Inclusion of Dietary Defatted Microalgae Dose-Dependently Enriches ω-3 Fatty Acids in Egg Yolk and Tissues of Laying Hens.

BACKGROUND: The potential for dietary microalgae to enrich eggs of laying hens with ω-3 (n-3) fatty acids, and the mechanisms involved, are unclear. OBJECTIVES: The aim of this study was to determine the effects and molecular regulation of a defatted Nannochloropsis oceanica microalgae (DNOM) biomass on the enrichment of the eggs and tissues of laying hens with ω-3 fatty acids. METHODS: Fifty Shaver-White Leghorn hens (46 wk of age, body weight: 1.70 ± 0.27 kg) were individually caged (n = 10) and fed a corn-soy-based diet supplemented with DNOM at 0% (control), 2.86%, 5.75%, 11.5%, and 23% for 6 wk. Fatty acid profiles, health status, and related gene expression in eggs, blood, and tissues were performed at weeks 0, 2, 4, and 6. Data were analyzed by a combination of 1-factor ANOVA and correlation between DNOM doses and measures. RESULTS: The DNOM produced linear (P < 0.01) enrichments of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and total ω-3 fatty acids in the egg yolk (R2 > 0.9) and of DHA in the liver, breast, and thigh (R2 = 0.66-0.82). Concentrations of EPA + DHA in the egg yolk and these 3 tissues of hens fed 11.5% and 23% DNOM were 1.4-2.1, 0.6-1, 3.3-5.3, and 6-7 times greater (P < 0.001) than those in the controls, respectively. The DNOM caused dose-dependent elevations (P < 0.01) of malic enzyme and elongases 3, 4, and 5 mRNA levels (R2 = 0.97, 0.78, 0.97, and 0.86, respectively), along with increased (P < 0.01) Δ5- and Δ6-desaturases and decreased (P < 0.01) Δ9-desaturase and acyl-coenzyme A thioesterase 4 mRNA levels in the liver. CONCLUSIONS: Feeding DNOM to laying hens produced dose-dependent enrichments of DHA in their eggs, liver, and muscles by regulating key genes involved in the elongation and desaturation of polyunsaturated fatty acids. Our findings will help produce DHA-enriched eggs.

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.

Effects of phytoestrogens on growth-related and lipogenic genes in rainbow trout (Oncorhynchus mykiss).

This study determined whether estradiol (E2) or the phytoestrogens genistein and daidzein regulate expression of growth-related and lipogenic genes in rainbow trout. Juvenile fish (5 mon, 65.8±1.8 g) received intraperitoneal injections of E2, genistein, or daidzein (5 µg/g body weight) or a higher dose of genistein (50 µg/g body weight). Liver and white muscle were harvested 24h post-injection. In liver, expression of vitellogenin (vtg) and estrogen receptor alpha (era1) increased in all treatments and reflected treatment estrogenicity (E2>genistein (50 µg/g)>genistein (5 µg/g)=daidzein (5 µg/g)). Estradiol and genistein (50 µg/g) reduced components of the growth hormone (GH)/insulin-like growth factor (IGF) axis in liver, including increased expression of IGF binding protein-2b1 (igfbp2b1) and reduced igfbp5b1. In liver E2 and genistein (50 µg/g) affected expression of components of the transforming growth factor beta signaling mechanism, reduced expression of ppar and rxr transcription factors, and increased expression of fatty acid synthesis genes srebp1, acly, fas, scd1, and gpat and lipid binding proteins fabp3 and lpl. In muscle E2 and genistein (50 µg/g) increased era1 and erb1 expression and decreased erb2 expression. Other genes responded to phytoestrogens in a manner that suggested regulation by estrogen receptor-independent mechanisms, including increased ghr2, igfbp2a, igfbp4, and igfbp5b1. Expression of muscle regulatory factors pax7 and myod was increased by E2 and genistein. These data indicate that genistein and daidzein affect expression of genes in rainbow trout that regulate physiological mechanisms central to growth and nutrient retention.

Effects of sexual maturation and feeding level on fatty acid metabolism gene expression in muscle, liver, and visceral adipose tissue of diploid and triploid rainbow trout, Oncorhynchus mykiss.

In many cultured fish species, such as salmonids, gonadal development occurs at the expense of stored energy and nutrients, including lipids. However, mechanisms regulating nutrient repartitioning during sexual maturation are not well understood. This study compared sexually maturing diploid (2N) and sterile triploid (3N) female rainbow trout to investigate effects of sexual maturation on expression of 35 genes involved in fatty acid metabolism, including genes within fatty acid synthesis, ß-oxidation, and cofactors of the mTOR and PPAR signaling pathways, in liver, white muscle, and visceral adipose tissue. Diploid fish were fed at different rations (0.25% and 0.50% tank biomass, and satiation) to determine effects of ration on gene expression. Gene expression was affected by ration level only in white muscle; erk and acat2 had higher expression in fish fed higher rations. On the other hand, sexual maturation affected gene expression across all three tissue types. Data indicate 2N fish have higher expression of ß-oxidation genes within white muscle and within visceral adipose tissue. These findings support enhanced fatty acid mobilization within these tissues during sexual maturation. Higher expression of fatty acid synthesis genes in 3N female liver is associated with higher expression of mTOR cofactors and pparγ, which reflects continued deposition of lipids in these fish. Furthermore, greater expression of genes involved in ß-oxidation pathways across ration levels in 2N females suggests that sexual maturation and the associated maturation-related signals are stronger regulators of lipid metabolism-related genes rather the rations applied in the current study.

Effect of sexual maturation on muscle gene expression of rainbow trout: RNA-Seq approach.

Muscle degradation occurs as a response to various physiological states that are regulated by specific molecular mechanisms. Previously, we characterized the metabolic changes of muscle deterioration of the female rainbow trout at full sexual maturity and spawning (Salem et al., Physiol. Genomics 2006;28:33-45; J. Proteomics 2010;73:778-789). Muscle deterioration in this model represents nutrient mobilization as a response to the energetic overdemands of the egg/ovarian growth phase. Our recent studies showed that most of the changes in muscle growth and quality start 2-3 months before spawning. Gravid fish exhibited reduced intramuscular fat that is lower in saturated and monounsaturated fatty acids and higher in polyunsaturated fatty acids compared to sterile fish. In this study, RNA-Seq was used to explain the mechanisms underlying changes during this phase of sexual maturity. Furthermore, to minimize changes due to nutrient deficits, fish were fed on a high-plane of nutrition. The RNA-Seq technique identified a gene expression signature that is consistent with metabolic changes of gravid fish. Gravid fish exhibited increased abundance of transcripts in metabolic pathways of fatty acid degradation and up-regulated expression of genes involved in biosynthesis of unsaturated fatty acids. In addition, increased expression of genes involved in the citric acid cycle and oxidative phosphorylation was observed for gravid fish. This muscle transcriptomic signature of fish fed on a high nutritional plane is quite distinct from that previously described for fish at terminal stages of maturity and suggest that female rainbow trout approaching spawning, on high nutritional planes, likely mobilize intramuscular fat rather than protein to support gonadal maturation.

Effect of sexual maturation on thermal stability, viscoelastic properties, and texture of female rainbow trout, Oncorhynchus mykiss, fillets.

UNLABELLED: The nutrient and energy demand of sexual maturation in many fish cultivars causes structural change to key contractile proteins and thereby, affects fillet firmness. Thermal denaturation and viscoelastic properties of white muscle from diploid (2N; fertile) and triploid (3N; sterile) female rainbow trout were investigated at 6 age endpoints from July 2008 through spawning in March 2009. Differential scanning calorimetry showed, in March, that the actin denaturation temperature (T(max,actin)) of 2N females was higher than that observed in 3N females (78.17 versus 77.27 °C). From 35 to 45 °C, viscoelastic measurement revealed that muscle from 2N females and younger fish (July, 16 mo) had greater elasticity (lower tan δ) than muscle from 3N females and older fish (November to March; 20 to 24 mo), respectively. The highest elastic response and the firmest fillets were observed in July. Raw fillets were softer (Allo-Kramer shear; P < 0.05) from September to January (288.77 g/g on average) than those collected in July (475.15 g/g) and March (366.79 g/g). Soft fillets became firmer after cooking except for January samples. Greater cook yield and softer fillets were observed in January compared to December. Lipid accumulation in 3N females may lubricate muscle fibers and protect them from losing functionality during the spawning season for animals on a high plane of nutrition. PRACTICAL APPLICATION: The relationship between fish maturation, measured as egg development, and chemical characteristics of fillets from fertile and sterile fish was evaluated. Thermal denaturation and viscoelastic characterization revealed changes in stability and gelling properties of muscle proteins that were related to changes in fillet texture.