Qinghui Ai

A comparative study: In vitro effects of EPA and DHA on immune functions of head-kidney macrophages isolated from large yellow croaker (Larmichthys crocea)

Comparative effects of different concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on immune responses of head-kidney macrophages isolated from large yellow croaker were studied in vitro. After exposing to serum-free medium for 1 day, cultured cells were incubated in medium supplemented with graded levels of EPA or DHA (0, 5, 25, 100, 200 and 1000 μM, respectively) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12 h, 24 h and 36 h, respectively. Control samples were incubated in the absence of EPA or DHA (2% bovine serum albumin, BSA). Following stimulation, cell viability, lipid peroxidation, secretary phopholipase A2 (sPLA2) and prostaglandin E2 (PGE2) production as well as some immune parameters including phagocytosis, respiratory burst activity and interleukin 1β (IL-1β) production were determined. Results showed that EPA and DHA affected cell viability in dose-dependent and time-dependent manners. In particular, cell viability was significantly decreased after 24 h and 36 h incubation with 1000 μM EPA or DHA (P < 0.05). Higher levels of EPA (200 and 1000 μM) caused a significant increase in the production of malondialdehyde (MDA) (P < 0.05), while DHA did not significantly affect the MDA production. EPA significantly increased the intracellular superoxide anion synthesis which, on the contrary, was significantly reduced by DHA. Phagocytosis percentage (PP) values were significantly higher in treatments with 5 μM DHA (P < 0.05), but significantly decreased by 200 and 1000 μM EPA and DHA compared to the control group (P < 0.05). Decreased PGE2 production was produced by cells treated with relatively low doses of EPA or DHA. When high levels of stimulants (1000 μM EPA or DHA) were used, PGE2 levels were elevated and reached a significant level (P < 0.05). Both EPA and DHA significantly inhibited the production of sPLA2, where DHA exerted the more potent inhibitory effects than EPA. No pronounced effect was observed on IL-1β production among all the treatments, and IL-1β level in cell culture supernatant was fairly low (only approximately 6 pg/ml). Those findings suggested that EPA and DHA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.

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 conjugated linoleic acid on growth, non-specific immunity, antioxidant capacity, lipid deposition and related gene expression in juvenile large yellow croaker ( Larmichthys crocea ) fed soyabean oil-based diets

The effects of conjugated linoleic acid (CLA) on growth performance, non-specific immunity, antioxidant capacity, lipid deposition and related gene expression were investigated in the large yellow croaker (Larmichthys crocea). Fish (7·56 (SEM 0·60) g) were fed soyabean oil-based diets with graded levels of CLA (0, 0·42, 0·83, 1·70%) for 70 d. Quantitative PCR was used to assess the effects of CLA on the transcription of inflammation- and fatty acid oxidation-related genes. Growth in fish fed the diet with 0·42% CLA was significantly higher. Also, phagocytic index and respiratory burst activity were significantly higher in fish fed the diets containing 0·42 and 0·83% CLA, respectively. Hepatic total antioxidative capacity and catalase activities increased significantly when CLA increased from 0 to 0·83%, and then decreased with further increase of CLA. However, hepatic malondialdehyde content decreased significantly as dietary CLA increased. Lipid concentration in the whole body and muscle increased significantly with increasing dietary CLA. Transcription of genes related to inflammation (cyclo-oxygenase-2 and IL-b) in the liver and kidney and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in the kidney decreased significantly as dietary CLA increased. PPAR alpha and acyl CoA oxidase expression in the liver decreased significantly as CLA increased from 0·42 to 1·70%. These results strongly suggest that dietary CLA could significantly affect growth performance, non-specific immunity, antioxidant capacity, lipid deposition and transcription of inflammation- and fatty acid oxidation-related genes of the large yellow croaker. This may contribute to our understanding of the mechanisms related to the physiological effects of dietary CLA in fish.

Dietary Lipid Levels Influence Lipid Deposition in the Liver of Large Yellow Croaker (Larimichthys crocea) by Regulating Lipoprotein Receptors, Fatty Acid Uptake and Triacylglycerol Synthesis and Catabolism at the Transcriptional Level

Ectopic lipid accumulation has been observed in fish fed a high-lipid diet. However, no information is available on the mechanism by which dietary lipid levels comprehensively regulate lipid transport, uptake, synthesis and catabolism in fish. Therefore, the present study aimed to gain further insight into how dietary lipids affect lipid deposition in the liver of large yellow croaker(Larimichthys crocea). Fish (150.00±4.95 g) were fed a diet with a low (6%), moderate (12%, the control diet) or high (18%) crude lipid content for 10 weeks. Growth performance, plasma biochemical indexes, lipid contents and gene expression related to lipid deposition, including lipoprotein assembly and clearance, fatty acid uptake and triacylglycerol synthesis and catabolism, were assessed. Growth performance was not significantly affected. However, the hepato-somatic and viscera-somatic indexes as well as plasma triacylglycerol, non-esterified fatty acids and LDL-cholesterol levels were significantly increased in fish fed the high-lipid diet. In the livers of fish fed the high-lipid diet, the expression of genes related to lipoprotein clearance (LDLR) and fatty acid uptake (FABP11) was significantly up-regulated, whereas the expression of genes involved in lipoprotein assembly (apoB100), triacylglycerol synthesis and catabolism (DGAT2, CPT I) was significantly down-regulated compared with fish fed the control diet, and hepatic lipid deposition increased. In fish fed the low-lipid diet, the expression of genes associated with lipoprotein assembly and clearance (apoB100, LDLR, LRP-1), fatty acid uptake (CD36, FATP1, FABP3) and triacylglycerol synthesis (FAS) was significantly increased, whereas the expression of triacylglycerol catabolism related genes (ATGL, CPT I) was reduced compared with fish fed the control diet. However, hepatic lipid content in fish fed the low-lipid diet decreased mainly due to low dietary lipid intake. In summary, findings of this study provide molecular insight into the role of lipid deposition in the liver in response to different dietary lipid contents.

Cloning and characterization of SREBP-1 and PPAR-α in Japanese seabass Lateolabrax japonicus, and their gene expressions in response to different dietary fatty acid profiles

In the present study, putative cDNA of sterol regulatory element-binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor α (PPAR-α), key regulators of lipid homoeostasis, were cloned and characterized from liver of Japanese seabass (Lateolabrax japonicus), and their expression in response to diets enriched with fish oil (FO) or fatty acids such as palmitic acid (PA), stearic acid (SA), oleic acid (OA), α-linolenic acid (ALA), and n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), was investigated following feeding. The SREBP-1 of Japanese seabass appeared to be equivalent to SREBP-1a of mammals in terms of sequence feature and tissue expression pattern. The stimulation of the mRNA expression level of SREBP-1 in liver of Japanese seabass by dietary fatty acids significantly ranked as follows: PA, OA>SA, ALA, and n-3 LC-PUFA>FO. A new PPAR-α subtype in Japanese seabass, PPAR-α2, was cloned in this study, which is not on the same branch with Japanese seabass PPAR-α1 and mammalian PPAR-α in the phylogenetic tree. Liver gene expression of PPAR-α1 of Japanese seabass was inhibited by diets enriched with ALA or FO compared to diets enriched with PA or OA, while the gene expression of PPAR-α2 of Japanese seabass was up-regulated by diets enriched with ALA or n-3 LC-PUFA compared to diets enriched with OA or FO. This was the first evidence for the great divergence in response to dietary fatty acids between PPAR-α1 and PPAR-α2 of fish, which indicated probable functional distribution between PPAR-α isotypes of fish.

Dietary ALA, But not LNA, Increase Growth, Reduce Inflammatory Processes, and Increase Anti‑Oxidant Capacity in the Marine Finfish Larimichthys crocea

Whilst aquaculture feed is increasingly formulated with the inclusion of plant oils replacing fish oil, and increasing research effort has been invested in understanding the metabolic effects of reduced dietary n-3 long chain poly unsaturated fatty acids (n-3 LC-PUFA), relatively little information is available on the potential direct metabolic roles of dietary alpha-linolenic acid (ALA, 18:3n-3) and alpha-linolenic acid/linoleic acid (LNA, 18:2n-6) ratio in cultured marine finfish species. In this study, four plant oil based diets, with varying ALA/LNA ratio (0.0, 0.5, 1.0 and 1.5) were fed to juvenile large yellow croakers (Larimichthys crocea) and compared to a fish oil-based control diet (CD) to evaluate the resulting effects on growth, nonspecific immunity, anti-oxidant capacity and related gene expression. High dietary LNA negatively impacted fish growth performance, nonspecific immunity and antioxidant capacity, but growth and immunity were maintained to levels comparable to CD by increasing the ratio of dietary ALA/LNA. The over-expression of genes associated with inflammation (cyclooxygenase-2 and interleukin-1β) and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in croakers fed high concentrations of LNA were reduced to levels comparable to those fed CD by increasing dietary ALA/LNA. This study showed that dietary ALA, by increasing the overall n-3/n-6 PUFA ratio, exerts direct anti-inflammatory and antioxidant effects, similar to those exerted by dietary n-3 LC-PUFA.

Replacement of dietary fish oil with vegetable oils improves the growth and flesh quality of large yellow croaker (Larmichthys crocea)

We investigated the effect of the replacement of dietary fish oil with vegetable oils on the growth and flesh quality of large yellow croaker (Larmichthys crocea). The basal diet (FO) was formulated to contain 66.5% fish meal and 6.4% menhaden fish oil; whereas the other 3 experimental diets were formulated by replacing the fish oil with 50% soybean oil (SO50), 100% soybean oil (SO100) and 100% palm oil (PO100), respectively. The 4 diets were randomly assigned to 4 floating sea cages (3.0 m × 3.0 m × 3.0 m), and each was stocked with 250 fish individuals with an initial average weight of 245.29 g ± 7.45 g. The fish were fed to apparent satiation twice a day at 5:00 and 17:00, respectively, for 12 weeks. Experimental analysis showed that the specific growth rate of fish fed SO50 or PO100 were significantly higher than that of fish fed FO or SO100 (P<0.05), and crude lipid contents of ventral muscle and viscera were significantly lower in fish fed FO than in those fed the other 3 diets (P<0.05). No significant differences in condition factor, viscerosomatic index, hepatosomatic index, gutted yield and colorimetric values of fish among the dietary treatments were observed (P>0.05). Compared to FO diet, SO50, SO100 and PO100 diets led to substantial decreases in the liquid loss and water loss from fresh fillets (1 d, 4°C) (P<0.05). Similarly, thiobarbituric acid reactive substance (TBARS) values of fillets under different storage conditions (1 d, 4°C; 7 d, 4°C; 4 weeks, −20°C; 8 weeks, −20°C) decreased significantly after partial or complete replacement of fish oil with vegetable oils. These findings indicated that the growth performance and selected flesh quality properties (liquid holding capacity and TBARS value) of large yellow croaker were substantially improved by replacing dietary fish oil with vegetable oils.

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.

Molecular Cloning, Functional Characterization and Nutritional Regulation of the Putative Elongase Elovl5 in the Orange-Spotted Grouper (Epinephelus coioides).

The enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs) are widely studied in fish species, as fish are the main source of n-3 LC-PUFAs for human beings. In the present study, a putative gene for elovl5, which encodes a key enzyme involved in LC-PUFA synthesis, was cloned and functionally characterized, and its transcription in response to dietary n-3 LC-PUFA exposure was investigated. Moreover, cell transfection and luciferase assays were used to explore the mechanism underlying the regulation of elovl5. The full-length cDNA of elovl5 was 1242 bp (excluding the polyA tail), including an 885 bp coding region encoding a 295 amino acid protein that possesses all of the characteristic features of elovl proteins. Functional characterization of heterologously expressed grouper Elovl5 indicated that it effectively elongates both C18 (18:2n-6, 18:3n-3, 18:3n-6 and 18:4n-3) and C20 (20:4n-6 and C20:5n-3) PUFAs, but not the C22 substrates. The expression of elovl5 was significantly affected by dietary n-3 LC-PUFA exposure: a high n-3 LC-PUFA level repressed the expression of elovl5 by slightly down-regulating the expression of sterol regulatory element-binding protein (SREBP)-1 and liver X receptor (LXR) α, which are major regulators of hepatic lipid metabolism. Promoter studies showed that grouper elovl5 reporter activity was induced by over-expression of LXRα but not SREBP-1. This finding suggests that elovl5 is a direct target of LXRα, which is involved in the biosynthesis of PUFAs via transcriptional regulation of elovl5. These findings may contribute to a further understanding of the mechanism underlying the regulation of LC-PUFA biosynthesis in marine fish species.

EFFECTS OF VITAMIN E ON ANTIOXIDANT ENZYME ACTIVITIES AND FATTY ACID COMPOSITIONS IN JUVENILE ABALONE HALIOTIS DISCUS HANNAI INO

Abstract A 240-day feeding trial was conducted in a recirculated water system to investigate the effects of dietary vitamin E on the activities of antioxidant enzymes (catalase, CAT; superoxide dismutase, SOD; glutathione peroxidase, GPX) and the composition of fatty acids in abalone, Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 0.71 ± 0.00 g; initial shell length: 15.49 ± 0.04 mm) were fed to satiation one of three semipurified diets containing 0, 50, and 5,000-mg/kg vitamin E, respectively. Abalone were sampled on the 120th day and the 240th day, respectively. There were no significant differences in activities of CAT and SOD in soft body of abalone fed with different levels of dietary vitamin E for 120 days (P > 0.05), but significantly higher activity of GPX was found with 5,000-mg/kg dietary vitamin E (P < 0.05). Activities of CAT and GPX were significantly elevated by dietary vitamin E on the 240th day. The lowest value of 18:1n-9, 18:2n-6 and the highest value of 22:6n-3 in soft body were found with 50 mg/kg dietary vitamin E supplement on the 120th day. On the 240th day, the content of monounsaturated fatty acids (MUFA) in abalone with 50-mg/kg dietary vitamin E supplement was significantly higher than those in the other two treatments (P < 0.05). There were no significant effects of dietary vitamin E on the content of polyunsaturated fatty acids (PUFA) in abalone during the two sampling periods (P > 0.05). In conclusion, 50-mg/kg dietary vitamin E supplement elevated the activities of antioxidant enzymes and could protect MUFA from peroxidation damage. Excessive dietary vitamin E (5,000 mg/kg) did not serve as an antioxidant any more, but tended to be a pro-oxidant in the soft body of abalone.