Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Development of two cell culture systems from Asian seabass Lates calcarifer (Bloch)

Two new cell culture systems namely epitheloid cells of Lates (LCE) and fibroblastic cells of Lates (LCF) have been developed from fry and fingerling of the economically important brackishwater fish Lates calcarifer. Primary cultures were initiated by explant technique using caudal fin of fingerling and whole body tissue of the fry. The nutritional requirements and the growth pattern in response to different culture environment were similar for the two cell cultures. The culture medium used was Leibovitz‐15 supplemented with 20% fetal bovine serum (FBS) and 1% fish serum. The LCE comprised of epithelioid cells and LCF cells were fibroblastic. With a split ratio of 1:2, the confluency of cells was attained in 8–10 days at an incubation temperature of 28°C. The cells were found to grow well in a wide range of temperature (24–32°C) and stable at 20 and 36°C. The growth rate of LCF and LCE cells increased proportionately with the concentration of FBS from 5% to 20%. A decrease of serum level to 10% after eight subcultures produced no apparent change in cell morphology and growth rate. The viability of cells was found to be 70% when revived after a month of storage in liquid nitrogen (?196°C).     

Establishment and characterization of a new cell line from koi brain (Cyprinus carpio L.)

A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L‐15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The cell line was cryopreserved in liquid nitrogen at ?196°C and was recovered from storage after 1 year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 10^5.73TCID₅₀/0.1 ml within 7 days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.     

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche)

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24‐h EC₅₀ < 10 mg L^?1) in their parasite‐killing effects: formalin, H₂O₂, bithionol, ClO₂ and bronopol. Moderately potent drugs (10 < 24‐h EC₅₀ < 100 mg L^?1) were quinine, fumagillin, amphotericin B, ketoconazole, povidone‐iodine, chloramine‐T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO₄, potassium monopersulphate and citric acid, exhibited EC₅₀ > 100 mg L^?1. When ascidians were artificially infected with A. hoyamushi, treated using 40 mg L^?1 formalin, bronopol, ClO₂, or H₂O₂ for 1 h and then monitored for 24 h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40 mg L^?1 formalin or ClO₂ for 1 h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.     

In Vitro Screening of Fungicidal Chemicals for Antifungal Activity against Saprolegnia

Saprolegnia is an important fish fungal pathogen that often results in significant economic losses to freshwater aquaculture. To find effective drugs to control saprolegniasis, 30 fungicidal chemicals used in agriculture were screened, in which kresoxim‐methyl and azoxystrobin, with minimum inhibitory concentration (MIC) values of 1.0 and 0.5 mg/L, respectively, showed good in vitro antifungal activities against Saprolegnia. Azoxystrobin has the most promising anti‐Saprolegnia activity with 50% effective concentration (EC₅₀) value of 0.212 mg/L against mycelial growth and minimum fungicidal concentration (MFC) value of 0.13 mg/L against spores, while EC₅₀ and MFC values to kresoxim‐methyl are 0.240 and 0.25 mg/L, respectively. Through the acute toxicity assay using goldfish, Carassius auratus, azoxystrobin exhibited wider margin of safety with a safe concentration (SC) value of 0.553 mg/L than kresoxim‐methyl with an SC value of 0.131 mg/L. These findings demonstrated that azoxystrobin has the potential for the development of therapy for the control of Saprolegnia in aquaculture. Both kresoxim‐methyl and azoxystrobin were tested with a post‐antifungal effects (PAFE) assay and the results revealed that the two chemicals had no significant effect on fungal growth inhibition after a 1‐hour exposure, indicating that the treatment needs to be carried out over an extended period.     

Enrichment of Adult Artemia Biomass and Squid Mantle Muscle,Dosidicus gigas,with Different Ascorbic Acid (L‐Ascorbyl‐2‐Monophosphate‐Na/Ca) Concentrations

L‐ascorbyl‐2‐monophosphate‐Na/Ca (AMP‐Na/Ca) was used as a vitamin C source to investigate its ascorbic acid (L‐AA) enrichment and retention in boosted Artemia biomass (AB) and squid mantle muscle (SM). Different doses of AMP‐Na/Ca (500, 1000, and 1500 AMP‐Na/Ca mg/kg) were gradually dissolved into the culture tanks at time 0 (T₀) and at each hour until Hour 6 (T₆). Samples of AB and SM were taken for AMP‐Na/Ca and L‐AA analysis at T₀, T₁, T₂, T₃, T_4, T₅, T₆, T₁₂, and T₂₄. There were no significant differences (P > 0.05) among the AB groups at T₁. The T₆ enrichment analysis for AB resulted in significant differences (P < 0.05) in the AMP‐Na/Ca content for the 1500 mg/kg treatment, in which the initial concentration (0.001 ± 0.002 mg/kg) increased by more than 16‐fold. For all AB enrichment treatments, the AMP‐Na/Ca content demonstrated a decrease (32–11%) for the T₆, T₁₂ and T₂₄ analysis. The T₁ analysis for SM at the higher AMP‐Na/Ca enrichment concentration registered 30 mg/kg of L‐AA and decreased (27.6%) at T₆. This study demonstrated that AB and SM can be boosted with AMP‐Na/Ca.     

Using Phage PSM‐1 to Control Shewanella marisflavi Infection in Juvenile Sea Cucumber,Apostichopus japonicus

Shewanella marisflavi is considered responsible for infection in juvenile sea cucumber, Apostichopus japonicas. A bacteriophage specific to S. marisflavi was isolated from seafood wholesale market sewage and designated as vB_SmaM_PSM (PSM‐1). The phage was classified as a member of the Myoviridae family by negative staining transmission electronic microscopy. The stability of phage PSM‐1 was observed after treatment with different temperature gradients and pH values. The results showed that the lytic activity of phage PSM‐1 was optimum at 28 C with a pH of 7.0 and ceased at temperatures higher than 60 C. A one‐step growth curve analysis of the phage revealed eclipse and latent periods of 20 and 40 min, respectively, with a burst size of 219 plaque forming units per infected cell. The ability of phage PSM‐1 to inhibit the growth of S. marisflavi AP629 was tested in vitro using the phage with three multiplicity of infection (MOI) values of 0.1, 1, and 10. All treated cultures that produced an inhibition of the growth of S. marisflavi AP629 were compared with the untreated culture, and the treatment group with a MOI value of 10 showed the highest inhibition effect among the three doses. In an in vivo performance experiment, the sea cucumbers (15–20 g) were randomly assigned to five groups (one negative control, one antibiotic treatment group, and three phage treatment groups) in a phage injection trial as well as a phage immersion trial. All groups were infected with S. marisflavi strain AP629 (3.3 × 10⁶ colony forming units/g, LD₅₀ [lethal dose 50%] level) by intraperitoneal injection. After 8 d of observation, the survival rates ranged from 40 to 80% in all phage treatment groups, while 10% in the negative control group. The optimal therapeutic time points of the two trials were also determined. The results showed that using phage prior to infection could be a good choice to protect sea cucumbers. The overall results of this experiment indicate that using phage may be a feasible biological control agent to prevent S. marisflavi infection in sea cucumbers.     

Amelioration of LPS‐induced inflammatory response and oxidative stress by astaxanthin in Channa argus lymphocyte via activating glucocorticoid receptor signalling pathways

The present study was conducted to evaluate the effects of astaxanthin (AST) against lipopolysaccharide (LPS)‐induced lymphocyte viability, ultrastructural lesions, apoptosis, oxidative stress and inflammatory responses in Channa argus. Lymphocytes exposed to more than 10 μg/ml LPS alone for 24 hr showed significantly decreased cell viability, elevated nitric oxide (NO) and malondialdehyde (MDA), lactate dehydrogenase (LDH) contents, and increased nuclear factor κB p65 (NF‐κB p65), myeloid differential protein‐88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐8 (IL‐8), caspase‐3, caspase‐8 and caspase‐9 gene expression. LPS at a concentration of 10 μg/ml could induce oxidative stress and inflammatory responses in lymphocytes. The activities of antioxidant enzymes (catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD)) were significantly decreased after exposure to 10 μg/ml LPS. Besides, AST strikingly antagonized the LPS‐induced negative effects. AST significantly increased the expression of HSP70, HSP90, IκB‐α, and glucocorticoid receptor (GR) and decreased inflammatory responses. Further study showed that AST can activate GR signalling pathway and inhibit p65 phosphorylation. In addition, AST attenuated LPS‐induced apoptosis, mitochondrial swelling, degeneration and vacuolization. Collectively, these findings suggest that AST has protective roles in LPS‐induced cell damage via modulating GR activation in C. argus lymphocytes.     

Toxicity of the aquaculture pesticide cypermethrin to planktonic marine copepods

The acute and sublethal toxicity of cypermethrin, the active ingredient in the sea lice treatment formulation Excis^®, to non‐target planktonic marine copepods was determined. The comparative sensitivities of three life stages (nauplii, copepodites, adults) of four common marine copepods (Acartia clausi, Pseudocalanus elongatus, Temora longicornis and Oithona similis) were assessed in 48‐h exposures, followed by a recovery period in toxicant‐free sea water. The cyclopoid copepod, O. similis, was most affected by cypermethrin, with EC₅₀ values ranging from 0.14 to 0.24 μg L^?1 for nauplii and adults respectively. With the exception of T. longicornis nauplii, the calanoid copepods (A. clausi, P. elongatus and T. longicornis) responded similarly to cypermethrin. Overall, 48‐h EC₅₀ values ranged from 0.12 μg L^?1 (T. longicornis nauplii) to >5 μg L^?1 (P. elongatus adults). For all species, nauplii and copepodite EC₅₀ values were lower than those of the adults. The primary toxic effect, immobilization, was generally irreversible. A sublethal test with adult A. clausi females, involving pulse exposures over 4 days measured a significant increase in egg production at the higher concentrations (1.58 and 5 μg L^?1). Concentrations causing acute toxicity to planktonic copepods were lower than the recommended sea lice treatment concentration of 5 μg L^?1 cypermethrin, indicating the potential for toxic effects in the field. However, acute toxicity values were higher than the Environmental Quality Standard of 0.016 μg L^?1 for dispersing treatment plumes, suggesting that cypermethrin released to the marine environment following sea lice treatments is unlikely to affect adversely planktonic copepods.     

Establishment and characterization of a fin tissue cell line derived from silver pomfret,Pampus argenteus

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT‐PCR and viral titre assays. In contrast, PaF cells were resistant to red‐spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune‐related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen–silver pomfret interaction.     

Dietary modulation of digestive enzymes by the administration of feed additives to rainbow trout,Oncorhynchus mykiss Walbaum

The aim of this study was to assess the metabolic and digestive enzyme activities in rainbow trout, Oncorhynchus mykiss Walbaum fed dietary supplements. The biometric indices and the following enzymes activities were measured: acid protease and pepsin from the stomach homogenates, alkaline phosphatase (AP) from the small intestine and the brush border membrane, total proteolytic enzymes and trypsin from the small intestine and liver/pancreas. Dietary garlic induced a higher pepsin activity in the stomach than lipopolysaccharide (LPS) and ginger. Conversely, dietary ginger and LPS showed a significant difference (P > 0.05) in acid protease activity compared to the controls. There was not any significant difference observed between treated groups and the controls for hepato‐pancreas proteolytic enzyme activity, except for trypsin. AP activity increased significantly with dietary garlic, ginger and LPS compared to the controls.     

Transactivational property of chemicals via medaka glucocorticoid receptor 1b using a stable reporter gene assay

The transactivational property of natural and synthetic chemicals via medaka GR1b was investigated after development of a stable cell line for the reporter gene assay. In our study, cortisol was the most potent agonist among the natural corticoids assayed for potency [EC₅₀ (concentration of agonist provoking a response halfway between the baseline and maximum response) 68 nM] and efficacy. Three artificial corticosteroids, namely, dexamethasone [EC₅₀ 16 nM, relative agonistic activity to cortisol (RAA) 144 %], prednisolone (EC₅₀ 81 nM, RAA 116 %) and clobetasol propionate (EC₅₀ 0.10 nM, RAA 220 %), showed strong agonistic activity and were more potent than the original corticoid, F. All synthetic corticoids used in our study were full agonists. Interestingly, melengestrol acetate, a synthetic progestogen, induced luciferase activity in a dose-dependent manner. Based on its EC₅₀ value and RAA of 29 nM and 57 %, respectively, this molecule was assessed as a partial agonist. None of the other steroids and chemicals assayed in our study induced an agonistic response. In conclusion, we successfully developed a stable reporter gene assay that can be used to assess the transactivational property of glucocorticoid-like chemicals toward medaka GR1b.     

胰蛋白酶酶解制备孔鳐软骨蛋白寡肽及其抗氧化活性

以孔鳐软骨为材料，采用盐酸胍抽提、丙酮分级沉淀，制备孔鳐软骨蛋白；以DPPH·和HO·清除活性为导向，采用胰蛋白酶酶解、膜超滤、DEAE-52阴离子交换层析、Sephadex G-15凝胶层析和反相高效液相色谱(RP-HPLC)等技术，制备抗氧化肽，并对其活性进行系统评价。结果显示，孔鳐软骨蛋白经胰蛋白酶酶解和分离纯化得到2个抗氧化肽RCPE-A和RCPE-B，经氨基酸序列分析确定其序列分别为Gly-Glu-Glu-Gly-Pro-Arg-Gly (GEEGPRG)和Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu (GEEGTMGL)，质谱(ESI-MS)测定其分子量分别为700.71和792.87 u。体外自由基清除实验结果显示，RCPE-A与RCPE-B对DPPH·(EC₅₀ 2.94和1.16 mg/mL)、HO·(EC₅₀ 0.34和0.54 mg/mL)、ABTS⁺·(EC₅₀ 0.34和0.10 mg/mL)和O₂^-·(EC₅₀ 0.11和0.03 mg/mL)具有良好的清除作用，RCPE-A与RCPE-B亦显示出较强的脂质过氧化抑制作用。研究表明，孔鳐软骨蛋白酶解物及制备多肽可用于抗氧化相关的功能食品开发，也可以用作抗氧化剂延长相关产品的货架期。     

Photosynthetic and Biochemical Effects of Cold Storage on Marine Benthic Diatoms of the Mexican Pacific Coast

We determined the effects of 16 wk of storage at low temperature (4 C) in darkness on the viability, growth, photosynthetic parameters, and biochemical composition of four diatom cultures. Significant differences in cell density and proximal composition were observed for all diatoms throughout storage. Cell density increased with time of storage for all diatoms. Protein content increased for Navicula incerta, Nitzschia laevis, and Navicula sp., whereas lipid content increased during storage in only N. incerta. When the stored diatoms were used as inocula in fresh medium, they increased their viability, generating a lag phase for Nitzschia thermalis var. minor, N. incerta, and Navicula sp. cultures. There were noted species‐specific modifications in proximal composition, ash‐free dry weight, and photosynthetic parameters in response to storage. We conclude that N. thermalis, N. incerta, N. laevis, and Navicula sp. can be stored at 4 C for 16 wk and are viable in new cultures.     

Beneficial effects of Bacillus subtilis on water quality,growth, immune responses,endotoxemia and protection against lipopolysaccharide‐induced damages in Oreochromis niloticus under biofloc technology system

The present research explored the effects of Bacillus subtilis on water quality, growth, immune responses, endotoxemia and protection against lipopolysaccharide (LPS) damages in Nile tilapia Oreochromis niloticus under biofloc system. B. subtilis was added at 0, 1, 2, 3 and 4 grams (1.19 × 10⁸ CFU/g) per kg of basal diet, named T1 (control), T2, T3, T4 and T5, respectively, and fed to fish (14.82 ± 0.42 g) for 50 days. The concentrations of TAN, NO₂ and NO₃ were significantly reduced, and fish fed probiotics displayed significantly better growth performances versus the control, concomitantly with significantly enhanced activities of digestive enzymes. They also showed significantly declined serum glucose and cholesterol vice versa significantly improved immune responses (total protein, albumin, globulin, lysozyme, alternative complement, protease, immunoglobulins, alkaline phosphatase and respiratory burst), antioxidant capacity (superoxide dismutase, total antioxidant capacity, malondialdehyde) and skin mucus parameters (total protein, lysozyme, alternative complement, protease, immunoglobulins). Meanwhile, significantly lower endotoxin (LPS) concentrations were detected in the intestines and serum of fish fed probiotics. LPS challenge induced profound oxidative stress and impaired immune responses. Interestingly, probiotic alleviated LPS‐induced damages and restored mentioned parameters. In conclusion, B. subtilis effectively enhanced fish production, immunity and protection against LPS‐induced damages in tilapia under biofloc system.     

Cold Storage of Six Marine Benthic Diatoms Native to the Mexico Pacific Coast

Cultures of six benthic diatoms were maintained in the dark to measure their viability and biochemical composition after 8 wk of storage at low temperature (4 C) in darkness by refrigeration. Cell density, growth rate, and viability for each benthic diatom changed significantly after storage. Significant differences were observed with regard to cell size (length and width) of Nitzschia laevis, Navicula sp., and Amphora tenerrima as a result of storage. In general, the proximal composition of the benthic diatom cultures changed after week 1 of storage and decreased after week 4 of storage for all the diatoms. These results demonstrate that under 1–4 wk of storage these diatoms maintain their viability and had changes in their proximal composition in species‐specific responses. Storage of preserved live microalgae cells is an alternative technique that can be used to reduce the need for continuous maintenance of live cultures and can provide live feed stock for aquaculture applications.     

Toxicity of a synthetic phenolic antioxidant,butyl hydroxytoluene (BHT), in vertebrate model zebrafish embryo (Danio rerio)

The synthetic antioxidant 3,5‐di‐tert‐butyl‐4‐hydroxytoluene (BHT) is widely used as an additive in the food, cosmetic and plastic industries to increase the tenability of food and plastic for the past 70 years. BHT is degraded to 3,5‐di‐test‐butyl‐4‐hydroxybenzaldehyde (BHT‐CHO) in mammals, as well as in the natural environment such as in river and water. The average daily intake of BHT for human being is estimated to be 0.3 mg/kg body weight. Even though it is considered safe for human at authorized level, but its ubiquitous presence in the aquatic environment and the controversial toxicological data are of great concern for human as well as aquatic life. The experimental findings of zebrafish embryo toxicity test (ZFET) showed that the acute toxicity of 96‐hr (LC₅₀) exposure during the embryogenic stage was found to be 4.388 mg/L and the effective concentration (EC₅₀) was 1.375 mg/L. The reduce heart rate from the sublethal concentrations indicates the chemical to be cardiotoxic but a further review is to be needed. The Teratogenic Index (TI) calculated to be 3.19, which implies the compound may be a potential teratogen in aquatic life. The findings obtained in this study will stretch more evidence regarding developmental toxicity of BHT, which will be of much importance in further risk assessment of ecotoxicological studies.     

Effects of dietary tea polyphenols on growth,immunity and lipid metabolism of juvenile black carp Mylopharyngodon piceus

An 8‐week feeding experiment was aimed to investigate the effect of dietary tea polyphenols (TP) on growth, immunity and lipid metabolism in juvenile black carp Mylopharyngodon piceus (initial weight 5.90 ± 0.03 g). Tea polyphenols were added at different levels (0, 25, 50, 100 and 500 mg/kg; TP₀, TP₂₅, TP₅₀, TP₁₀₀ and TP₅₀₀). The results are as follows: the highest specific growth rate (SGR) and condition factor (CF) and activity of trypsin (TRS) of intestine in TP₅₀, but SGR and activities of lipase (LPS)and TRS of intestine and content of whole body crude protein in TP₅₀₀ were remarkably lower than TP₀. Compared with TP₀, content of serum superoxide dismutase (SOD) and glutamic oxalacetic transaminase (GOT) remarkably increased (p < .05), but contents of glutathione (GSH), glutathione peroxidase (GSH‐Pox), malondialdehyde (MDA), triglyceride (TG) and low‐density lipoprotein cholesterol (LDL‐C) were significantly decreased (p < .05), content of cortisol was remarkably lower in TP₅₀ and TP₁₀₀ (p < .05), expression of growth hormone (GH) and melanocortin 4 receptor (MC4R) in liver and GH in muscle were remarkably up‐regulated in TP₅₀, but expression of apolipoprotein A‐1 (ApoA1), GH and MC4R of intestine, ApoA1 of liver and MC4R of muscle in TP₅₀₀ were remarkably down‐regulated, contents of complement 3 (C3), complement 4 (C4), high‐density lipoprotein cholesterol (HDL‐C) and LDL‐C were significantly reduced in TP₅₀₀ (p < .05). In conclusion, TP could improve growth performance and oxidative capacity on juvenile black carp, and its optimal dosage was 50 mg/kg.     

Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC₅₀) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log₁₀ CFU mL^?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log₁₀ CFU g^?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log₁₀CFU mL^?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.     

L‐carnitine exerts a cytoprotective effect against H2O2‐induced oxidative stress in the fathead minnow muscle cell line

This study was conducted to investigate the protective effect of L‐carnitine (LC) against H₂O₂‐induced oxidative stress in the fathead minnow muscle cell line (FHM). The FHM cells were stimulated with 1 mM H₂O₂ for 1 h after LC pre‐treatment, and the cell viability and the activity and mRNA relative expression of antioxidant enzyme were measured to assess the antioxidant properties of LC. The results showed that the toxic effect of H₂O₂ on the viability of FHM cells was both dose‐ and time‐dependent. Furthermore, the viability of the 0.01–1 LC mM groups was significantly higher than those of the 1 mM H₂O₂ group. L‐carnitine protected the cells from H₂O₂‐induced oxidative damage, which was demonstrated by a significant reduction in the malondialdehyde and reactive oxygen species levels and increases in the intracellular total glutathione levels and the activities of total superoxide dismutase, catalase, glutathione peroxidase (GPx) and gamma‐glutamyl‐cysteine synthetase (γ‐GCS) in FHM cells pre‐treated with LC for 6 h compared with the 1 mM H₂O₂ group. In addition, the mRNA relative expression levels of the γ‐GCS catalytic subunit and nuclear factor nuclear factor erythroid 2‐related factor 2 were significantly higher than those of the 1 mM H₂O₂ group. It could be concluded that LC exerts a beneficial antioxidant effect against oxidative stress induced by H₂O₂ in FHM cells and that the appropriate treatment is 0.1–1 mM for 6 h in this study.