Wei-Na Xu

Dietary carbohydrate/lipid ratios affect stress, oxidative status and non-specific immune responses of fingerling blunt snout bream, Megalobrama amblycephala.

This study aimed to evaluate the effects of dietary carbohydrate/lipid (CHO:L) ratios on stress, liver oxidative status and non-specific immune responses of fingerling blunt snout bream. Fish were fed six isonitrogenous and isoenergetic diets containing various CHO:L ratios for 10 weeks. After the feeding trial, fish were challenged by Aeromonas hydrophila and survival rate was recorded for the next 10 days. The lowest plasma cortisol, lactate, aspartate aminotransferase and alanine aminotransferase were all observed in fish fed a CHO:L ratio of 5.64. They were significantly (P < 0.05) lower than those of fish offered the lowest CHO:L ratio, but showed little difference (P > 0.05) with those of fish fed the highest CHO:L ratio. This also held true for liver catalase and glutathione peroxidase activities, whereas the opposite was true for liver reduced glutathione contents, plasma lysozyme and alternative complement (ACH50) activities. Contrary to leucocyte counts, plasma glucose levels, liver malondialdehyde contents, blood haemoglobin contents and erythrocyte numbers all increased significantly (P < 0.05) with decreasing dietary CHO:L ratios. The highest plasma total protein and globulin content both observed in fish fed a CHO:L ratio of 2.45 was significantly (P < 0.01) higher than that of fish offered the lowest CHO:L ratio, but showed no statistical difference (P > 0.05) with that of the other groups. After challenge, fish fed the lowest CHO:L ratio obtained significantly (P < 0.05) low survival rate. However, survival rate showed little difference (P > 0.05) as dietary CHO:L ratios ranged from 3.67 to 24.20. The results of this study indicated that high dietary lipid may cause metabolic stress of fingerling blunt snout bream, as might consequently lead to the elevated liver oxidation rates, impaired liver function, depressed immunity and reduced resistance to A. hydrophila infection of this species, whereas the opposite was true for carbohydrate enriched diets.

Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate immunity, antioxidant capability and disease resistance of triangular bream (Megalobrama terminalis)

This study was conducted to investigate the effects of fructooligosaccharide (FOS) and Bacillus licheniformis (B. licheniformis) and their interaction on innate immunity, antioxidant capability and disease resistance of triangular bream Megalobrama terminalis (average initial weight 30.5 ± 0.5 g). Nine experimental diets were formulated to contain three FOS levels (0, 0.3% and 0.6%) and three B. licheniformis levels (0, 1 × 10(7), 5 × 10(7) CFU g(-1)) according to a 3 × 3 factorial design. At the end of the 8-week feeding trial, fish were challenged by Aeromonas hydrophila (A. hydrophila) and survival rate was recorded for the next 7 days. The results showed that leucocyte counts, alternative complement activity as well as total serum protein and globulin contents all increased significantly (P < 0.05) as dietary B. licheniformis levels increased from 0 to 1 × 10(7) CFU g(-1), while little difference (P > 0.05) was observed in these parameters in terms of dietary FOS levels. Both plasma alkaline phosphatase and phenoloxidase activities were significantly (P < 0.05) affected only by dietary FOS levels with the highest values observed in fish fed 0.6 and 0.3% FOS, respectively. Both immunoglobulin M content and liver superoxide dismutase (SOD) activity were significantly affected (P > 0.05) by both FOS and B. licheniformis. Liver catalase, glutathione peroxidase as well as plasma SOD activities of fish fed 1 × 10(7) CFU g(-1)B. licheniformis were all significantly (P < 0.05) higher than that of the other groups, whereas the opposite was true for malondialdehyde content. After A. hydrophila challenge, survival rate was not affected (P > 0.05) by either FOS levels or B. licheniformis contents, whereas a significant (P < 0.05) interaction between these two substances was observed with the highest value observed in fish fed 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis. The results of this study indicated that dietary FOS and B. licheniformis could significantly enhance the innate immunity and antioxidant capability of triangular bream, as well as improve its disease resistance. The best combination of these two prebiotics and/or probiotics was 0.3% FOS and 1 × 10(7) CFU g(-1)B. licheniformis.

Hepatic β-Oxidation and Regulation of Carnitine Palmitoyltransferase (CPT) I in Blunt Snout Bream Megalobrama amblycephala Fed a High Fat Diet

High-fat diets may promote growth, partly through their protein-sparing effects. However, high-fat diets often lead to excessive fat deposition, which may have a negative impact on fish such as poor growth and suppressive immune. Therefore, this study investigated the effects of a fat-rich diet on the mechanisms of fat deposition in the liver. Three-hundred blunt snout bream (Megalobrama amblycephala) juveniles (initial mass 18.00±0.05 g) were fed with one of two diets (5% or 15% fat) for 8 weeks. β-Oxidation capacity and regulation of rate-limiting enzymes were assessed. Large fat droplets were present in hepatocytes of fish fed the high-fat diet. This observation is thought to be largely owing to the reduced capacity for mitochondrial and peroxisomal β-oxidation in the livers of fish fed the high-fat diet, as well as the decreased activities of carnitine palmitoyltransferase (CPT) I and acyl-CoA oxidase (ACO), which are enzymes involved in fatty-acid metabolism. Study of CPT I kinetics showed that CPT I had a low affinity for its substrates and a low catalytic efficiency in fish fed the high-fat diet. Expression of both CPT I and ACO was significantly down-regulated in fish fed the high-fat diet. Moreover, the fatty-acid composition of the mitochondrial membrane varied between the two groups. In conclusion, the attenuated β-oxidation capacity observed in fish fed a high-fat diet is proposed to be owing to decreased activity and/or catalytic efficiency of the rate-limiting enzymes CPT I and ACO, via both genetic and non-genetic mechanisms.

The Effect of Exposure to a High-Fat Diet on MicroRNA Expression in the Liver of Blunt Snout Bream (Megalobrama amblycephala)

Blunt snout bream (Megalobrama amblycephala) are susceptible to hepatic steatosis when maintained in modern intensive culture systems. The aim of this study was to investigate the potential roles of microRNAs (miRNAs) in diet-induced hepatic steatosis in this species. MiRNAs, small non-coding RNAs that regulate gene expression at the posttranscriptional level, are involved in diverse biological processes, including lipid metabolism. Deep sequencing of hepatic small RNA libraries from blunt snout bream fed normal-fat and high-fat diets identified 202 (193 known and 9 novel) miRNAs, of which 12 were differentially expressed between the normal-fat and high-fat diet groups. Quantitative stem-loop reverse transcriptase-polymerase chain reaction analyses confirmed the upregulation of miR-30c and miR-30e-3p and the downregulation of miR-145 and miR-15a-5p in high-fat diet-fed fish. Bioinformatics tools were used to predict the targets of these verified miRNAs and to explore potential downstream gene ontology biological process categories and Kyoto Encyclopedia of Genes and Genomes pathways. Six putative lipid metabolism-related target genes (fetuin-B, Cyp7a1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 2, 3-oxoacid CoA transferase 1b, stearoyl-CoA desaturase, and fatty-acid synthase) were identified as having potential important roles in the development of diet-induced hepatic steatosis in blunt snout bream. The results presented here are a foundation for future studies of miRNA-controlled lipid metabolism regulatory networks in blunt snout bream.

Effect of Trichlorfon on Hepatic Lipid Accumulation in Crucian Carp Carassius auratus gibelio

This study evaluated the toxic effects of the organophosphate pesticide trichlorfon on hepatic lipid accumulation in crucian carp Carassius auratus gibelio. Seventy-five fish were divided into five groups (each group in triplicate), and then exposed to 0, 0.5, 1.0, 2.0, and 4.0 mg/L of trichlorfon and fed with commercial feed for 30 d. At the end of the experiment, plasma and hepatic lipid metabolic biochemical status were analyzed. Triglyceride contents were significantly (P < 0.05) increased in liver but decreased in plasma after 1.0, 2.0, and 4.0 mg/L trichlorfon treatments. Plasma insulin contents were markedly (P < 0.05) increased when trichlorfon concentrations were 0.5, 1.0, and 4.0 mg/L. There were no significant differences in hepatic hormone-sensitive lipase contents between the trichlorfon-treated fish and the controls. Hepatic cyclic adenosine 3′, 5′-monophosphate, very-low-density lipoprotein, and apolipoprotein B100 contents were decreased in the fish when trichlorfon concentration was 2.0 mg/L. Furthermore, electron microscope observations showed rough endoplasmic reticulum dilatation and mitochondrial vacuolization in hepatocytes with trichlorfon exposure. On the basis of morphological and physiological evidence, trichlorfon influenced crucian carp hepatic pathways of lipid metabolism and hepatocellular ultrastructure, which resulted in lipid accumulation in the liver.

Association of Mitochondrial Dysfunction with Oxidative Stress and Immune Suppression in Blunt Snout Bream Megalobrama amblycephala Fed a High-Fat Diet

High-fat diets may have favorable effects on growth, partly based on protein sparing, but high-fat diets often lead to fatty liver (excessive fat deposition in the liver), which may be deleterious to fish growth and health. The goal of this study was therefore to investigate possible adverse effects and how they develop. Juvenile Blunt Snout Bream Megalobrama amblycephala (initial weight ± SE = 17.70 ± 0.10 g) were fed two diets (5% fat [control] or 15% fat). After 8 weeks, fish that were fed the 15% fat diet showed a high rate of mortality and poor growth. The histological results clearly showed that the high fat intake resulted in fat and glycogen accumulation and structural alterations of the hepatocytes, mitochondria, and nuclei. In the high-fat group, impairments of the mitochondria included mitochondrial swelling and the loss of cristae and matrix. Fish that were given the 15% fat diet exhibited low succinate dehydrogenase and Na(+),K(+)-ATPase activities and increased cytochrome-c release from the mitochondria. Expression of genes for complex I and III subunits of the mitochondrial respiratory chain were down-regulated in fish that received the high-fat diet. Increases in malondialdehyde level and the ratio of oxidized glutathione to reduced glutathione suggested oxidative stress in the livers of fish from the high-fat diet group. Moreover, the lower leukocyte count, lysozyme and alternative complement activities, and globulin level in fish that received the high-fat diet indicated suppressive immune responses. Overall, the intake of excessive fat impaired mitochondrial bioenergetics and physiological functions. The dysfunction of the mitochondria subsequently mediated oxidative stress and hepatocyte apoptosis, which in turn led to the reduced efficacy of the immune system.

Identification and differential expression of hepatopancreas microRNAs in red swamp crayfish fed with emodin diet

Using high-throughput Illumina Solexa system, the differential miRNA expressions from hepatopancreas in red swamp crayfish (Procambarus clarkii) fed with diets containing 0 (control) and 75 mg emodin kg(-1) (trial) were identified, respectively. As a result, 13,335,928 raw reads from the control sample and 14,938,951 raw reads from the trial sample were obtained while 13,053,344 (98.77%) and 14,517,522 (98.34%) small RNA were identified, respectively. 106 mature miRNAs (belonging to 68 miRNA gene families) were identified. 35 miRNAs displayed significantly differential expressions between two libraries. Of these, comparing to the control library, 6 miRNAs were significantly up-regulated and 29 miRNAs were significantly down-regulated. Moreover, 5 novel miRNAs (2 from control sample, 3 from trial sample) and target genes were predicted. GO analysis suggested that these miRNAs might be involved in innate immune response, growth, metabolism, cellular process, biological regulation and stimulus response. Our knowledge from this study could contribute to a better understanding of the miRNAs roles in regulating innate immune response and the study of miRNA function in crayfish.

Molecular characterization of carnitine palmitoyltransferase IA in Megalobrama amblycephala and effects on its expression of feeding status and dietary lipid and berberine.

Carnitine palmitoyltransferase I (CPT I, EC 2.3.1.21) controls the main regulatory step of fatty acid oxidation, and hence studies of its molecular characterization are useful to understand lipid metabolism in cultured fish. Here, a full-length cDNA coding CPT I was cloned from liver of blunt snout bream Megalobrama amblycephala. This cDNA obtained covered 2499bp with an open reading frame of 2181bp encoding 726 amino acids. This CPT I mRNA predominantly expressed in heart and white muscle, while little in eye and spleen. The phylogenetic tree constructed on the basis of sequence alignments among several vertebrate species suggests that this blunt snout bream CPT I sequence belongs to the CPT IA family. In order to investigate the characterization of CPT IA mRNA expression, post-prandial experiment and feeding trial were conducted. The results showed that CPT IA mRNA expression was unchanged from 2 to 12h, and then significantly increased at 24h post-feeding in liver and heart. Berberine, an alkaloid, was identified as a promising lipid-lowering drug. In order to elucidate the effect of berberine on CPT I expression, fish were fed for 8 weeks with three diets (low-fat diet (LFD, 5% fat), high-fat diet (HFD, 15% fat), and berberine-supplemented diet (BSD, 15% fat). The results showed that HFD could decrease the expression of CPT IA and PPARα, while BSD increased those expressions.

Cloning and characterization of microsomal triglyceride transfer protein gene and its potential connection with peroxisome proliferator-activated receptor (PPAR) in blunt snout bream (Megalobrama amblycephala)

Microsomal triglyceride transfer protein (MTTP), a major intracellular protein capable of transferring neutral lipids, plays a pivotal role in the assembly and secretion of apolipoprotein B-containing lipoproteins. In this study, MTTP cDNA was firstly cloned from the liver of blunt snout bream (Megalobrama amblycephala), the full-length cDNA covered 3457-bp with an open reading frame of 2661-bp, which encodes 886 amino acids, including a putative signal peptide of 24 amino acids long. After the feeding trial, a graded tissue-specific expression pattern of MTTP was observed and high expression abundance in the liver and intestine indicated its major function in lipid transport in this fish species. In addition, expression of genes encoding MTTP as well as peroxisome proliferator-activated receptor (PPAR), which are transcription factors and serve as key regulators in lipid homoeostasis, was all affected by dietary lipid and choline supplementations. Elevated dietary lipid levels significantly increased the liver, intestinal and muscle MTTP mRNA abundance. Additionally, the down-regulation of MTTP expression in the liver and muscle was observed when fish were fed with inadequate choline supplementation in high-fat diet, yet up-regulated as supplementing extra choline in diet. Expressions of PPARα and PPARβ in the liver and muscle showed similar trend of MTTP expression. The results suggested the potential connection of MTTP and PPAR in response to different dietary nutritional factors. Furthermore, extra choline supplementations could promote lipid transfer and enhance fatty acid oxidation, which indicated a molecular mechanism of choline on diminishing fat accumulation in blunt snout bream.