Exposure of farmed fish to petroleum hydrocarbon pollution and the recovery process: A simulation experiment with tiger puffer Takifugu rubripes.

Petroleum hydrocarbon (PH) pollution threatens both wild and farmed marine fish. How this pollution affects the nutrient metabolism in fish and whether this effect can be recovered have not been well-known. The present study aimed to evaluate these effects with a feeding trial on tiger puffer, an important farmed species in Asia. In a 6-week feeding trial conducted in indoor flow-through water, fish were fed a control diet (C) or diets supplemented with diesel oil (0.02 % and 0.2 % of dry matter, named LD and HD, respectively). Following this feeding trial was a 4-week recovery period, during which all fish were fed a same normal commercial feed. At the end of the 6-week feeding trial, dietary PH significantly decreased the fish growth and lipid content. The PH significantly accumulated in fish tissues, in particular the liver, and caused damages in all tissues examined in terms of histology, anti-oxidation status, and serum biochemical changes. Dietary PH also changed the volatile flavor compound profile in the muscle. The hepatic transcriptome assay showed that the HD diet tended to inhibit the DNA replication, cell cycle and lipid synthesis, but to stimulate the transcription of genes related to liver protection/repair and lipid catabolism. The 4-week recovery period to some extent mitigated the damage caused by PH. After the recovery period, the inter-group differences in some parameters disappeared. However, the differences in lipid content, anti-oxidase activity, liver PH concentration, and histological structure still existed. In addition, differences in cellular chemical homeostasis and cytokine-cytokine receptor interaction at the transcriptional level can still be observed, indicated by the hepatic transcriptome assay. In conclusion, 6 weeks of dietary PH exposure significantly impaired the growth performance and health status of farmed tiger puffer, and a short-term recovery period (4 weeks) was not sufficient to completely mitigate this impairment.

Recovery of Fatty Acid and Volatile Flavor Compound Composition in Farmed Tiger Puffer (Takifugu rubripes) with a Fish Oil-Finishing Strategy.

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.

Effects of Lysophosphatidylcholine on Intestinal Health of Turbot Fed High-Lipid Diets.

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-1ß, 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.

Additional supplementation of sulfur-containing amino acids in the diets improves the intestinal health of turbot fed high-lipid diets.

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-1ß, 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.

Response of lipid and fatty acid composition of turbot to starvation under different dietary lipid levels in the previous feeding period.

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.

Effects of lysine and leucine in free and different dipeptide forms on the growth, amino acid profile and transcription of intestinal peptide, and amino acid transporters in turbot (Scophthalmus maximus).

This study was conducted to evaluate the effects of different dipeptides (lysine-leucine, lysine-glycine, and leucine-glycine) and free amino acids (lysine and leucine) on the growth, gene expression of intestinal peptide and amino acid transporters, and serum free amino acid concentrations in turbot. Fish (11.98 ± 0.03 g) were fed four experimental diets supplementing with crystalline amino acids (CAA), lysine-leucine (Lys-Leu), lysine-glycine (Lys-Gly), and leucine-glycine (Gly-Leu). Fish protein hydrolysate (FPH) containing a mixture of free amino acids and small peptides was designed as a positive control diet. There was no significant difference in the growth and feed utilization among three dipeptide diets (Lys-Leu, Lys-Gly, and Gly-Leu). Compared with the CAA group, feed efficiency ratio was significantly higher in the Lys-Leu and Lys-Gly groups, and protein efficiency ratio was significantly higher in the Lys-Leu group. For peptide transporter, oligopeptide transporter 1 (PepT1) mRNA level was not affected by dietary treatments. For amino acid transporters, lower expression of B0 neutral amino acid transporter 1 (B0AT1) and proton-coupled amino acid transporter 1 (PAT1) were observed in fish fed the dipeptide and FPH diets compared with the CAA diet. In conclusion, juvenile turbot fed Lys-Leu, Gly-Leu, and Lys-Gly had a similar growth performance, whereas lysine and leucine in the Lys-Leu form can be utilized more efficiently for feed utilization than those in free amino acid from. In addition, compared to free amino acids, dipeptides and fish protein hydrolysate in diets may down-regulate the expression of amino acid transporters but did not affect the expression of PepT1.

Tissue distribution of transcription for 29 lipid metabolism-related genes in Takifugu rubripes, a marine teleost storing lipid predominantly in liver.

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 ß-oxidation-related genes was low in the muscle. The peroxisomal fatty acid oxidation may dominate over mitochondrial ß-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.

Dietary taurine stimulates the hepatic biosynthesis of both bile acids and cholesterol in the marine teleost, tiger puffer (Takifugu rubripes).

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.

Dietary methionine increased the lipid accumulation in juvenile tiger puffer Takifugu rubripes.

Methionine (Met) is one of the most important amino acids in fish feed. The effects of dietary Met on lipid deposition in fish varied a lot among different studies. The present study was aimed at investigating the effects of dietary Met supplementation on the lipid accumulation in tiger puffer, which have a unique lipid storage pattern. Crystalline L-Met was supplemented to a low-fishmeal control diet to obtain two experimental diets with a low (1.1% of dry weight, L-MET) or high Met level (1.6% of dry weight, H-MET). A 67-day feeding trial was conducted with juvenile tiger puffer (average initial weight, 13.83 g). Each diet was fed to triplicate tanks (30 fish in each tank). The results showed that the total lipid contents in whole-body and liver significantly increased with increasing dietary Met levels. The hepatosomatic index, weight gain, and total bile acid content in serum showed similar patterns in response to dietary Met treatments, while the lipid content in muscle was not affected. The hepatic contents of 18-carbon fatty acids were elevated by dietary Met supplementation. The Hepatic mRNA expression of lipogenetic gene such as FAS, GPAT, PPARγ, ACLY, and SCD1 was down-regulated, while the gene expression of lipolytic genes ACOX1 and HSL, as well as that of ApoB100, were up-regulated by increasing dietary Met levels. The hepatic lipidomics of experimental fish was also analyzed. In conclusion, increasing dietary Met levels (0.61%, 1.10%, and 1.60%) increased the hepatic lipid accumulation in tiger puffer. The mechanisms involved warrant further studies.

Regulation of FADS2 transcription by SREBP-1 and PPAR-α influences LC-PUFA biosynthesis in fish.

The present study was conducted to explore the mechanisms leading to differences among fishes in the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs). Replacement of fish oil with vegetable oil caused varied degrees of increase in 18-carbon fatty acid content and decrease in n-3 LC-PUFA content in the muscle and liver of rainbow trout (Oncorhynchus mykiss), Japanese seabass (Lateolabrax japonicus) and large yellow croaker (Larimichthys crocea), suggesting that these fishes have differing abilities to biosynthesize LC-PUFAs. Fish oil replacement also led to significantly up-regulated expression of FADS2 and SREBP-1 but different responses of the two PPAR-α homologues in the livers of these three fishes. An in vitro experiment indicated that the basic transcription activity of the FADS2 promoter was significantly higher in rainbow trout than in Japanese seabass or large yellow croaker, which was consistent with their LC-PUFA biosynthetic abilities. In addition, SREBP-1 and PPAR-α up-regulated FADS2 promoter activity. These regulatory effects varied considerably between SREBP-1 and PPAR-α, as well as among the three fishes. Taken together, the differences in regulatory activities of the two transcription factors targeting FADS2 may be responsible for the different LC-PUFA biosynthetic abilities in these three fishes that have adapted to different ambient salinity.

Effects of oxidised dietary fish oil and high-dose vitamin E supplementation on growth performance, feed utilisation and antioxidant defence enzyme activities of juvenile large yellow croaker (Larmichthys crocea).

This study was conducted to elucidate the effects of oxidised dietary lipids and high-dose vitamin E (VE) on growth performance and immune responses of large yellow croaker. Juvenile fish (initial average body weight of 7·82 (sem 0·68) g) were fed diets containing either fresh fish oil (fresh diet, peroxide value (POV)=1·72 mEq/kg) or fish oil oxidised to varying degrees (oxidised diets, POV=28·29-104·21 mEq/kg), with or without supplementary 600 mg VE/kg diet, for 10 weeks in floating cages. Growth was significantly lower and feed intake (g/100 g body weight per d) was higher in fish fed the oxidised diet. Supplementation with VE increased the growth of fish fed the oxidised diets, but significantly decreased the growth of fish fed the fresh diet. Hepatosomatic index increased with increasing dietary POV and decreased with VE supplementation. Hepatic catalase activity, superoxide dismutase (SOD) activity and malondialdehyde content were significantly higher in fish fed the oxidised diets, and these values decreased significantly following VE supplementation. However, hepatic SOD activity was enhanced by VE supplementation in fish fed the fresh diet. Air-exposure mortality was significantly increased by dietary POV, and this effect was inhibited by VE supplementation. These results suggest that dietary oxidised fish oil could stimulate the activities of antioxidant defence enzymes in stressed large yellow croaker. High-dose VE supplementation can alleviate oxidative stress of large yellow croaker fed oxidised fish oil, but can exert deleterious effects on fish in the absence of oxidative stress.

Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

Effects of dietary n-3 highly unsaturated fatty acids on growth, nonspecific immunity, expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans).

The study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3 HUFA) on growth, nonspecific immunity, expression of some immune related genes and disease resistance of juvenile large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans). Six isoproteic and isolipidic diets were formulated with graded levels of n-3 HUFA ranging from 0.15% to 2.25% of the dry weight and the DHA/EPA was approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (initial average weight 9.79 ± 0.6 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 58 days. Results showed that moderate n-3 HUFA level (0.98%) significantly enhanced growth compared with the control group (0.15% HUFA) (P < 0.05), while higher n-3 HUFA levels (1.37%, 1.79% and 2.25%) had detrimental effects on the growth though no significance was found (P > 0.05). Nitro blue tetrazolium (NBT) positive leucocytes percentage of head kidney and serum superoxide dismutase (SOD) activity increased with increasing n-3 HUFA from 0.15% to 0.60%, and decreased with further increase of n-3 HUFA from 0.60% to 2.25% (P < 0.05). Serum lysozyme activity increased significantly as n-3 HUFA increased from 0.15% to 1.37%, and then decreased with n-3 HUFA from 1.37% to 2.25% (P > 0.05). There were no significant differences in phagocytosis index (PI) of head kidney leucocytes among dietary treatments (P > 0.05). The hepatic mRNA expression of Toll-like receptor 22 (TLR22) and Myeloid differentiation factor 88 (MyD88) was significantly up-regulated in fish fed the diets with low or moderate levels, while in kidney this increment was only found at specific sampling time during the natural infestation of parasites. The 13 d cumulative mortality rate following natural infestation of parasites decreased with n-3 HUFA increased from 0.15% to 0.60% (P < 0.05), and significantly increased with n-3 HUFA from 0.60% to 2.25% (P < 0.05). Results of this study suggested that fish fed low or moderate dietary n-3 HUFA had higher growth, nonspecific immune responses, expression levels of some immune related genes and disease resistance of large yellow croaker following natural infestation of parasites and dietary n-3 HUFA may regulate fish immunity and disease resistance by altering the mRNA expression levels of TLR22 and MyD88.