Universal PCR primers for ribosomal protein gene introns of fish

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.     

Effects of Dietary Protein and Lipid Levels with Different Protein‐to‐Energy Ratios on Growth,Feed Utilization,and Plasma Biochemical Parameters of Dianchi Golden‐line Barbell,Sinocyclocheilus grahami

An 8‐wk feeding trial was conducted to evaluate the effects of varying dietary protein and lipid levels with different protein‐to‐energy (P/E) ratios on growth, feed utilization, and plasma biochemical parameters of Sinocyclocheilus graham. Nine experimental diets were formulated to contain three protein levels (35, 40, and 45%), and each with three lipid levels (4, 8, and 12%), to produce a range of P/E ratios from 71.31 to 98.64 mg protein/kcal. Each diet was randomly assigned to triplicate groups of 25 fish. Fish grew best when fed the diet containing 45% protein and 12% lipid. However, fish fed the diet with 40% protein and 8% lipid had similar weight gain and specific growth rate as those fed the above diet but showed relatively better feed conversion ratio, protein efficiency ratio, and protein and energy retentions. Additionally, plasma total amino acids and blood urea nitrogen contents and γ‐glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase activities in fish fed the diet containing 40% protein and 8% lipid were relatively low among the dietary treatments. These results may indicate that a diet containing 40% protein and 8% lipid with P/E ratio of 84.05 mg protein/kcal would be suitable for the optimal growth, feed utilization, and health of S. graham.     

Effects of long-term feed deprivation on body weight loss,muscle composition,plasma metabolites,and intermediate metabolism of meagre (<Emphasis Type="Italic">Argyrosomus regius</Emphasis>) under different water temperatures

The effect of feed deprivation at four water temperatures (17, 20, 23, 26 °C) was investigated in meagre (Argyrosomus regius) of initial mean weight ± SD, 116.16?±?4.74 g, in triplicate groups. Fish were deprived of feed for a period of 60 days and sampled on days 0, 14, 41, and 60, during which body weight, specific growth rate, somatic indices, muscle proximate composition, plasma metabolite levels (total lipids, proteins, cholesterol, triglycerides, glucose), and liver and muscle enzymatic activities [L-lactate dehydrogenase (L-LDH), citrate synthase (CS), malate dehydrogenase (MDH)] were evaluated. Long-term feed deprivation resulted in a significant decrease in body weight, condition factor (CF), hepatosomatic index (HSI), muscle lipids, and plasma metabolites (all except proteins) and increase in muscle moisture. Plasma glucose concentration decreased with time and became significantly lower at 41 and 60 days. Glucose concentration and weight loss expressed a different response in the short term (14 days) than in the long term (14 and 60 days) of feed deprivation, suggesting a change in glucose metabolic profile. After 60 days of feed deprivation, there was an increase in the L-LDH activity in the liver at all temperature levels, which reflects a rising glycolytic potential by activating the carbohydrate metabolism and an ATP-dependent demand. MDH activity increased in the liver and muscle, except at 17 °C in the muscle, which indicates aerobic glycolysis and lipolysis. CS activity in the liver increased after the 60 days, whereas that in the muscle decreased, indicating the muscle is less dependent on aerobic oxidation for energy reserves.     

Nutritional history does not modulate hepatic oxidative status of European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) submitted to handling stress

The aim of the present study was to assess the impact of an acute handling stress on hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed diets differing in lipid so urce and carbohydrate content. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as lipid source and with 20 or 0% gelatinized starch as carbohydrate source. Triplicate groups of fish with 74 g were fed each diet during 13 weeks and then subjected to an acute handling stress. Stress exposure decreased hematocrit (Ht) and hemoglobin (Hb) levels. Independent of dietary treatment, stress exposure increased hepatic lipid peroxidation (LPO). Stressed fish exhibited lower glucose 6-phosphate dehydrogenase (G6PD), catalase (CAT), and superoxide dismutase (SOD) activities, independent of previous nutritional history. In the VO groups, stress exposure increased glutathione peroxidase (GPX) activity. Diet composition had no effect on Ht and Hb levels. In contrast, dietary carbohydrate decreased hepatic LPO and CAT activity and increased glutathione reductase (GR) and G6PD activities. Dietary lipids had no effect on LPO. Fish fed the VO diets exhibited higher G6PD activity than fish fed the FO diets. In conclusion, dietary carbohydrates contributed to the reduction of oxidative stress in fish. However, under the imposed handling stress conditions, liver enzymatic antioxidant mechanisms were not enhanced, which may explain the overall increased oxidative stress.     

Rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) anesthesia with myrcene: efficacy and physiological responses in comparison with eugenol

The aim of the present study was to investigate anesthetic efficacy of myrcene in rainbow trout (Oncorhynchus mykiss) along with the fish biochemical response to anesthesia in comparison with eugenol. In the first experiment, 240 fish were stocked in 12 tanks and acclimatized to experimental conditions for 2 weeks. Then, the fish of each tank were subjected to one concentration of either eugenol (12, 20, 30, 50, 80, and 130 μL/L) or myrcene (100, 150, 200, 300, 400, and 500 μL/L) concentrations. Induction time of and recovery time from anesthesia were recorded for each fish separately. Using these results, desired concentrations to induce anesthesia within 60, 180, 300, and 600 s were determined, being 81, 30, 19, and 10 μL/L eugenol and 531, 251, 177, and 111 μL/L myrcene. In the second experiment, 96 fish were stocked in 8 tanks. Six fish were netted from each tank and exposed to the calculated eugenol or myrcene concentrations. Blood samples were taken after the fish reached anesthesia. The results showed that there was no significant difference in serum lactate, alanine transaminase, and alkaline phosphatase. Increase in the induction time of anesthesia resulted in increased serum glucose with no significant difference between the anesthetics. Increase in induction time of anesthesia led to increase in serum lactate dehydrogenase activity in the eugenol-anesthetized fish and aspartate transaminase activity in myrcene-anesthetized fish. In conclusion, myrcene is capable to anesthetize rainbow trout, but at higher concentrations compared to eugenol. In addition, biochemical analysis showed that increase in induction time of anesthesia leads to hyperglycemia and increase in AST and LDH activities depending on anesthetic type.     

Upstream migration mechanisms of juvenile temperate sea bass <Emphasis Type="Italic">Lateolabrax japonicus</Emphasis> in the stratified Yura River estuary

Hydrographic conditions and distributions of juvenile temperate sea bass Lateolabrax japonicus were observed in early spring from 2009 to 2012 in the Yura River estuary, which is highly stratified due to its small tides and consequent seawater intrusion into the bottom layer of the river as a salt wedge. In all four studied years, the upstream expansion of their distribution coincided with the timing of the salt wedge intrusion from the lower to upper estuary, indicating that juvenile fish used salt wedge intrusions to ascend the estuary in early spring. However, juveniles sometimes remained in the nearshore area even when the salt wedge intrusion had already occurred, indicating that other triggers are also likely to be necessary. We therefore evaluated the effects of temperature on upstream migration behaviors. The relationship between the mean temperature they experienced from hatch until starting the ascent and mean age of each cohort at the upstream migration fitted with the law of effective cumulative temperature. Most cohorts ascended the river at an effective cumulative temperature of approximately 500 °C–days. This suggests that higher temperatures would lead to a shorter period prior to the upstream migration.     

Amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> interleukin-10 negatively suppresses host cell-mediated immunity

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC)?+?rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM⁺ cells among spleen leukocytes in N. seriolae?+?rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.     

Effect of body weight on the chemical composition and collagen content of snakehead fish <Emphasis Type="Italic">Channa striata</Emphasis> skin

Snakehead fish (Channa striata) skin is a fishery by-product that has the potential for further processing because it contains a high amount of organic matter. This study investigates the effect of body weight on the chemical composition and collagen content of snakehead fish skin. This study used fresh snakehead fish of either gender. Their body weights were divided into three groups: small, medium, and large size. The characteristics of snakehead fish skin included proximate composition, amino acid content, collagen content, microstructure, and minerals. Snakehead fish skin from fish of different body weights indicated that the moisture content and ash tended to decrease, the protein content was relatively stable, and the lipid content tended to increase with increasing body weight. Glycine and proline comprised the highest percentages of amino acids at all levels of body weight, and the presence of hydroxyproline showed that snakehead fish skin was the source of collagen. There was no significant difference in body weight observed on collagen protein content of the skin. This study on the microstructure and mineral content of snakehead fish skin can be used as supporting information to promote the potential utility and economic value of the skin.