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Booming fish farming results in relative shortage of fish oil (FO), making it urgent to explore alternative lipid sources. This study comprehensively investigated the efficacy of FO replacement with poultry oil (PO) in diets of tiger puffer (average initial body weight, 12.28 g). An 8-week feeding trial was conducted with experimental diets, in which graded levels (0, 25, 50, 75, and 100%, named FO-C, 25PO, 50PO, 75PO, and 100PO, respectively) of FO were replaced with PO. The feeding trial was conducted in a flow-through seawater system. Each diet was fed to triplicate tanks. The results showed that FO replacement with PO did not significantly affect the growth performance of tiger puffer. FO replacement with PO at 50-100% even slightly increased the growth. PO feeding also had marginal effects on fish body composition, except that it increased the liver moisture content. Dietary PO tended to decrease the serum cholesterol and malondialdehyde content but increase the bile acid content. Increasing levels of dietary PO linearly upregulated the hepatic mRNA expression of the cholesterol biosynthesis enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase, whereas high levels of dietary PO significantly upregulated the expression of the critical regulatory enzyme of bile acid biosynthesis, cholesterol 7-alpha-hydroxylase. In conclusion, poultry oil is a good substitution for fish oil in the diets of tiger puffer. Poultry oil could replace 100% added fish oil in the diet of tiger puffer, without adverse effects on growth and body composition.

A nine-week feeding trial was conducted to investigate changes in the intestinal microbiota of turbot in response to alternate feeding between terrestrially sourced oil (TSO)- and fish oil (FO)-based diets. The following three feeding strategies were designed: (1) continuous feeding with the FO-based diet (FO group); (2) weekly alternate feeding between soybean oil (SO)- and FO-based diets (SO/FO group); and (3) weekly alternate feeding between beef tallow (BT)- and FO-based diets (BT/FO group). An intestinal bacterial community analysis showed that alternate feeding reshaped the intestinal microbial composition. Higher species richness and diversity of the intestinal microbiota were observed in the alternate-feeding groups. A PCoA analysis showed that the samples clustered separately according to the feeding strategy, and among the three groups, the SO/FO group clustered relatively closer to the BT/FO group. The alternate feeding significantly decreased the abundance of Mycoplasma and selectively enriched specific microorganisms, including short-chain fatty acid (SCFA)-producing bacteria, digestive bacteria (Corynebacterium and Sphingomonas), and several potential pathogens (Desulfovibrio and Mycobacterium). Alternate feeding may maintain the intestinal microbiota balance by improving the connectivity of the ecological network and increasing the competitive interactions within the ecological network. The alternate feeding significantly upregulated the KEGG pathways of fatty acid and lipid metabolism, glycan biosynthesis, and amino acid metabolism in the intestinal microbiota. Meanwhile, the upregulation of the KEGG pathway of lipopolysaccharide biosynthesis indicates a potential risk for intestinal health. In conclusion, short-term alternate feeding between dietary lipid sources reshapes the intestinal microecology of the juvenile turbot, possibly resulting in both positive and negative effects.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.

An 8-week feeding trial was conducted, where turbot were fed four experimental diets, containing different LPC levels (0%, 0.1%, 0.25%, and 0.5%, named LPC0, LPC0.1, LPC0.25, and LPC0.5, respectively). The intestinal morphology results showed that there were no widened lamina propria and mixed inflammatory cells in the LPC-supplemented groups. Dietary LPC remarkably decreased the expression of TLRs (TLR3, TLR8, TLR9, and TLR22), MyD88, and signaling molecules (NF-κB, JNK, and AP-1). Similarly, diets with LPC supplementation markedly depressed the gene expression of NF-κB and JNK signaling pathway downstream genes (TNF-α, IL-1β, Bax, Caspase9, and Caspase-3). Furthermore, dietary LPC modified the intestinal microbial profiles, increasing the relative abundance of short-chain fatty acids-producers, lactic acid bacteria, and digestive enzyme-producing bacteria. Predictive functions of intestinal microbiota showed that turbot fed LPC diets had a relatively higher abundance of functions, such as lipid metabolism and immune system, but a lower abundance of functions, such as metabolic diseases and immune system diseases. The activities of intestinal acid phosphatase and alkaline phosphatase were also increased by dietary LPC. In conclusion, LPC supplementation could regulate the intestinal mucosal barrier via the TLR signaling pathway and alter the intestinal microbiota profile of turbot fed high-lipid diets.

Exogenous cholesterol has been supplemented into aqua-feeds due to the reduced proportions of fishmeal and fish oil. This study aimed to investigate the effects of dietary cholesterol supplementation on the muscle lipidomics of two marine fish species, turbot and tiger puffer. A 70-day feeding trial was conducted, where two low-fishmeal diets supplemented with 0 or 1% cholesterol were used. The lipidomic analysis with targeted tandem mass spectrometry showed that, in turbot, a total of 49 individual lipids exhibited significant differences in their abundance in response to dietary cholesterol, whereas the number was 30 for tiger puffer. Dietary cholesterol up-regulated the abundance of cholesterol and cholesterol ester in both species. In turbot, the dietary cholesterol also increased the abundance of triacylglycerol and acylcarnitine, whereas in tiger puffer, it primarily regulated the abundance of phospholipids and BMP. This was the first time the responses of marine fish muscle lipidomics to dietary cholesterol supplementation have been investigated.

An eight-week feeding trial was conducted to investigate the effects of diets supplemented with three sulfur-containing amino acids (SAA), namely, methionine, cysteine, and taurine, on the intestinal health status of juvenile turbot (Scophthalmus maximus) fed high-lipid diets. Four diets were formulated, namely, a high-lipid control diet (16% lipid, HL) and three SAA-supplemented diets, which were formulated by supplementing 1.5% methionine (HLM), 1.5% cysteine (HLC), and 1.5% taurine (HLT) into the HL control diet, respectively. Each diet was assigned to triplicate tanks, and each tank was stocked with 30 juvenile fish (appr. initial weight, 8 g). The histological and morphometric results showed that dietary SAA supplementation obviously improved the intestinal morphology and integrity, in particular as reflected by higher height of microvilli and mucosal folds. Dietary SAA supplementation, in particular cysteine, up-regulated the gene expression of mucin-2 and tight junction proteins (ZO-1, Tricellilun and JAM). Dietary SAA supplementation remarkably down-regulated the gene expression of apoptosis-related factors such as p38, JNK, and Bax, expression of pro-inflammatory factors (e.g., NF-κB, AP-1 IL-1β, IL-8, and TNF-α). SAA supplementation resulted in higher antioxidative abilities in the intestine. Additionally, dietary SAA supplementation largely altered the communities of intestinal microbiota. Compared with the HL group, higher relative abundance of potential beneficial bacteria, and lower relative abundance of opportunistic pathogens were observed in SAA-supplemented groups. Dietary taurine supplementation significantly increased the relative abundance of Ligilactobacillus (in particular Lactobacillus murinus) and Limosilactobacillus (especially Lactobacillus reuteri). In conclusion, dietary sulfur-containing amino acids supplementation have promising potential in ameliorating the intestinal inflammation of turbot fed high-lipid diets. Especially dietary cysteine and taurine supplementation have more positive effects on the communities of the intestinal microbiota of turbot.

This study investigated the sex difference in fatty acid (FA) composition of six wild marine fish species, namely, Cleisthenes herzensteini, Platichthys bicoloratus, Pseudosciaena polyactics, Platycephalus indicus, Alosa sapidissima and Scomberomorus niphonius. The coefficient of distance value between sexes (D

Acute hypoxia is a common stress in aquaculture, and causes energy deficiency, oxidative damage and death in fish. Many studies have confirmed that acute hypoxia activated hif1α expression, anaerobic glycolysis and antioxidant system in fish, but the effects of acute hypoxia on lipid and protein metabolism, organelle damage, and the functions of hif2α and hif3α in economic fishes have not been well evaluated. In the present study, turbot was exposed to acute hypoxia (2.0 ± 0.5 mg/L) for 6 h, 12 h, and 24 h, respectively. Then, the contents of hemoglobin (HB), metabolite, gene expressions of hifα isoforms, energy homeostasis, endoplasmic reticulum (ER) stress, and apoptosis were measured. The results suggested that turbot is intolerant to acute hypoxia and the asphyxiation point is about 1.5 mg/L. Acute hypoxia induced perk-mediated ER stress, and increased lipid peroxidation and liver injury in turbot. The blood HB level and liver vegfab expression were increased under hypoxia, which enhances oxygen transport. At hypoxia stress, hif3α, anaerobic glycolysis-related genes expression, and lactate content were increased in the liver, and glycogen was broken down to ensure ATP supply. Meanwhile, hif2α, lipid synthesis-related genes expression, and TG content were increased in the liver, but the lipid catabolism and protein synthesis were suppressed during hypoxia, which reduced the oxygen consumption and ROS generation. Our results systematically illustrate the metabolic and physiological changes under acute hypoxia in turbot, and provide important guidance to improve hypoxia tolerance in fish.

In order to find out the respective influences of chemical reactivity and physical transport of hydrogen additive on nonpremixed flame, two fabricated hydrogen additions were introduced into nonpremixed methane/air flame modeling. Hydrogen addition was assumed as inert gas or partial reactivity fuel to respectively explore the kinetic reasons by the three aspects: the elementary reaction route, heat release, and physical diffusion of hydrogen addition. The analyses were implemented in terms of OH and H production. Results showed that, hydrogen addition can enhance OH and H production via elementary reactions, and causes flame reaction zone migration through the coupling interaction between the low-temperature heat enthalpy release and diffusion behavior of hydrogen addition. R84 (OH + H2=H + H2O) and R38 (H + O2=O + OH) are the most important elementary reactions related to OH and H production. The physical incentive of hydrogen addition can hardly work without the chemical effects of hydrogen addition.

Booming fish farming results in a relative shortage of fish oil (FO) supply, meaning that alternative oils are increasingly used in fish feeds, which leads to reduction of long-chain polyunsaturated fatty acids (LC-PUFAs) and other relevant changes in fish products. This study investigated the efficacy of an FO-finishing strategy in recovering the muscle quality of farmed tiger puffer. An eight-week feeding trial (growing-out period) was conducted with five experimental diets, in which graded levels (0 (control), 25, 50, 75, and 100%) of added FO were replaced by poultry oil (PO). Following the growing-out period was a four-week FO-finishing period, during which fish in all groups were fed the control diet. Dietary PO significantly decreased the muscle LC-PUFA content, whereas in general, the FO-finishing strategy recovered it to a level comparable with that of the group fed FO continuously. The recovery efficiency of EPA was higher than that of DHA. Dietary PO also led to changes of volatile flavor compounds in the muscle, such as butanol, pentenal, and hexenal, whereas the FO-finishing strategy mitigated the changes. In conclusion, the FO-finishing strategy is promising in recovering the LC-PUFA and volatile-flavor-compound composition in farmed tiger puffer after the feeding of PO-based diets.

The tissue distribution pattern of lipid is highly diverse among different fish species. Tiger puffer has a special lipid storage pattern, storing lipid predominantly in liver. In order to better understand the lipid physiology in fish storing lipid in liver, the present study preliminarily investigated the tissue distribution of transcription for 29 lipid metabolism-related genes in tiger puffer, which are involved in lipogenesis, fatty acid oxidation, biosynthesis and hydrolysis of glycerides, lipid transport, and relevant transcription regulation. Samples of eight tissues, brain, eye, heart, spleen, liver, intestine, skin, and muscle, from fifteen juvenile tiger puffer were used in the qRT-PCR analysis. The intestine and brain had high transcription of lipogenic genes, whereas the liver and muscle had low expression levels. The intestine also had the highest transcription level of most apolipoproteins and lipid metabolism-related transcription factors. The transcription of fatty acid β-oxidation-related genes was low in the muscle. The peroxisomal fatty acid oxidation may dominate over mitochondrial β-oxidation in the liver and intestine of tiger puffer, and the MAG pathway probably predominates over the G3P pathway in re-acylation of absorbed lipids in the intestine. The intracellular glyceridases were highly transcribed in the brain, eye, and heart. In conclusion, in tiger puffer, the intestine could be a center of lipid metabolism whereas the liver is more likely a pure storage organ for lipid. The lipid metabolism in the muscle could also be inactive, possibly due to the very low level of intramuscular lipid.

Taurine (TAU) plays important roles in the metabolism of bile acids, cholesterol and lipids. However, little relevant information has been available in fish where TAU has been identified as a conditionally essential nutrient. The present study aimed to investigate the effects of dietary TAU on the metabolism of bile acids, cholesterol and lipids in tiger puffer, which is both an important aquaculture species and a good research model, having a unique lipid storage pattern. An 8-week feeding trial was conducted in a flow-through seawater system. Three experimental diets differed only in TAU level, that is, 1·7, 8·2 and 14·0 mg/kg. TAU supplementation increased the total bile acid content in liver but decreased the content in serum. TAU supplementation also increased the contents of total cholesterol and HDL-cholesterol in both liver and serum. The hepatic bile acid profile mainly includes taurocholic acid (94·48 %), taurochenodeoxycholic acid (4·17 %) and taurodeoxycholic acid (1·35 %), and the contents of all these conjugated bile acids were increased by dietary TAU. The hepatic lipidomics analysis showed that TAU tended to decrease the abundance of individual phospholipids and increase those of some individual TAG and ceramides. The hepatic mRNA expression study showed that TAU stimulated the biosynthesis of both bile acids and cholesterol, possibly via regulation of farnesoid X receptor and HDL metabolism. TAU also stimulated the hepatic expression of lipogenic genes. In conclusion, dietary TAU stimulated the hepatic biosynthesis of both bile acids and cholesterol and tended to regulate lipid metabolism in multiple ways.

A 56-day feeding trial was conducted in a flow-through seawater system to investigate the effects of lysophosphatidylcholine (LPC) on growth performance and lipid metabolism of turbot. Four experimental diets were prepared, differing only in the LPC supplementation, namely, 0 (LPC-0, control), 0.1 (LPC-0.1), 0.25 (LPC-0.25), and 0.5% (LPC-0.5) of dry matter. Each diet was randomly fed to triplicate tanks. LPC-0.1 and LPC-0.25 led to significantly higher weight gain than the control diet, and the highest weight gain was observed in LPC-0.1. Compared to the control group, the LPC-supplemented groups had higher survival and lower hepatosomatic index and viscerosomatic index. LPC-0.25 led to significantly lower contents of crude lipid and ash in whole fish. Dietary LPC supplementation led to a basic decrease in the lipid metabolism-related biochemical parameters in serum but had only very minor influence on the fatty acid composition in the liver and subcutaneous tissue around the fin (STF). High LPC levels upregulated the mRNA expression of BSAL and ApoEα in both the liver and STF. In conclusion, dietary LPC supplementation (0.1-0.25%) enhanced the growth, lowered the lipid accumulation in juvenile turbot, and significantly regulated the lipid metabolism. However, it seldom influenced the fatty acid composition.

Fatty acid-binding proteins (fabps) play important roles in lipid homeostasis. In the present study, 7 fabp isoforms, namely, fabp1, fabp2, fabp3, fabp4, fabp6, fabp7, and fabp10, in two marine teleosts, were characterized. In general, turbot and tiger puffer fabp genes showed high identity to their orthologs in other fish species and mammals, but tiger puffer Fabp6 shared the lowest identity to its known orthologs in zebrafish and human. The tissue distribution patterns of fabps were generally in accordance with their function features. However, tiger puffer fabps, in particular Fabp1, Fabp2, Fabp6 and Fabp7, may have functions distinct from other teleosts, as indicated by the phylogenetic tree and tissue distribution patterns. In both species, high dietary lipid levels downregulated the expression of fabp2, fabp3, fabp6, and fabp7a but tended to upregulate the fabp1 expression. Starvation downregulated the expression of most fabps in both fish species, but the downregulation of fabp expression in turbot was much more drastic and earlier compared to tiger puffer. Long-term (30-day) starvation increased the fabp7 expression in tiger puffer and tended to increase the fabp6 expression in turbot. Results of this study contribute to fish fabp physiology and its nutritional regulation.

Two 70-day feeding trials were conducted to reevaluate the necessity of cholesterol supplementation in diets of two important marine fish species, turbot and tiger puffer. Graded levels (0, 0.5, 1.0, 2.0, and 4.0% of dry matter) of cholesterol were added into the basal diet (with 30% fishmeal but no fish oil) to make five experimental diets, which were designated as control, CHO-0.5, CHO-1.0, CHO-2.0, and CHO-4.0, respectively. The dietary cholesterol concentration was measured to be 0.11%, 0.65%, 1.10%, 2.32%, and 4.59%, respectively. Each group had triplicate tanks, and each tank was stocked with 30 fish. The results showed that compared to the control, dietary cholesterol supplementation had no significant effect on the growth of both turbot and tiger puffer. Nevertheless, excess dietary cholesterol (CHO-2.0 and CHO-4.0) led to significant lower weight gain of tiger puffer compared to CHO-1.0. The feed intake decreased with increasing levels of dietary cholesterol. In general, the cholesterol supplementation decreased the crude lipid content in the liver, whereas 0.5% dietary cholesterol supplementation tended to increase the crude lipid content in the muscle of both species. Dietary cholesterol supplementation significantly regulated the lipid-related biochemical parameters in serum, liver, and muscle, and compared to turbot, tiger puffer lipid compositions had a higher buffering capacity in response to changes in dietary cholesterol level. Dietary cholesterol supplementation increased the 20:4n-6 content in the liver. Dietary cholesterol supplementation significantly downregulated the gene expression of HMG-COAr, upregulated the CYP7A1 expression, and downregulated the expression of lipid absorption and biosynthesis genes, but had no significant effect on gene expression of CPT1 and lipid transport-related genes (ApoA1, ApoA4, ApoB100, and ApoEα) in the liver. In conclusion, under the present experimental condition, dietary cholesterol supplementation had no significant effect on the growth of turbot and tiger puffer. In both species, dietary cholesterol supplementation significantly regulated the lipid accumulation and metabolism.

The fish oil finishing (FOF) strategy, that is, re-feeding fish with fish oil (FO)-based diet after a certain period of feeding with alternative lipid source-based diets. On tiger puffer, the present study investigated the response of intestinal microbiota to FOF. Fish were fed four diets based on FO, soybean oil, palm oil and beef tallow as lipid sources, respectively, firstly for 50 days (growing-out period), and then fed the FO-based diet for 30 more days (FOF period). The results showed that dietary terrestrially sourced oils impaired the intestinal function in the growing-out period. However, the activities of amylase, trypsin and anti-oxidative enzymes (SOD, CAT, T-AOC), as well as gene expression of inflammatory cytokines (IL-1β, TNF-α, TGF-β) and tight junction protein (Claudin4, Claudin7, Claudin18, JAM, ZO-1) in the intestine were significantly recovered by FOF. The 16S rDNA sequencing analysis showed that FOF improved the similarity of bacterial community among the groups. The MetaStat analysis confirmed that FOF regulated the abundance of butyric acid-producing bacteria (Lachnospiraceae, Eubacterium, Butyricicoccus, Clostridium and Roseburia) and bacteria related to digestion and absorption (Sphingomonas, Romboutsia and Brevibacillus). In conclusion, FOF can recover the intestine function. The intestinal microbiota probably participated in and played a key role in the recovery process.

The study aimed to investigate the effects of crystalline Lys and Leu, Lys‐Leu dipeptide and Lys‐Leu‐Lys/Leu‐Lys‐Leu tripeptide, on growth, postprandial Lys and Leu concentrations, gene expression of peptide and amino acid (AA) transporters and related gene expression of protein synthesis pathway in turbot. Three diets (CAA, Di and Tri) contained Lys and Leu as free (Lys and Leu), dipeptide (Lys‐Leu) and tripeptide (Lys‐Leu‐Lys/Leu‐Lys‐Leu) forms, respectively. Improved growth was observed in the Di group compared with the CAA group. For peptide and AAs transporters, PepT1, B⁰AT1 and y⁺LAT2 mRNA levels were affected in proximal intestine by dietary treatments. Free Lys and Leu concentrations in the CAA group were significantly higher than that of the Di and Tri groups at 6 hr postfeeding in serum and at 2, 6 and 24 hr postfeeding in muscle. For protein synthesis pathway in muscle, Akt2, TOR and S6k1 gene expression were the highest in the Di group and the lowest in the CAA group, whereas MuRF1 relative expression was the highest in the Tri group. In conclusion, dietary Lys‐Leu dipeptide was utilized more efficiently than free Lys and Leu or Lys‐Leu‐Lys/Leu‐Lys‐Leu tripeptide for turbot by regulating AAs transport, postprandial AAs concentration and the synthesis of muscle proteins.

Lysophosphatidylcholine (LPC) has been widely used as emulsifier in animal feeds to enhance the lipid utilization. However, the effects of LPC on fillet quality has rarely been known. The present study was the first time to investigate the response of fish muscle lipidomics to dietary LPC supplementation. Turbot muscle samples were collected after a 56-day feeding trial where the experimental diet contained 0 or 0.25% LPC. Targeted tandem mass spectrometry was used in the lipidomic analysis. A total of 62 individual lipids (58 up-regulated and 7 down-regulated by LPC) showed significant difference in concentration in response to dietary LPC. Most of these differentially abundant lipids were diacylglycerol, free fatty acid and cardiolipin, and they all were up-regulated by dietary LPC. However, LPC exerted only marginal effects on muscle fatty acid composition and lipid content. The effects of dietary LPC on fillet lipid composition cannot be neglected in fish product evaluation.

A nine-week feeding trial was conducted to comprehensively investigate the effects of different levels of dietary lipid on intestinal physiology of juvenile turbot. Three diets with different lipid levels (8%, 12% and 16%) were formulated, which were designated as the low-lipid group (LL), medium-lipid group (ML) and high-lipid group (HL), respectively. Each diet was fed to six replicate tanks, and each tank was stocked with 35 fish. The results revealed that medium dietary lipid (12%) increased the activities of intestinal digestive enzymes and brush border enzymes. Excessive dietary lipid (16%) decreased the intestinal antioxidative enzyme levels and increased the lipid peroxidation pressure. In addition, HL stimulated the occurrence of intestinal inflammation and significantly up-regulated the mRNA expression level of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β). Dietary LL and HL induced the apoptosis of intestinal epithelial cells. Sequencing of bacterial 16 s rRNA V4 region indicated that the abundance and diversity of intestinal microflora in fish fed with medium lipid diet (12%) were significantly higher than those in other groups, indicating the intestinal microflora ecology in group ML was more balanced. MetaStat analysis indicated that both low- and high-lipid diets significantly reduced the relative abundance of intestinal beneficial bacteria. In conclusion, results of this study demonstrated the sensitivity of intestinal health and microbiota to dietary lipid levels. From the perspective of microecological balance, medium dietary lipid (12%) was more conducive to maintaining the intestinal microflora stability of turbot.

The present study was aimed at investigating the interactive effects of starvation and dietary lipid level in the previous feeding period on lipid-related composition of turbot. Turbot with an average initial body weight of 26 g were firstly fed diets with different lipid levels, namely, 8%, 12%, and 16%, for 9 weeks, and then subjected to starvation for 30 days. Each diet was fed to sextuplicate tanks of 35 fish in the feeding trial. Tissue samples were collected at the end of the feeding trial and at 10, 20, and 30 days after starvation. The results showed that 30-day starvation decreased the lipid content in the liver and the subcutaneous tissue around the fin (STF), but increased the lipid content in the muscle. A synergetic increase of muscle lipid by starvation and dietary lipid level was observed. Starvation mobilized different fatty acids among the three tissues, namely, MUFA (16:1n-7 and 18:1n-9) in the muscle, SFA (14:0 and 16:0), MUFA (16:1n-7, 18:1n-9 and 20:1n-9), and 18C-PUFA (18:2n-6 and 18:3n-3) in the liver, and unexpectedly n-3 PUFA (18:3n-3, EPA, and DHA) in the STF, respectively. The 30-day starvation decreased the muscle hardness and resilience, but affected other texture parameters in a starvation time-dependent manner. Up-regulation of expression of lipolytic genes by starvation occurred later in the STF than in the liver. Interactive effects of starvation and dietary lipid level were observed mainly on tissue fatty acid compositions. Results of this study suggested that combined manipulation of starvation time and dietary lipid level could be used as an effective means of fish quality regulation in terms of lipid/fatty acid-related composition.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.

Omics approaches provide more metabolic information to explain the relationship between dietary nutrition and fish growth. This study aimed to explore the metabolome and proteome response of turbot (Scophthalmus maximus) fed diets containing lysine and leucine in free and dipeptide forms by the approaches of integrated liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics. Plant protein-based diets were formulated to contain the equivalent of lysine and leucine in free amino acid [crystalline amino acid (CAA)] and synthetic Lys-Leu (Lys-Leu) forms. The metabolome and proteome profiles of the liver were screened in fish fed either the CAA diet or the Lys-Leu diet after an 8-week feeding trial. Fish fed the Lys-Leu diet showed a significantly higher final body weight and a specific growth rate compared with fish fed the CAA diet. Protein- and amino acid-related metabolic processes in the liver were identified between the Lys-Leu and CAA groups based on differential metabolites and proteins. The proteolytic enzymes and amino acid transporters from differential proteins of the liver showed that the process of protein digestion and absorption may be affected by the different forms of lysine and leucine in the feed. A mechanistic target of rapamycin complex 1 and ubiquitin proteasome pathways were identified by differential proteins, which were involved in the processes of protein synthesis and degradation in the liver. Lysine degradation, tryptophan metabolism, alanine, aspartate, and glutamate metabolism, arginine biosynthesis, arginine and proline metabolism, and glycine, serine, and threonine metabolism were identified based on differential metabolites and proteins, which showed that the metabolism of various amino acids, including lysine, had been affected by both the CAA and Lys-Leu groups. In conclusion, the data of integrated metabonomics and proteomics suggested that different forms of lysine and leucine in the feed may affect liver metabolic processes including protein digestion and absorption, protein synthesis and degradation, and amino acid metabolism. In addition, a good correlation between differential metabolites and proteins was observed in amino acid metabolism by using the approaches of integrated LC-MS/MS-based metabolomics and iTRAQ-based proteomics.

The effects of fish protein hydrolysate (FPH) on growth, peptide and amino acid (AA) transporters, postprandial free AA and related gene expression of IGF‐1/AKT pathway were evaluated in turbot (Scophthalmus maximus). Three diets were formulated to contain the same low level of fishmeal; meanwhile 0, 45 and 180 g/kg FPH were, respectively, supplemented to the FF (FPH‐free), FL (FPH‐Low) and FH (FPH‐High) diets. Fish fed the FH diet improved the growth compared with the other groups. For peptide and AA transporters, PepT1, B⁰AT1, CAT1 and PAT1 mRNA levels in proximal or distal intestine decreased in fish fed the FH diet. The concentration of free total essential AAs in serum was higher in the FH treatment than that in the FF treatment at 2 and 6 hr after feeding. For IGF‐1/AKT pathway in muscle, IGF‐1, 4E‐BP1 and FoxO1 mRNA levels were the highest in the FH group, whereas IGF‐1R mRNA levels were the highest expression level in the FF group. In conclusion, down‐regulated AAs transport, alleviated the delayed postprandial peak of serum‐free AAs and increased muscle protein synthesis were observed to improve the growth when turbot was fed high FPH level diets containing a high plant protein.

Our previous studies have indicated that dietary arachidonic acid (ARA) significantly affects the gonadal steroidogenesis in the marine teleost tongue sole Cynoglossus semilaevis, and this effect was more positive in male fish than in female fish (Aquaculture, 468, 378–385). As a following up study, the present study was further aimed at investigating the possible mechanisms in the brain mediating the effects of dietary ARA on gonadal steroidogenesis. A 70‐day feed trial was repeated with two‐year‐old tongue sole, using three experimental diets with graded levels of ARA, 0.34%, 2.53%, and 9.63% of total fatty acids. Each diet was fed to triplicate groups of 23 fish (15 males and 8 females). The results confirmed the positive effect of dietary ARA on testosterone production in male fish. The concentration of gonadotrophin‐releasing hormone (GnRH) in serum responded to dietary ARA in a similar pattern with the testosterone concentration, but the concentration of gonadotrophin in serum was not affected by dietary ARA. The response of gene expression of PKCβ, ARRB1, ARRB2, ERK2 and ATF3 in the brain to dietary ARA was in good agreement with those of GnRH and testosterone, indicating the possible involvement of PKC‐ARRB‐ERK‐ATF3 pathway in signalling transduction of GnRH. However, the PI3K/Akt and TLR/NF‐κB pathways may not be directly involved in the regulation of GnRH metabolism by ARA. This is the first study reporting the possible involvement of PKC/MAPK pathways in regulation of reproductive endocrine processes by long‐chain polyunsaturated fatty acid in brain of marine fish.

A 74‐day feeding trial was conducted to investigate the efficacy of n‐6 polyunsaturated fatty acid (n‐6 PUFA) in diets for tiger puffer. Six experimental diets with graded levels of n‐6 PUFA (from 6.01 to 37.98 g/kg) replacing saturated fatty acids (SFAs) were used in this study. Increasing levels of n‐6 PUFA resulted in decreased weight gain (from 472.6% to 349.4%), feed efficiency and crude protein content in fish, but increased lipid accumulation in whole fish and liver. Liver samples from fish fed the lowest and highest level of n‐6 PUFA were subjected to transcriptomic assay. Analysis of the differentially expressed genes showed that high dietary levels of n‐6 PUFA tended to induce glycolysis and triacylglycerol biosynthesis, but reduce synthesis of amino acid and protein, and cause impairment to liver health. The mitochondrial DNA copy number, activity of mitochondrial respiratory chain complex I and glycogen content in the liver, which indicate energy supply status, significantly decreased with increasing levels of dietary n‐6 PUFA. In conclusion, high n‐6 PUFA/SFA ratios in diet led to reduced energy supply (by approximately 50%) and increased hepatic lipid accumulation (by 55%), which probably consequently negatively affect protein synthesis, liver health and growth of tiger puffer.

To investigate the different effects of dietary α-linolenic acid (ALA) and linoleic acid (LA) on the euryhaline fish Japanese seabass, a feeding trial followed by hepatic transcriptome assay was conducted. Two experimental diets containing 10% LA-rich sunflower seed oil (diet LA) or 10% ALA-rich perilla oil (diet ALA) were used in the feeding trial. LA and ALA in diets were characteristically incorporated into fish tissues while no significant difference was observed in growth performance and body proximate composition between groups LA and ALA. Compared to LA, ALA up-regulated transcription of 49 unigenes and down-regulated those of 311 unigenes. Quantitative RT-PCR studies on eight lipid metabolism-related genes and seven randomly selected genes were conducted to validate the transcriptomic results. Lipid metabolism-related genes ApoA1, ApoA4, ApoE, FABP1, FABP3, FABP4, FATP6, and DGAT1, as well as ribosomal proteins L9e, L13e, and S4e, were transcriptionally down-regulated by ALA. The differentially expressed genes (DEGs) were primarily enriched in Gene Ontology terms such as Lipid transport, Protein metabolic process, and Ribosome biogenesis, as well as in KEGG pathways such as Complement and coagulation cascades and Ribosome. The Protein-Protein Interaction (PPI) network based on the peptide biosynthesis-related DEGs showed that ribosomal proteins such as SAe, L4e, S4e, L15e, L9e, and L13Ae had high betweenness centrality in the dietary regulation of peptide biosynthetic processes. In conclusion, under the present experimental conditions, a high level of dietary α-linolenic acid tended to suppress lipid transport and protein biosynthetic processes in the liver of Japanese seabass at the gene expression level.

A 74-day feeding trial was conducted to investigate the effects of different levels of dietary arachidonic acid (ARA) on mucosal barrier and microbiota in the intestine of tiger puffer Takifugu rubripes. Three isonitrogenous and isolipidic experimental diets were formulated to contain different ARA levels, i.e., 0.33 (Control), 2.45 (L-ARA), and 23.41 (H-ARA) g kg−1 dry matter. Each diet was fed to triplicate tanks. The results showed that the integrity of intestinal epithelial cells was impaired by increasing ARA levels. The leucocyte infiltration in the lamina propria was also enhanced by dietary ARA supplementation. Compared to the control group, group H-ARA had significantly down-regulated gene expression of intestinal tight junction proteins (claudin-4, claudin-7 and zonula occludens-1), but significantly up-regulated expression of myosin light chain kinase (MLCK). Gene expression of claudin-7 was significantly down-regulated also in group L-ARA. The intestinal lysozyme activity significantly decreased with increasing dietary ARA levels. Compared to the Control, diet H-ARA significantly up-regulated the gene expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 8 (IL-8), and down-regulated that of anti-inflammatory cytokines transforming growth factor-β1 (TGF-β1) in the hind intestine. Intestinal gene expression of Toll-like receptor 3 (TLR-3) and nuclear factor kappa B p100 (NF-κB p100) was significantly up-regulated by H-ARA. Sequencing of bacterial 16 s rRNA V4 region showed that Proteobacteria, Tenericutes and Firmicutes were dominant in the intestine of tiger puffer. Fish fed diet H-ARA showed significantly decreased alpha diversity of microbiota and increased relative abundance of Arcobacter, a potential pathogenic genus. These results suggested that dietary ARA tended to induce the dysfunction of intestinal mucosal barrier and modulate the intestinal bacterial community, and MLCK and TLR3/NF-κB signaling were possibly involved in these processes. The ARA content in diets for tiger puffer needs to be carefully managed considering the intestinal sensitivity to dietary ARA of this fish.

Tiger puffer have a unique lipid storage pattern. They store lipid predominantly in liver. To investigate the hepatic physiology of tiger puffer in response to a moderately high level of dietary lipid, a 74-day feeding trial was conducted, followed by a hepatic transcriptome assay. Two experimental diets which had an optimal (10.44%, Control) or moderately higher (14.64%, high-lipid diet, HL) dietary lipid level was formulated. Each diet was fed to triplicate tanks. No significant difference was observed in growth and feed efficiency between the two groups, but the survival was significantly lower in group HL. The HL diet also resulted in significantly higher lipid accumulation in liver, which was reflected in the hepatic histology. The biochemical parameters in serum confirmed the existence of stress in liver caused by high lipid intake. A total of 63 differentially expressed genes between groups were observed in the hepatic transcriptome assay, which were primarily enriched in Gene Ontology (GO) terms such as Endopeptidase activity, Secretion, and Organic acid transport, as well as in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways such as Protein processing in endoplasmic reticulum and DNA replication. The HL diet tended to inhibit the protein synthesis and secretion in liver, and this was evidenced by activities/concentrations of selected proteins secreted into intestine. Genes related to liver protection and repair were transcriptionally stimulated by diet HL. In conclusion, compared to the optimal lipid requirement, a moderately higher lipid level in the diet for tiger puffer did not compromise the growth performance, but tended to inhibit the protein secretion function of liver and stimulate the processes related to liver protection and repair. The present results will be beneficial to both feed management in tiger puffer farming and elucidation of lipid metabolism characteristics in fish which store lipid in liver.

A 10‐week feeding experiment in indoor flow‐through seawater system was conducted to investigate the effects of dietary krill hydrolysate on the expression of growth‐related genes in juvenile turbot (Scophthalmus maximus L.; initial body weight 9.45 ± 0.01 g). Three isonitrogenous and isolipidic experimental diets containing high plant protein were formulated to contain 0 (control), 50 g/kg (LKH) and 100 g/kg (HKH) krill protein hydrolysate (KH) to replace fishmeal, respectively. Triplicate groups of 30 fish were fed for 10 weeks to apparent satiation twice daily. At the end of the feeding trial, the mRNA expressions of insulin‐like growth factor (IGF‐1) gene in liver, peptide transporters (PepT1) gene in pyloric caeca and proximal intestine and neuropeptide Y (NPY) gene in brain in all groups were determined. IGF‐1, PepT1 and NPY expression levels in HKH group were significantly increased compared with those of LKH and control (p

In fish, it has been debatable whether branched-chain amino acid (BCAA) imbalance will show antagonistic effects on the growth and the concentrations of plasma or tissue BCAAs. The objective of this study were to investigate the effects of incremental levels of dietary leucine on the growth (trial 1) and potential antagonistic effects between BCAAs (trial 2) in tiger puffer. This study was composed of two separate trials. In trial 1, six experimental diets were prepared by the basal diet containing incremental levels of leucine (19.3, 24.9, 28.6, 32.6, 36.3 and 40.7 g kg−1 of dry diet). In trial 2, five experimental diets were formulated to contain a proper amount of leucine (29.0 g kg−1 of dry diet), a deficiency of leucine (12.6 g kg−1 of dry diet), an excess of leucine (55.9 g kg−1 of dry diet), an excess of isoleucine (43.2 g kg−1 of dry diet) and an excess of valine (49.6 g kg−1 of dry diet). All diets in two trials were fed to fish in triplicate net cages and polyethylene tanks, respectively. In trial 1, growth increase relative to initial body weight was 206.2–227.5%, while growth performance and body condition indices were not significantly affected by different levels of dietary leucine and did not show significantly linear or quadratic relationships with incremental levels of dietary leucine. In trial 2, growth performance also did not exhibit significant differences in fish fed imbalanced BCAAs diets. Compared with a proper amount of leucine group, the activities of branched-chain α-keto acid dehydrogenase enzyme complex (BCKDH) and BCKDH kinase increased in the leucine or valine excessive diet. The expressions of amino acid transporters (B0AT1 and y+LAT2) and peptide transporter 1 (PepT1) were down-regulated by excessive dietary leucine, isoleucine or valine. Free valine concentration in plasma and muscle increased in the leucine deficient diet compared to a proper amount and an excess of leucine in diets. In conclusion, the growth of tiger puffer was not affected by the diets containing 12.6–55.9 g kg−1 leucine. An excess of dietary leucine, isoleucine or valine possibly resulted in antagonism between BCAAs based on the activities of enzymes related to BCAA catabolism and the expressions of amino acid transporters, although the growth was not significantly depressed by one excessive BCAA when the other two BCAAs met the requirements for tiger puffer. In addition, plasma and muscle valine levels indicated that the antagonism of valine to leucine were observed in the leucine deficient diet, but not in the leucine excessive diet.

Fish are the main source of long-chain polyunsaturated fatty acids (LC-PUFA, >C18) for human consumption. In general, it has been widely observed that the fatty acid (FA) profiles of farmed fish are reflective of the diet. However, the degree of tissue FA "distortion" based on incorporation of different dietary FA into fish tissues varies greatly depending on FA type, fish species and environmental factors. In terms of fish FA composition, this variation has not been comprehensively reviewed, raising the question: "Are fish what they eat?". To date, this remains unanswered in detail. To this end, the present review quantitatively summarized the 'diet-fish' FA relationship via an analysis of FA composition in diets and fish tissues from 290 articles published between 1998 and 2018. Comparison of this relationship among different fish species, tissue types or individual FA was summarized. Furthermore, the influence of environmental factors such as temperature and salinity, as well as of experimental conditions such as fish size and trophic level, feeding duration, and dietary lipid level on this relationship are discussed herein. Moreover, as a means of restoring LC-PUFA in fish, an emphasis was paid to the fish oil finishing strategy after long-term feeding with alternative lipid sources. It is envisaged that the present review will be beneficial in providing a more comprehensive understanding of the fundamental relationship between the FA composition in diets, and subsequently, in the farmed fish. Such information is integral to maintaining the quality of farmed fish fillets from the perspective of FA composition.

The tissue distribution pattern of lipid is highly diverse among different fish species. Tiger puffer has a special lipid storage pattern, storing lipid predominantly in liver. In order to better understand the lipid physiology in fish storing lipid in liver, the present study preliminarily investigated the tissue distribution of transcription for 29 lipid metabolism-related genes in tiger puffer, which are involved in lipogenesis, fatty acid oxidation, biosynthesis and hydrolysis of glycerides, lipid transport, and relevant transcription regulation. Samples of eight tissues, brain, eye, heart, spleen, liver, intestine, skin, and muscle, from fifteen juvenile tiger puffer were used in the qRT-PCR analysis. The intestine and brain had high transcription of lipogenic genes, whereas the liver and muscle had low expression levels. The intestine also had the highest transcription level of most apolipoproteins and lipid metabolism-related transcription factors. The transcription of fatty acid β-oxidation-related genes was low in the muscle. The peroxisomal fatty acid oxidation may dominate over mitochondrial β-oxidation in the liver and intestine of tiger puffer, and the MAG pathway probably predominates over the G3P pathway in re-acylation of absorbed lipids in the intestine. The intracellular glyceridases were highly transcribed in the brain, eye, and heart. In conclusion, in tiger puffer, the intestine could be a center of lipid metabolism whereas the liver is more likely a pure storage organ for lipid. The lipid metabolism in the muscle could also be inactive, possibly due to the very low level of intramuscular lipid.

Taurine (TAU) plays important roles in the metabolism of bile acids, cholesterol and lipids. However, little relevant information has been available in fish where TAU has been identified as a conditionally essential nutrient. The present study aimed to investigate the effects of dietary TAU on the metabolism of bile acids, cholesterol and lipids in tiger puffer, which is both an important aquaculture species and a good research model, having a unique lipid storage pattern. An 8-week feeding trial was conducted in a flow-through seawater system. Three experimental diets differed only in TAU level, that is, 1·7, 8·2 and 14·0 mg/kg. TAU supplementation increased the total bile acid content in liver but decreased the content in serum. TAU supplementation also increased the contents of total cholesterol and HDL-cholesterol in both liver and serum. The hepatic bile acid profile mainly includes taurocholic acid (94·48 %), taurochenodeoxycholic acid (4·17 %) and taurodeoxycholic acid (1·35 %), and the contents of all these conjugated bile acids were increased by dietary TAU. The hepatic lipidomics analysis showed that TAU tended to decrease the abundance of individual phospholipids and increase those of some individual TAG and ceramides. The hepatic mRNA expression study showed that TAU stimulated the biosynthesis of both bile acids and cholesterol, possibly via regulation of farnesoid X receptor and HDL metabolism. TAU also stimulated the hepatic expression of lipogenic genes. In conclusion, dietary TAU stimulated the hepatic biosynthesis of both bile acids and cholesterol and tended to regulate lipid metabolism in multiple ways.

Our previous studies have shown that ARA regulated the gonadal steroidogenesis and the synthesis of gonadotrophin-releasing hormone (GnRH) in the brain in a gender-dependent manner in Chinese tongue sole, a marine teleost with typical sex dimorphism. As a following up-study, the current study aimed to clone and characterize a gene responding to dietary ARA differently between males and females, cholesterol 25-hydroxylase (ch25h), which has important roles in cholesterol and lipid metabolism of mammals, but is rarely investigated in fish. The full-length cDNA of Chinese tongue sole ch25h was cloned and characterized, and its transcription in the gonads, brain and liver of both males and females in response to dietary ARA levels (0.34%, 2.53%, and 9.63% of TFA) was investigated. The Chinese tongue sole Ch25h, which putatively is Ch25h subtype B, shared moderate identity to its known orthologs of other teleost and lower identity to human Ch25h. It has high transcription levels in the gonads, followed by skin and muscle, but low levels in the intestine, spleen, and kidney. High ARA levels significantly increased the ch25h transcription in the gonads and brain of male fish, but did not affect the transcription in the females. However, the effect of dietary ARA on ch25h transcription in the liver showed an opposite gender-difference pattern to those in the gonads and brain. To our knowledge, this is the first study in marine teleost investigating the nutritional regulation of ch25h expression.