Kangsen Mai

Interaction of dietary Bacillus subtilis and fructooligosaccharide on the growth performance, non-specific immunity of sea cucumber, Apostichopus japonicus.

A feeding experiment was conducted to investigate the interaction of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on the growth performance, immunity, intestinal microflora and disease resistance of sea cucumber (Apostichopus japonicus). Five hundred and forty individuals (initial body weight: 5.06 +/- 0.10 g, mean +/- S.E) were fed nine practical diets according to a 3 x 3 factorial design: the basal diet as the control diet supplemented with three levels of B. subtilis (0, 1.82 x 10(7) or 4.95 x 10(7) CFU g(-1) diet), crossed with 0, 0.25% or 0.50% FOS. After 8 weeks, three sea cucumbers per tank were sampled for bacterial quantification and immunity determination. Then all the sea cucumbers left were challenged by Vibrio splendidus. The results showed that dietary B. subtilis significantly increased the specific growth rate (SGR), total coelomocytes counts (TCC), phagocytosis of sea cucumbers, the counts of total viable bacteria and disease resistance to V. splendidus (P < 0.05), whereas the counts of Vibrio decreased. However, dietary B. subtilis had no significant effect on phenoloxidase (PO) activity in coelomocyte lysate supernatant (CLS) (P > 0.05). The SGR, PO activity, total viable bacterial counts (TBC) and Vibrio counts (VBC) were significantly affected by dietary FOS. In the group with 0.50% FOS, TCC, phagocytosis and PO activity significantly increased compared to the group fed without FOS in diet (P < 0.05). In the groups with 1.82 x 10(7) CFU B. subtilis/g diet, FOS supplementation remarkably decreased VBC. And higher level of FOS (0.50%) resulted in significantly higher TCC and PO activity compared with 0.25% FOS (P < 0.05). Moreover, the animals fed with diets supplemented with 0.25% and 0.50% FOS at each B. subtilis level had notably lower cumulative mortality after 14 days following V. splendidus exposure (P < 0.05). Under the experimental conditions, dietary B. subtilis and FOS had a synergistic effect on enhancing immunity and disease resistance of sea cucumber (P < 0.05).

Effects of potential probiotic Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine influences of potential probiotic Bacillus subtilis T13 (isolated from intestine of healthy sea cucumbers) on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Animals were fed with diets containing B. subtilis T13 at 0, 10(5), 10(7) and 10(9) CFU/g for 30 days, respectively. At the end of the growth trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers from each replicate were challenged with V. splendidus. Results showed that administration of B. subtilis T13 had significant effect on the specific growth rates (SGR) of sea cucumbers (P < 0.05). Phagocytosis, respiratory burst activity and total nitric oxide synthase (T-NOS) activity were significantly improved in coelomocytes of sea cucumbers fed with T13 at 10(9) CFU/g diet (P < 0.05). The highest values of the total coelomocytes counts (TCC) and superoxide dismutase (SOD) activity were found in sea cucumbers fed diet containing T13 at 10(9) CFU/g. The cumulative mortality after V. splendidus challenge decreased significantly in sea cucumbers fed with T13 at dose of 10(9) CFU/g (P < 0.05). The present study confirmed the potential beneficial effects of B. subtilis T13 as dietary probiotic in juvenile A. japonicus.

Effects of dietary zinc on gene expression of antioxidant enzymes and heat shock proteins in hepatopancreas of abalone Haliotis discus hannai.

The expression patterns of different genes encoding antioxidant enzymes and heat shock proteins were investigated, in present study, by real-time quantitative PCR in the hepatopancreas of abalone Haliotis discus hannai fed with different levels of dietary zinc (6.69, 33.8, 710.6 and 3462.5 mg/kg) for 20 weeks. The antioxidant enzymes include Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT), mu-glutathione-s-transferase (mu-GST) and thioredoxin peroxidase (TPx). The results showed that the mRNA expression of these antioxidant enzymes increased and reached the maximum at the dietary zinc level of 33.8 mg/kg, and then dropped progressively. Expression levels of the heat shock proteins (HSP26, HSP70 and HSP90) firstly increased at 33.8 mg/kg dietary Zn level, and reached to the maximum at 710.6 mg/kg, then dropped at 3462.5 mg/kg (p<0.05). Excessive dietary Zn (710.6 and 3462.5 mg/kg) significantly increases the Zn content and significantly decreases the total antioxidant capacity (T-AOC) in hepatopancreas (p<0.05). These findings showed that dietary Zn (33.8 mg/kg) could highly trigger the expression levels of antioxidant enzymes and heat shock proteins, but excessive dietary Zn (710.6 and 3462.5 mg/kg) induces a high oxidative stress in abalone.

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.

Identification, organ expression and ligand-dependent expression levels of peroxisome proliferator activated receptors in grass carp (Ctenopharyngodon idella)

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors belonging to the nuclear receptor family, and can regulate various genes involved in lipid metabolism. The aim of the present study was to investigate the tissue distribution patterns of PPARs and their ligand specificities in grass carp. We cloned three PPAR isotypes of the species and evaluated their organ distribution patterns using real-time PCR. Through analyzing the deduced amino acid sequences identities between the products cloned in grass carp and those described in other species, we concluded that the same type of PPAR amino acid sequences in different species were with high homology, and different subtypes of PPAR in the same species were with low homology. The mRNA constitutive expression level of PPARα predominated in the liver, but was weak in other tested tissues. PPARβ was present in all tested organs, and particularly abundant in heart, liver and muscle. PPARγ was only detected in the liver, and to a lesser extent in brain, muscle and visceral adipose tissue. Grass carp were intraperitoneally injected with 50 mg kg(-1) body mass (bw) dose of clofibrate, 42 mg kg(-1) bw dose of 2-bromo palmitate and 1 mg kg(-1) bw dose of 15-deoxy-Δ(12,14) prostaglandin J2 (15d-PGJ2), respectively, and the relative changes of the mRNA abundance of PPARs in liver were analyzed by real-time PCR. Clofibrate was able to increase the expressions of both PPARα and β, but was not able to for PPARγ. 2-bromo palmitate could affect the expressions of both PPARβ and γ, but was not able to for PPARα. 15d-PGJ2 was able to induce PPARβ expression, but PPARα and γ were not enhanced. Consequently, these results indicate that clofibrate, 2-bromo palmitate and 15d-PGJ2 could be applied as the activators of grass carp PPARs.

Molecular cloning, characterization and expression analysis of heat shock protein 90 from Pacific abalone, Haliotis discus hannai Ino in response to dietary selenium.

In the present study, the cDNA of heat shock protein 90 from Pacific abalone Haliotis discus hannai Ino (HdhHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of HdhHSP90 was of 2660 bp, including an open reading frame (ORF) of 2187 bp encoding a polypeptide of 728 amino acids with predicted molecular weight of 84.134 kDa and theoretical isoelectric point of 4.619. BLAST analysis revealed that HdhHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in HdhHSP90, which indicated that HdhHSP90 should be a cytosolic member of the HSP90 family. The expression levels of HdhHSP90 in haemocytes and hepatopancreas were measured by real-time PCR after Pacific abalone were fed with semi-purified diets containing graded levels of selenium (0.15, 1.32 and 48.70 mg Kg(-1)) for 20 weeks, respectively. The results showed that the expression levels of HdhHSP90 transcript were significantly up-regulated and reached the maximum (0.47-fold) in hepatopancreas of Pacific abalone fed with optimal dietary Se (1.32 mg Kg(-1)) (p < 0.05). However, these levels significantly decreased in hepatopancreas at the excessive dietary Se (48.70 mg Kg(-1)) (p < 0.01). In haemocytes, the expression of HdhHSP90 mRNA increased and reached the maximum (0.96-fold) at the excessive dietary Se (48.70 mg Kg(-1)) (p < 0.01). It is implied that the expression levels of HdhHSP90 could be affected by dietary Se in hepatopancreas and haemocytes, and HdhHSP90 was potentially involved in the anti-oxidation responses in Pacific abalone H. discus hannai.

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.