (growth line, GL) and a contemporary randomly mated control line (synthetic control, SC) from the same selective breeding program. A third genetic line from a commercial egg supplier (commercial A, CA) was also included to characterize differences in gene expression profiles between populations. Body weight of the GL at harvest was approximately 20% and 8% heavier (p < 0.05) than SC and CA, respectively. There were 145 and 36 differentially expressed genes (DEG) in liver and white muscle, respectively, between the GL and SC that were enriched for the growth hormone/insulin-like growth factor axis (GH/IGF) and PI3K-Akt, JAK-STAT, MAPK, and cAMP signal transduction pathways. A greater concentration of plasma IGF-I was detected in the GL compared with SC (p < 0.05). A unique gene profile was detected in CA, with 11 and 210 DEG in liver and white muscle; these genes associated with innate immunity, complement systems, and metabolic pathways. Collectively, these findings provide a more extensive characterization of the fast-growth phenotype in fish that furthers knowledge of the physiological basis for genetic variation in growth performance in selectively bred rainbow trout.

Supplemental Microalgal DHA and Astaxanthin Affect Astaxanthin Metabolism and Redox Status of Juvenile Rainbow Trout.

Microalgal docosahexaenoic acid (DHA) and astaxanthin (AST) may substitute for fish oil and synthetic AST in aquafeeds. This study explored the effects and mechanisms of those substitutions on AST metabolism and redox status of rainbow trout fed plant protein meal (PM)- or fishmeal (FM)-based diets. Two parallel experiments (PM vs. FM) were performed with 612 juvenile rainbow trout for 16 weeks as a 2 × 3 factorial arrangement of treatments with two AST sources (synthetic (SA) vs. microalgal (AA), at 80 mg/kg) and three levels (0, 50, and 100%) of fish oil substitutions with DHA-rich microalgae. The fish oil substitutions exhibit main effects (p < 0.05) and/or interactive effects (p < 0.05) with the source of AST on AST deposition, malondialdehyde and glutathione concentrations, and mRNA levels and activities of major redox enzymes (glutathione reductase (GR), glutathione peroxidase (GPX), glutathione S-transferase (GST), and superoxide dismutase (SOD)) in the muscle and liver of trout fed both diet series. The AST source produced only differences in tissue AST deposition (p < 0.05) and number of metabolites. In conclusion, the substitutions of fish oil by the DHA-rich microalgae exerted more impacts than those of SA by AA on redox status and functional expression of antioxidant enzymes in the tissues of rainbow trout.

Compensatory Response of the Somatotropic Axis from IGFBP-2b Gene Editing in Rainbow Trout (Oncorhynchus mykiss).

Rainbow trout with gene editing-induced reductions in serum insulin-like growth factor binding protein (IGFBP)-2b exhibit similar growth performance compared to fish without IGFBP-2b gene disruption. The objective of this study is to determine how the components of the insulin-like growth factor (IGF)/IGFBP system respond to a reduction in serum IGFBP-2b abundance. Editing the IGFBP-2b genes in rainbow trout resulted in an 83% decrease in serum IGFBP-2b in mutants. This resulted in a 35% reduction in serum IGF-I, which was offset by reduced expression of hepatic igfbp-1a2 and increased muscle igfr-1a; these responses suggest that an increased IGF-I signaling capacity offset reductions in serum IGF-I. During feed deprivation, the differential expression of igfbp genes supports the attenuation of the growth inhibitory response, likely due to the further reduction in serum IGF-I that alleviated the need for an IGF-inhibitory response. Unique igfbp expression patterns occurred during refeeding, suggesting an enhanced IGF-I signaling capacity in controls. Collectively, these findings support that the role of IGFBP-2b is to regulate serum IGF-I concentrations. The compensatory regulation of IGF/IGFBP system genes indicates that adjustments in other IGFBP, both circulating and at the local level, maintain IGF-I signaling at a level appropriate for the nutritional state of the fish.

Diet-Induced Physiological Responses in the Liver of Atlantic Salmon (Salmo salar) Inferred Using Multiplex PCR Platforms.

The simultaneous quantification of several transcripts via multiplex PCR can accelerate research in fish physiological responses to diet and enable the development of superior aquafeeds for farmed fish. We designed two multiplex PCR panels that included assays for 40 biomarker genes representing key aspects of fish physiology (growth, metabolism, oxidative stress, and inflammation) and 3 normalizer genes. We used both panels to assess the physiological effects of replacing fish meal and fish oil by terrestrial alternatives on Atlantic salmon smolts. In a 14-week trial, we tested three diets based on marine ingredients (MAR), animal by-products and vegetable oil (ABP), and plant protein and vegetable oil (VEG). Dietary treatments affected the expression of genes involved in hepatic glucose and lipid metabolism (e.g., srebp1, elovl2), cell redox status (e.g., txna, prdx1b), and inflammation (e.g., pgds, 5loxa). At the multivariate level, gene expression profiles were more divergent between fish fed the marine and terrestrial diets (MAR vs. ABP/VEG) than between the two terrestrial diets (ABP vs. VEG). Liver ARA was inversely related to glucose metabolism (gck)- and growth (igfbp-5b1, htra1b)-related biomarkers and hepatosomatic index. Liver DHA and EPA levels correlated negatively with elovl2, whereas ARA levels correlated positively with fadsd5. Lower hepatic EPA/ARA in ABP-fed fish correlated with the increased expression of biomarkers related to mitochondrial function (fabp3a), oxidative stress (txna, prdx1b), and inflammation (pgds, 5loxa). The analysis of hepatic biomarker gene expression via multiplex PCR revealed potential physiological impacts and nutrient-gene interactions in Atlantic salmon fed lower levels of marine-sourced nutrients.