Proteolytic digestive enzyme response in Japanese flounder larvae <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> fed two types of rotifers <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> species complex

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p?<?0.05) at 50 kDa in 1.5- and 3-h incubation whereas L-type treatment had not completely hydrolyzed the proteins after 3 h at the same molecular weight. Larvae fed SS-type had significantly higher (p?<?0.05) trypsin activity at 3, 5, 6, 7 DAH. Contribution of live prey to trypsin fraction in larvae showed significantly higher (p?<?0.05) fraction for SS-type at 5 DAH (2.18?±?0.44%) and 6 DAH (2.04?±?0.29%) and the effect of exogenous trypsin from live prey was relatively low when compared to the total trypsin activity in larvae. This study discusses the differences in ability to digest proteins in Japanese flounder when fed different rotifer morphotypes and highlights the adaptability of this species to alternative rotifer morphotypes during its early developmental stages.     

Optimization of Antioxidant Peptides Production from the Mantle of Cuttlefish (Sepia pharaonis) Using RSM and Fractionation

Persian Gulf cuttlefish mantles were hydrolyzed (CPH) using alcalase, and the optimal hydrolysis parameters were obtained for the highest degree of hydrolysis (DH) and strongest antioxidant (based on their ability to quench 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals) activity using response surface methodology (RSM). The predicted optimal parameters of DH and quenching DPPH radicals was: pH of 7.88, 50.2°C, 150 min, and enzyme to substrate ratio of 1.5%. The reducing power (RP) and ability of optimized peptides to quench ABTS radicals in a gastro-intestinal track model system increased during the intestinal stage, while scavenging ability against DPPH radicals dropped (P < 0.05). The oxidation of lipid was retarded in a lecithin-liposome model added with optimized CPH in a concentration dependent response. Ultrafiltration of optimized CPH showed that the 3–10 KDa fraction had the greatest DPPH radical scavenging activity, the 10–30 KDa fraction had the highest reducing power, and the <3 KDa fraction had the greatest ABTS radical scavenging activity.     

Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro

Glucose 6-phosphate dehydrogenase (G6PD) is a key enzyme catalyzing the first step of the pentose phosphate pathway which generates NADPH for anabolic pathways and protection systems in various organisms, including fish. In the present study, G6PD was purified from grass carp (Ctenopharyngodon idella) hepatopancreas using the methods of 2′,5′-ADP-Sepharose 4B affinity chromatography followed by DEAE Sepharose Fast Flow ion exchange chromatography. The characterization of G6PD and inhibition effects of several metal ions on G6PD activity in vitro were also determined. Grass carp hepatopancreas G6PD, with a specific activity of 18 U/mg protein, was purified 1,066-fold with a yield of 19.5 % and Mr of 71.85 kDa. The enzyme had a temperature optimum of 42 °C, pH optimum of 7.5 and 9.0. The K _m values for G6-P and NADP⁺ were determined to be 0.026, 0.0068 mM, respectively. The V _max values for G6-P and NADP⁺ were 2.20 and 2.27 μM min^?1 mg protein^?1, respectively. The catalytic efficiency for G6-P and NADP as the substrates was 0.085 and 0.334 × 10^?6 min^?1 mg protein^?1, respectively. Inhibition effects of metal ions on the purified G6PD activity indicated that IC₅₀ values of Zn⁺², Mn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.42, 0.54, 0.94, 1.20, and 4.17 mM, respectively. The Ki constants of Zn⁺², Al⁺³, Cu⁺², and Cd⁺² were 0.52, 1.12, 0.26, and 4.8 mM, respectively. Zn⁺², Al⁺³, and Cd⁺² showed competitive inhibition, while Cu⁺² inhibited the G6PD in a noncompetitive inhibition manner. Our study provided important information about the control of the grass carp liver PPP, the biosynthesis of several important related biomolecules, and the status of detoxification systems in grass carp liver in relation to metabolism.     

Optimization of Hydrolysis Conditions for Production of Gelatin Hydrolysates from Shark Skin Byproduct and Evaluation of Their Antioxidant Activities

ABSTRACT

The effect of Alcalase hydrolysis conditions on antioxidant activities of shark skin gelatin hydrolysate (SSGH) byproduct was studied. Optimal hydrolysis condition for SSGH at 60°C, pH 7.5, and 2.70 AU kg^?1 gelatin protein was used for varied hydrolysis times (10–90 min). The IC₅₀ against DPPH radicals of SSGH hydrolyzed for 90 min (SSGH-90) was 27.39 mg ml^?1, which is greater than ascorbic acid. SSGH-90 was predominantly composed of 15–20 kDa protein fragments that regulated increases in peroxide value and thiobarbituric acid reactive substances values and suppressed decrease in DHA of a fish oil-in-water emulsion during storage at 30°C for 7 days.     

Assessment of the Nutritive,Biochemical, Antioxidant and Antibacterial Potential of Eight Tropical Macro algae Along Kachchh Coast,India as Human Food Supplements

Eight tropical marine macro algae were investigated for in-vitro antioxidant, antibacterial, and biochemical properties. The moisture content [% DW (dry weight)] ranged from 7.21 to 14.72%; ash, 24.92 to 47.04%; lipids, 0.73 to 2.67%; protein, 4.56 to 12.59%; and carbohydrate, 30.1 to 48.51%. The % sum of polyunsaturated fatty acids (PUFAs) ranged from 21.71 to 78.22%, while omega-6/omega-3 (ω6/ω3) ratio was from 0 to 2.08, which remained within the prescribed World Health Organization (WHO) standards (<10). The % sum of essential amino acids (EAAs) ranged from 86.18 to 204.66, and Na/K ratio ranged from 0.37 to 2.85. The extracts exhibited significant (P < 0.05) values for total phenol (7.25–16.0 µg PGE mg^–1 DW) and total flavonoid (4.06–15.63 µg QE mg^–1 DW) at 200 µg/ml. Antibacterial activity was tested against the selected food spoilage and pathogenic bacteria. The principal component analysis (PCA) confirmed a positive correlation between total phenolic content, 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical-scavenging activities, and ferric reducing antioxidant power (FRAP), which supports the integration of seaweeds as ingredients in functional foods.     

Effects of dietary Bacillus pumilus on growth performance,innate immunity and digestive enzymes of giant freshwater prawns (Macrobrachium rosenbergii)

The present study was conducted to investigate the effects of Bacillus pumilus on the growth performance, innate immunity and digestive enzymes of the giant freshwater prawn, Macrobrachium rosenbergii. Prawns (mean weight: 1.81 ± 0.01 g) were fed different levels of B. pumilus at doses of 0, 1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ CFU/g to produce one control group and three experimental groups (G1, G2 and G3), respectively. After a 60‐day experimental feeding period, the results showed that the final weight, weight gain rate (WGR) and specific growth rate (SGR) were significantly higher in experimental groups than in the control (p < 0.05), whereas these improvements did not differ significantly (p > 0.05) among experimental groups. The improvements in phagocytic activity, respiratory burst (RBs) activity and the activity of catalase (CAT), nitric oxide synthase (NOS) and acid phosphatase (ACP) were significantly higher in G2 and G3 than in the control and G1 (p < 0.05). Further, the activity of CAT, NOS and ACP significantly increased with an increase in the dietary level of B. pumilus (p < 0.05). The activity of phenoloxidase (PO) in G3 was significantly higher than that in the control (p < 0.05). The activity of amylase was significantly higher in the groups G2 and G3 than that in the control group. G3 improved the activity of protease compared with the control (p < 0.05). In conclusion, the results of this study indicated that the potential use of B. pumilusat a dose of 1 × 10⁸ CFU/g feed could improve the growth, immunity and digestive enzymes of M. rosenbergii.     

Effect of dietary supplementation of Bacillus licheniformis on gut microbiota, growth and immune response in giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879)

The present study was conducted to evaluate the effect of dietary supplementation of probiotic bacterium Bacillus licheniformis on the gut microbiota, growth and immune response in Macrobrachium rosenbergii juveniles (4.0 ± 0.02 g). Five isocaloric experimental diets supplemented with different concentrations of B. licheniformis, viz. Control (without probiotic supplementation), T₁ (1.0 × 10⁶ cfu g^?1feed), T₂ (1.0 × 10⁷ cfu g^?1feed), T₃ (1.0 × 10⁸ cfu g^?1 feed) and T₄ (1.0 × 10⁹ cfu g^?1 feed) were used for the experiment. After 60 days of feeding trial, the growth performance increases significantly in all the experimental groups compared with control, and highest weight gain, specific growth rate and lowest feed conversion ratio were recorded from T₄ (P > 0.05). However, juvenile survival was not found to be significantly affected by probiotic supplementation (P > 0.05). The total bacterial count and Bacillus spp. count in the gut were significantly increased with concurrent decrease in pathogenic Aeromonas spp. and Pseudomonas spp. in all the experimental groups compared with control (P < 0.05). Compared with control, immune parameters were increased significantly in all the experimental groups (P < 0.05). Highest haemocyte count, phenol oxidase, superoxide dismutase and antibacterial activity in haemolymph were recorded from T₄ (P < 0.05). When prawns were challenged with Vibrio alginolyticus, the cumulative mortality was significantly lower in T₄ (P < 0.05). These results indicate that dietary supplementation of B. licheniformis can manipulate the gut microbiota and increase the growth and immune response of M. rosenbergii. In the present study, concentration response on the probiotic effect of B. licheniformis was observed which showed higher effects at a concentration of 1.0 × 10⁹ cfu g^?1 feed in the laboratory condition.     

Impact of microwave-assisted enzymatic hydrolysis on functional and antioxidant properties of rainbow trout <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis> by-products

Fishery by-products can be better utilized following enzymatic hydrolysis treatment to produce fish protein hydrolysates (FPH) with potentially enhanced interface-stabilizing properties (e.g. functionality). The production of FPH could be accelerated through the application of rapid heating methods [e.g. microwave-assisted heating (MW)] rather than slower conventional heating (CH) treatments. The objective of this study was to investigate the effects of microwave heating during enzymatic hydrolysis on the functionality and antioxidant properties of FPH. Trout by-products were hydrolyzed with Alcalase at an enzyme substrate ratio (E:S) of 0.5, 1.7, and 3.0% (w/v), respectively, for 3, 5 and 15 min using a microwave system (1200 W, 20% power with 50% duty cycle at 50–55 °C) and a conventional heating method (water bath at 50 °C). The degree of hydrolysis and protein solubility was higher (P < 0.05) for the MW-FPH than for the CH-FPH. MW-FPH at 5 min (0.5% E:S) demonstrated higher (P < 0.05) emulsifying activity and emulsion stability than CH-FPH with the same treatment. Foam capacity and stability were also greater (P < 0.05) for MW-FPH samples that were treated 15 min by microwave-assisted heating (0.5% E:S) when compared to CH. Overall, MW-FPH exhibited higher (P < 0.05) 2,2-diphenyl-1-picryhydrazyl and ferric ion reducing capacity than CH-FPH. We therefore conclude that microwave-assisted hydrolysis is an alternative method to produce FPH with improved solubility, emulsifying activity, foaming properties and antioxidant activity.     

Dietary ascorbic acid influences the intestinal morphology and hematology of hybrid sorubim catfish (<Emphasis Type="Italic">Pseudoplatystoma reticulatum</Emphasis> × <Emphasis Type="Italic">P. corruscans</Emphasis>)

This study evaluated the effect of graded levels of dietary ascorbic acid (AA) (12.47, 20.27, 115.44, 475.50, 737.72, and 850.70 mg kg^?1) on growth, hematology, intestinal morphometry, and phagocyte activity of hybrid sorubim Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans. Fish (n = 420, 14.57 ± 2.71 g, 15.11 ± 0.90 cm) were distributed in 30 polyethylene tanks (80 l) (5 replicates per treatment with 14 fish per tank) and fed for 45 days. Dietary treatment did not have a significant effect on growth metrics (P > 0.05). Fish fed 737.72 mg AA kg^?1 had a higher villi height (289.80 ± 19.96 μm) (P < 0.05) than fish fed 850.70 mg AA kg^?1 (245.4 ± 18.25 μm). Hemoglobin in fish fed 850.70 mg AA kg^?1 (5.34 ± 0.96 g dl^?1) was higher (P < 0.05) than fish fed 12.47 mg AA kg^?1 (3.42 ± 0.55 g dl^?1) and 20.27 mg AA kg^?1 (3.06 ± 1.26 g dl^?1). The erythrocyte number of hybrid sorubim fed 115.40 mg AA kg^?1 (1.73 ± 0.27 × 10⁶ μl^?1) and 475.50 mg AA kg^?1 (1.70 ± 0.28 × 10⁶ μl^?1) were higher (P < 0.05) than in those fed diets containing 20.27 mg AA kg^?1 (1.11 ± 0.34 × 10⁶ μl^?1). There was no significant effect (P > 0.05) of dietary AA on leukocyte and thrombocyte and on phagocyte activity and phagocyte index. Inclusion of AA in feed seems to increase the integrity of the intestinal mucosa and stimulate erythropoiesis in hybrid sorubim catfish.     

Evaluation of the Antioxidant Activity In Vitro and in Hippocampal HT-22 Cells System of Protein Hydrolysates of Common Carp (Cyprinus carpio) By-Product

Fish processing by-products may become more than 50% of the starting material. If mismanaged, these large quantities of discarded fish can create serious pollution problems and can also generate cost associated with their disposal. Enzymatic hydrolysis is one of the techniques that is currently being developed in order to recover and add value to these biomolecules. There is an increasing interest in natural antioxidants that are safer for consumers compared with synthetic antioxidants. In this study, common carp by-product was hydrolyzed using the enzymes Alcalase (A) and Protamex (P) to reach degrees of hydrolysis (DH) of 10 and 15%, respectively. Antioxidant capacity against peroxyl radicals (ACAP); 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging; and the measurement of intracellular reactive oxygen species (ROS) concentration after exposure to common carp protein hydrolysates were investigated. The results revealed that the hydrolysate A15 exhibited significantly (p < 0.05) higher antioxidant activity against the DPPH radical. A15 showed the highest in vitro antioxidant competence against peroxyl radicals, whereas P15 showed the lowest activity against peroxyl radicals (p < 0.05). Hydrolysates having the highest and the lowest in vitro antioxidant activity (A15 and P15, respectively) were selected for the determination of antioxidant activity in the HT-22 cells system. Measurement of intracellular ROS concentration revealed that P15 at the concentration of 1.25 mg/mL significantly (p < 0.05) reduced the intracellular ROS concentration. These results showed that common carp by-product protein hydrolysates are a source of antioxidant peptides with a high potential for food and pharmaceutical industries to develop new nutraceuticals and functional foods.     

Identification of haematological markers in shrimp health assessment from the immune profile of Fenneropenaeus indicus on β-1,3-glucan administration and white spot syndrome virus challenge

Haematological alterations in Fenneropenaeus indicus fed marine yeast glucan-incorporated diet and challenged with white spot syndrome virus were analysed. Adult F. indicus 16.45 ± 2.12 g (mean ± SD) were reared in 25 ‰ sea water and allowed to acclimate for a period of 7 days. Diet was prepared incorporating glucan (0.2 %) extracted from marine yeast, Debaryomyces hansenii S169. The shrimps were reared on experimental diet for 28 days and then challenged with white spot syndrome virus through oral administration. Random sampling was done (n = 6 shrimps) on 1st, 15th and 28th day of the experiment and on post-challenge day 2 (PCD 2) and PCD 7 for haematological analysis. The immune parameters viz. plasma protein, total haemocyte count, phenoloxidase activity, superoxide anion production, alkaline phosphatase and acid phosphatase activities were estimated in the haemolymph. Superoxide dismutase, catalase, glutathione peroxidase, hydroperoxide, conjugated dienes and malondialdehyde concentrations in the haemolymph of shrimps were also analysed. Analysis of variance showed that there are significant differences (p < 0.05) in the immune and antioxidant parameters in different treatment groups of F. indicus. The shrimps fed glucan-incorporated diet showed higher survival rate with comparatively lower accumulation of lipid peroxidation products. Correlation coefficients showed that all haematological variables except hydroperoxides and conjugated dienes exhibited positive correlation with the survival rate. The immune variables and antioxidant parameters exhibited a greater degree of correlation with each other. When multiple regression of survival rate on all immune parameters was considered, the amount of variability explained was 93 % (R Square = 0.932). When significant regression coefficients among the immune parameters were taken into account, it was found that total haemocyte count (p < 0.001), phenoloxidase activity (p < 0.05), alkaline phosphatase activity (p < 0.05) and plasma protein concentration (p < 0.05) together are explaining 84 % (R ² = 0.842) of the variability, indicating that these four parameters are mainly supporting the survival. The amount of variability explained by the antioxidant parameters was 94 % (R ² = 0.938). When significant regression coefficients among the antioxidant parameters were taken into account, it was found that superoxide dismutase activity (p < 0.01) and catalase activity (p < 0.05) together are explaining 87.4 % (R ² = 0.874) of the variability, indicating that these two antioxidants act as the major immune effectors supporting the survival in shrimps.     

Dietary supplementation effects of Pediococcus acidilactici as probiotic on growth performance,digestive enzyme activities and immunity response in zebrafish (Danio rerio)

This study was carried out to assess the effects of Pediococcus acidilactici on zebrafish (Danio rerio). Different levels of P. acidilactici including 0, 1 × 10⁶, 2 × 10⁶, 4 × 10⁶ and 8 × 10⁶ colony‐forming unit per g of the diet (cfug^?1) were examined in fish with 120 ± 10 mg weight for 60 days in a completely randomized design. The results showed that the best growth indices were recorded in group 4 × 10⁶ cfug^?1 (p < 0.05). The highest number of total viable count and lactic acid bacteria of intestine were found in group 4 × 10⁶ cfug^?1 (p < 0.05). The maximum activity of digestive enzymes including amylase, lipase, protease and alkaline phosphatase was observed in group 4 × 10⁶ cfug^?1. The highest activity for superoxide dismutase was recorded in group 4 × 10⁶ cfug^?1 while catalase, glutathione peroxidase and glutathione reductase showed the highest activity in group 8 × 10⁶ cfug^?1. The most growth inhibition zone of Aeromonas hydrophylla, Flavobacterium columnare, Vibrio anguillarum and Edwardsiella tarda was found in group 4 × 10⁶ cfug^‐1 (p < 0.05). Therefore, P. acidilactici as a probiotic improved growth and immunity of the zebrafish and could be used by zebrafish farmers.     

Study of digestive proteinases and proteinase inhibitors of Daphnia carinata

Quantification of proteases activities and their class structure have been studied in a cladoceran, Daphnia carinata. Protease activity ranged from 0.28 to 0.55 Unit mg⁻¹ protein min⁻¹ with an average value of 0.42±0.06 Unit mg⁻¹ protein min⁻¹. Chymotrypsin activity was more than twofold higher (0.49±0.09 Unit mg⁻¹ protein min⁻¹) than the trypsin activity (0.21±0.02 Unit mg⁻¹ protein min⁻¹). Protease activity and reduction of activity in bands of samples treated with specific inhibitors were documented in photometric assay and substrate SDS–PAGE. Proteinase activity against azocasein was inhibited (91.4±1.5%) with SBTI. PMSF reduced the enzyme activity by 53.1±6.5%, and the azocasein hydrolysis was reduced up to 64.6±3.8% by the specific inhibitor of trypsin, TLCK. In the present investigation, the molecular weight of various activity bands ranged from 16.3 to 51.1 kDa. The molecular weights of several protein bands are similar to protease activity zones. The knowledge of digestive enzyme profiles of fish food organisms generated in the present study may assist in the formulation of age-specific feed.     

Physiological response,blood chemistry profile and mucus secretion of red sea bream (<Emphasis Type="Italic">Pagrus major</Emphasis>) fed diets supplemented with <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> under low salinity stress

Environmental stressors caused by inadequate aquaculture management strategies suppress the immune response of fish and make them more susceptible to diseases. Therefore, efforts have been made to relieve stress in fish by using various functional feed additives in the diet, including probiotics. The present work evaluates the effects of Lactobacillus rhamnosus (LR) on physiological stress response, blood chemistry and mucus secretion of red sea bream (Pagrus major) under low salinity stress. Fish were fed four diets supplemented with LR at [0 (LR0), 1 × 10² (LR1), 1 × 10⁴ (LR2) and 1 × 10⁶ (LR3) cells g^?1] for 56 days. Before stress, blood cortisol, urea nitrogen (BUN) and total bilirubin (T-BIL) showed no significant difference (P > 0.05), whereas plasma glucose and triglyceride (TG) of fish-fed LR2 and LR3 diets were significantly lower (P < 0.05) than those of the other groups. Plasma total cholesterol (T-CHO) of fish-fed LR3 diet was significantly (P < 0.05) lower than that of the other groups. Furthermore, total plasma protein, mucus myeloperoxidase activity and the amount of mucus secretion were significantly enhanced in LR-supplemented groups when compared with the control group (P < 0.05). After the application of the low salinity stress test, plasma cortisol, glucose, T-CHO and TG contents in all groups showed an increased trend significantly (P < 0.01) compared to the fish before the stress challenge. However, plasma total protein and the amount of secreted mucus showed a decreased trend in all groups. On the other hand, BUN, T-BIL and mucus myeloperoxidase activity showed no significant difference after exposure to the low salinity stress (P > 0.05). In addition, the fish that received LR-supplemented diets showed significantly higher tolerance against low salinity stress than the fish-fed LR-free diet (P < 0.05). The physiological status and the detected immune responses, including total plasma protein and mucus myeloperoxidase activity in red sea bream, will provide a more comprehensive outlook of the effects of probiotics to relieve stress in fish.     

Common Kilka Hydrolysis: Investigation of Functional and In Vitro Antioxidant Properties of Hydrolysates Obtained by Kiwifruit and Ginger Proteases

The functional and in vitro antioxidant properties of common kilka fish protein hydrolysates with different degree of hydrolysis (DH) obtained by kiwifruit protease (KP) and ginger protease (GP) were evaluated. The electrophoretic patterns of hydrolysates showed the presence of one major band in different DHs with a molecular weight of less than 25 kDa. Protein solubility was positively correlated with DH, and the solubility of KP and GP hydrolysates (HKP and HGP, respectively) at 15% DH was higher than other DHs (p ≤ 0.05). Higher emulsifying and foaming properties were observed in HKP over a pH range of 2–10 (p ≤ 0.05), and in vitro antioxidant activity was higher in HKP at 15% DH as seen from 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric-reducing power. Thus, the results reveal that DH and enzyme type affects functional properties and antioxidant activity of the hydrolysates.     

Effect of treatment with probiotics as water additives on tilapia (Oreochromis niloticus) growth performance and immune response

A feeding trial was conducted for 40 days to delineate the effect of treatment with probiotics as water additives on tilapia (Oreochromis niloticus) growth performance and immune response. About 360 juveniles were randomly distributed into four treatment groups, each with three replicates. Different probiotics (T-1, Bacillus subtilis B10; T-2, Bacillus coagulans B16; T-3, Rhodopseudomonas palustris G06) were added to the water of tanks at final concentration of 1 × 10⁷ cfu ml^?1 every 2 days, with no probiotic added to control tanks. At the end of the feeding trial, fish treated with B. coagulans B16 (T-2) and R. palustris G06 (T-3) had significantly (P < 0.05) higher final weight, daily weight gain, and specific growth rate compared with those treated with B. subtilis B10 (T-1) and those without probiotics (control). The highest (P < 0.05) content of total serum protein was found in T-2 compared with that in T-1, T-3, and the control. However, albumin concentration and albumin/globulin ratio were not affected by the probiotics treatments. Compared with the control, probiotic supplementation remarkably improved activities of superoxide dismutase and catalase (P < 0.05). T-2 fish exhibited higher average myeloperoxidase activity than the control, T-1, and T-3 groups. Regarding serum lysozyme content in tilapia, assays showed no difference (P > 0.05) among the treatment groups. Furthermore, probiotics treatments remarkably increased respiratory burst activity compared with control, with T-2 showing higher values than T-1 and T-3. This indicated that treatment with probiotics, B. coagulans B16 and R. palustris G06, as water additives could be used to enhance immune and health status, thereby improving growth performance of O. niloticus.     

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg²⁺, whereas inhibited by Zn²⁺, Ca²⁺ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO₄ ^2? and product HPO₄ ^2? showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the V _max and K _m values were 24.45 μmol/L min and 5.76 mM, respectively.     

Effects of practical dietary protein to lipid levels on growth,digestive enzyme activities and body composition of juvenile rice field eel (Monopterus albus)

A 10-week feeding experiment was conducted to evaluate the effects of dietary protein to lipid ratios (P/L) on growth, intestinal digestive enzyme activities and body composition in juvenile rice field eel (Monopterus albus) (initial mean body weight of 65.76 ± 1.07 g, mean ± SEM). Nine test diets were formulated in a 3 × 3 factorial design to contain three protein levels (350, 400 and 450 g kg^?1) for each of three lipid levels (40, 80 and 120 g kg^?1), respectively. Each diet was randomly assigned to triplicate groups of 60 fish per net cage (1.5 × 2.0 × 1.5 m). Results showed the survival was above 96 % and was not affected by dietary treatments. Fish fed the diet with 450 g kg^?1 protein and 40 g kg^?1 lipid showed the best weight gain (WG) (103.95 %) and feed conversion ratio (1.60) (P < 0.05). WG, protein efficiency ratio and energy retention increased with the increasing in lipid at 350 g kg^?1 protein level (P < 0.05). However, WG showed a little decline with increasing dietary lipid when fish fed the diets with 400 and 450 g kg^?1 protein level, but no significant difference was observed (P < 0.05). Hepatosomatic index, visceralsomatic index and intestinal lipase activity increased with the increasing of dietary lipid level irrespective protein level. Intestinal trypsin activity increased with the increasing of dietary lipid level when fish fed the diets with 350 g kg^?1 protein, but showed converse trend when fish fed the diets with 400 and 450 g kg^?1 protein. Serum triglyceride, body lipid and energy were positively correlated with the dietary lipid. Results of the present study showed that the dietary protein/lipid ratio of 450/40 g kg^?1 is considered optimum for rice field eel under culture conditions, and the increase in dietary lipid level has no efficient protein-sparing effect when fish fed the diets with 400 and 450 g kg^?1 protein level.     

Oxidative stress and antioxidant enzymes activities in the African catfish,Clarias gariepinus,experimentally challenged with Escherichia coli and Vibrio fischeri

The impacts of bacterial infection on cultivated fish species, African catfish, were investigated using oxidative stress biomarkers [lipid peroxidation (LPO) and protein carbonylation] and the activities of important antioxidant/detoxifying enzymes [catalase and glutathione S-transferase (GST)]. Fish were inoculated via oral gavage with one of the following treatments: 1 × 10⁵ CFU/ml of Escherichia coli (EC1), 2 × 10⁵ CFU/ml of E. coli (EC2), 1 × 10⁵ CFU/ml of Vibrio fischeri (V1), 2 × 10⁵ CFU/ml of V. fischeri (V2), gavaged with distilled water and not gavaged. Fish were maintained in the laboratory for 7 days after the bacterial inoculation, and the levels of LPO, protein carbonylation, GST, and catalase activities were determined in the muscle, gills, and liver of fish. Fish inoculated with bacteria (either E. coli or V. fischeri) had a significant higher levels of tissue LPO, protein carbonylation, and GST activities in a tissue-specific pattern (liver > muscle > gills). This appears to be related with the levels of bacterial inoculation, with effects more pronounced in fish inoculated with either EC2 or V2. The catalase activity did not differ significantly between the inoculated and fish that were not inoculated. The results of this study indicate that bacterial inoculation could result in oxidative stress in fish, and liver has a higher rate of oxidative stress per mg tissue compared to the gills and the muscle.     

Development of phototaxis in the early life stages of Pacific bluefin tuna <Emphasis Type="Italic">Thunnus orientalis</Emphasis>

We investigated the development of phototaxis in larval and early juvenile stages of Pacific bluefin tuna Thunnus orientalis to detail behavior development in relation to light. We observed the distribution of Pacific bluefin tuna in an illumination gradient tank from 1 to 26 days after hatching (DAH). Two long rectangular tanks (100 × 10 × 10 cm) were used for experiments; each tank was divided into four sections for observations and one tank was equally illuminated at 1.0 × 10³ lx as the control tank, and the other was at 4.0 × 10², 1.0 × 10³, 1.0 × 10⁴ and 7.0 × 10⁴ lx as the illumination gradient tank. Laboratory-reared larvae and juvenile were released into each tank and acclimatized for 10 min. Thereafter, the number of individuals in each section was counted for 5 times with 10-min intervals. Until 2 DAH (3.6 ± 0.1 mm BL), larvae distributed homogeneously in each section in both tanks. After 3 DAH (3.7 ± 0.1 mm BL) when the eyes of the larvae were pigmented, most fish moved into the 7.0 × 10⁴ lx section in the illumination gradient tank, while fish in the control tank maintained a uniform distribution during the experimental period (Chi-square test, P < 0.05). The results suggest that the post-larvae and juvenile stages show strong positive phototaxis.