Effects of <Emphasis Type="SmallCaps">l</Emphasis>-carnitine against H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced oxidative stress in grass carp ovary cells (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

This study was designed in vitro to investigate the effects of l-carnitine against H₂O₂-induced oxidative stress in a grass carp (Ctenopharyngodon idellus) ovary cell line (GCO). GCO cells were pre-treated with different concentrations of l-carnitine, followed by incubation with 2.5 mM H₂O₂ for 1 h to induce oxidative damage. The results indicated that adding l-carnitine at concentrations of 0.01–1 mM into the medium for 12 h significantly increased cell viability. Pre-treatment with l-carnitine at concentrations of 0.1–5 mM for 12 h significantly inhibited 2.5 mM H₂O₂-induced cell viability loss. The significant decreases in the level of reactive oxygen species and cell apoptosis were observed in 0.5 mM l-carnitine group compared to the H₂O₂ group. Malondialdehyde values of all of the l-carnitine groups were significantly lower than those of the H₂O₂ group, while total glutathione levels of all of the l-carnitine groups were significantly higher than of the H₂O₂ group. The activity of antioxidant enzymes, such as total superoxide dismutase (0.1 and 0.5 mM l-carnitine), catalase (0.5 mM l-carnitine) and γ-glutamyl cysteine synthetase (0.5 and 1 mM l-carnitine), was significantly increased. In addition, pre-treatment of l-carnitine in GCO cells exposed to 2.5 mM H₂O₂ significantly increased the mRNA expression of copper, zinc superoxide dismutase, catalase (0.5 mM l-carnitine), glutamate cysteine ligase catalytic subunit (0.1–1 mM) and glutathione peroxidase (0.1 mM l-carnitine). In conclusion, l-carnitine promotes GCO cell growth and improves antioxidant function, it plays a protective role against oxidative stress induced by H₂O₂ in GCO cells, and the appropriate supplemental amount of l-carnitine is 0.1–1 mM.     

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress,but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.     

Zebrafish fin‐derived fibroblast cell line: A model for in vitro wound healing

The goal of this study was to develop and characterize a cell line from the caudal fin tissue of zebrafish and also its application as an in vitro model to study the effect of H₂O₂ in wound healing. Fibroblastic cell line was developed using explant culture method from caudal fin tissue of zebrafish and characterized. This cell line was named as DrF cell line. The DrF cells treated with 0–10 µM/ml H₂O₂ were tested for viability, proliferation and motility by MTT assay, trypan blue assay and chemotaxis assay, respectively. Among the different concentrations of H₂O₂, 4 µM was found to be nontoxic to study cell migration in in vitro scratch wound assay. Furthermore, the expression of proliferating cell nuclear antigen (PCNA) and chemokine receptor (CXCR4) genes was carried by qPCR. The cell survival, proliferation and migration were extremely enriched at 4 µM level of H₂O₂. We observed accelerated wound closure in DrF cells treated with H₂O_2. The qPCR results indicated that H₂O₂ markedly up‐regulated mRNA expression of PCNA and CXCR4. The findings from our study suggest that H₂O₂ at low levels promotes cell survival, proliferation, migration and wound healing in DrF cells.     

Evaluation of the immune response in rainbow trout fry,Oncorhynchus mykiss (Walbaum), after waterborne exposure to Flavobacterium psychrophilum and/or hydrogen peroxide

The immune response in rainbow trout fry against Flavobacterium psychrophilum was elucidated using an immersion‐based challenge with or without prior exposure to hydrogen peroxide (H₂O₂). Samples were taken from the head kidney 4, 48, 125 and 192 h after immersion, and the regulation of several genes was examined. Bacterial load was assessed based on the presence of 16S rRNA and correlated with gene expression, and the levels of specific antibodies in the blood were measured 50 days post‐infection. Separately, both H₂O₂ and F. psychrophilum influenced gene expression, and pre‐treatment with H₂O₂ influenced the response to infection with F. psychrophilum. Pre‐treatment with H₂O₂ also affected correlation between gene regulation and pathogen load for several genes. A delay in antibody production in H₂O₂‐treated fish in the early phase of infection was indicated, but H₂O₂ exposure did not affect antibody levels 50 days post‐infection. An increasing amount of F. psychrophilum 16S rRNA was found in the head kidneys of infected fish pre‐treated with H₂O₂ relative to the F. psychrophilum group. The results show that a single pre‐treatment with H₂O₂ impairs the response against F. psychrophilum and may intensify infection.     

Effect of dietary l‐carnitine on growth performance and antioxidant response in Amur minnow (Phoxinus lagowskii Dybowskii)

l ‐carnitine (LC) is required for transporting long‐chain fatty acids into the mitochondria, where β‐oxidation takes place, and it works as an antioxidant molecule against reactive oxygen species. This study evaluated the effects of LC on the growth and antioxidant function of Amur minnow (Phoxinus lagowskii Dybowskii). Five isonitrogenous (380.4 g/kg) and isoenergetic (17.63 MJ/kg) diets were supplemented with five LC levels: control level (0 mg/kg) and treatment levels (50, 400, 750, or 1,100 mg/kg) were fed to fish (18.19 ± 0.56 g) for 120 days. The results showed that the growth performance of fish fed a diet containing 400 mg/kg of LC was significantly higher than that of the control and those fed other LC level treatments. Similarly, the 400 mg/kg treatment had the best feed efficiency. Further, the levels of total antioxidant capacity and total glutathione in the serum and hepatopancreas of fish fed a diet containing 750 mg/kg of LC were significantly increased; however, malondialdehyde levels were significantly reduced compared to those of the control group. The activities of antioxidant enzymes of 750 mg/kg treatments in the serum and hepatopancreas were significantly higher than those of the control group, including total superoxide dismutase, catalase, glutathione peroxidase and gamma‐glutamyl‐cysteine synthetase. Finally, 750 mg/kg treatment significantly upregulated the mRNA relative expression of antioxidant enzymes and nuclear factor erythroid‐2‐related factor 2 and inhibited the mRNA level of kelch‐like ECH‐associated protein 1 in the hepatopancreas. In conclusion, the dietary LC level of 400–750 mg/kg could improve the growth performance, feed utilization and antioxidant defense system of Amur minnow under the culture conditions.     

The protective effects of Vitamin C and Trolox on kinematic and oxidative stress indices in rainbow trout sperm cells against flower‐like ZnO nanoparticles

This study investigated the protective effects of 1 mM Vitamin C and 1 mM Trolox on kinematic and oxidative stresses in rainbow trout (Oncorhynchus mykiss) sperm cells against flower‐like zinc oxide nanoparticles (ZnO‐NP, 1 mg/L) in vitro, during 2 hr. Trolox showed protective effects on all kinematic parameters measured for sperm cells against ZnO‐NPs, whereas Vitamin C only affected angular path velocity (VAP), linearity (LIN) and amplitude of lateral displacement of the sperm cell head (ALH). Although ZnO‐NPs reduced total glutathione (TGSH) and catalase (CAT) compared with the control, there were positive treatment effects for both Trolox and Vitamin C on antioxidant capacity. Conversely, while ZnO‐NP increased the rate of malondialdehyde (MDA) lipid peroxidation, it decreased following antioxidant therapy. This research suggests that Trolox and Vitamin C play a protective role for rainbow trout sperm exposed to ZnO‐NP.     

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

Nocardia seriolae, a facultative intracellular bacterium, is the main pathogen of fish nocardiosis. Bioinformatic analysis showed that the histone‐like DNA‐binding protein (HLP) gene of N. seriolae (nshlp) encoded a secreted protein and might target the mitochondria in the host cell. To further study the preliminary function of HLP in N. seriolae (NsHLP), the gene cloning, extracellular products identification, subcellular localization, overexpression and apoptosis detection assay were carried out in this study. Mass spectrometry analysis of the extracellular products from N. seriolae showed that NsHLP was a secreted protein. Subcellular localization of HLP‐GFP fusion proteins mainly assembled in the nucleus, which indicated that the NsHLP was co‐located with the nucleus rather than mitochondria in fathead minnow (FHM) cells. Notably, the expression of NsHLP had changed the distribution of mitochondria into lumps in the FHM cell. In addition, apoptotic features were found in the transfected FHM cells by overexpression of NsHLP. Quantitative assays of mitochondrial membrane potential value, caspase‐3 activity and pro‐apoptotic genes mRNA (Bad, Bid and Bax) expression level demonstrated that the cell apoptosis was induced in the transfected FHM cells. All the results presented in this study provided insight on the function of NsHLP, which suggested that it may participate in the cell apoptosis regulation and play an important role in the pathogenesis of N. seriolae.     

Effect of dietary L‐carnitine supplementation on growth performance and lipid metabolism in Rhynchocypris lagowski Dybowski fed oxidized fish oil

Lipid content of a diet is very susceptible to oxidation, which has many negative effects on farmed animals. Therefore, this study studied the protective effect of L‐carnitine (LC) on fish body stimulated by oxidized fish oil (OFO) from lipid metabolism. Lipid content of the diet was replaced by OFO in 0, 100 and 400 meq/kg. L‐carnitine was added to the diet in two levels, 500 and 1,000 mg/kg, giving a total of seven experimental diets. A total of 735 healthy Rhynchocypris lagowski Dybowski with an initial weight of 4.48 ± 0.14 g after 2‐week adaptation randomly divided into 15 glass aquariums. Fish were fed satiated three times daily. After 8 weeks, biometry was done to evaluate growth performance, and hepatopancreas and muscle samples were taken for biochemical analysis. The result showed that feeding with OFO had negative growth. However, in fish received both OFO and LC, growth indices improved slightly (p > .05). Feeding with OFO and LC, the content of EPA, DHA and PUFA in the muscle of R. lagowski was significantly higher than that in the control group (p < .05), which reached the maximum value in the OFO100 + LC500 group. The content of SFA, MUFA, ∑n‐6 and PUFA in hepatopancreas increased significantly (p < .05), and the content of SFA reached the maximum in OFO100 + LC500 group. Feeding with OFO increased hepatopancreas total cholesterol, triacylglycerol, HDL/LDL ratio, FAS and ACCα that involved in lipid synthesis enzymes, while reduced HL and HSLα enzyme activity and gene expression that associated with lipid decomposition. Dietary LC moderated the effects of OFO on lipid metabolism. According to the result of the present study, it can be argued that feeding of R. lagowski with OFO has negative effects on growth performance and lipid metabolism, whereas LC dosages used in this study have increased the oxidation rate of fatty acids in the hepatopancreas of R. lagowski and improved the accumulation of fat in hepatopancreas cells induced by oxidized fish oil.     

Hepatoprotective and antioxidant effects of dietary Angelica sinensis extract against carbon tetrachloride‐induced hepatic injury in Jian Carp (Cyprinus carpio var. Jian)

The present study aimed to evaluate the hepatoprotective effects of Angelica sinensis extract (ASE) against carbon tetrachloride (CCl₄)‐induced hepatotoxicity in Jian carp (Cyprinus carpio var.Jian). Fish were fed diets containing four doses of ASE (0%, 0.1%, 0.5% and 1.0%) for 60 days, and then given an intraperitoneal injection of 30% CCl₄ in olive oil at a volume of 0.05 mL/10 g body weight. At 72 h post injection, blood and liver samples were collected for biochemical analysis, comet assay, histopathological examination and CYP3A mRNA expression. Results showed that the increases of glutamate pyruvate transaminase (GPT) and glutamate oxalate transaminase (GOT) induced by CCl₄ were significantly inhibited by pre‐treating the fish with 0.1%, 0.5% and 1.0% ASE in the diets. The elevation of lactate dehydrogenase (LDH) and the reductions of the total protein (TP) and albumin (Alb) in the serum induced by CCl₄ were also inhibited by pre‐treatments with 0.5 and 1.0% ASE. In the liver tissue, pre‐treatment with 1.0% ASE significantly inhibited the malondialdehyde (MDA) formation and the reductions of the total antioxidant capacity (T‐AOC), superoxide dismutase (SOD), glutathione (GSH) and CYP3A mRNA expression induced by CCl₄. Comet assay showed that tail moment, olive tail moment, tail length and tail DNA% were positively changed in fish pretreated with 0.5 and 1.0% ASE. CCl₄‐induced histological changes were obviously reduced by 0.5% and 1.0% ASE. Overall results prove the hepatoprotective effect of ASE in a dose‐dependent manner and support the use of ASE (1.0%) as a hepatoprotective and antioxidant agent in fish.     

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.     

Dietary profile of n‐3 series LC‐PUFAs in rainbow trout under regular stripping condition: Semen production and quality,hepato‐somatic index,haemato‐immunologic values,oxidative stress and fatty acid composition of liver,muscle and semen

In this study, the semen production and quality, hepato‐somatic index, haemato‐immunologic values, oxidative stress and the fatty acid contents in liver, muscle and semen of rainbow trout fed diets supplemented n‐3 series long‐chain polyunsaturated fatty acids (LC‐PUFAs) under regular stripping condition were investigated. For this aim, three diets (Control, D1 and D2) were prepared. These diets were contained n‐3 LC‐PUFAs (as a percentage of dietary total fatty acid) at 3.14%, 7.84% and 13.63% respectively. Experimental fish were fed with the control and test diets. The highest hepato‐somatic index, spermatologic (semen pH and volume, sperm motility and density), haematologic (haematocrit value, haemoglobin, erythrocyte count, corpuscular volume, haemoglobin and its concentration in corpuscular), immunologic (nitroblue tetrazolium activity, leucocyte count, phagocytic index, protein and immunoglobulin [IgM] in total plasma) and antioxidants (reduced glutathione, catalase, glutathione peroxidase and superoxide dismutase) values were found in fish fed the D2, D1 and control diets respectively (p < 0.01). Increase in the dietary n‐3 LC‐PUFAs was not significantly (p > 0.01) increased the oxidative stress (malondialdehyde) in fish. The results indicated that the n‐3 LC‐PUFAs at 13.63% level of total fatty acid in the diet could increase the semen production and quality, hepato‐somatic index, haematologic and immunologic values, and the n‐3 LC‐PUFA contents in liver, muscle and semen of rainbow trout broodstock under regular stripping condition.     

Antioxidant and protective effects of a peptide (VTAL) derived from simulated gastrointestinal digestion of protein hydrolysates of Magallana gigas against acetaminophen-induced HepG2 cells

Oxidative stress is an automatic mechanism responsible for the commencement and continuance of liver injury. In this study, an antioxidative peptide Val-Thr-Ala-Leu (VTAL) was purified from simulated gastrointestinal digestion of protein hydrolysates of the triploid oyster Magallana gigas. Significant antioxidant activity was identified, as well as a protective effect against acetaminophen (APAP)-induced human liver cancer (HepG2) cells. The results suggested that the antioxidant activity improved in a dose-dependent manner. The highest cell viability (88.105?±?3.62%) was observed in 15 mM APAP-induced cells when treated with 25 μg/mL M. gigas peptide [M.g (pep)]. The peptide sequences include hydrophobic amino acids, which could be responsible for its chemoprotective and antioxidant activities. Treatment with M.g (pep) significantly promoted the proliferation of HepG2 cells, thus protecting them against APAP and imbuing them with significant antioxidant capacity. M.g (pep) could be beneficial for treating drug-induced oxidative stress and liver damage. Additionally, M.g (pep) could serve as an alternative to synthetic antioxidant drugs.

     

Improvement in wild endangered Persian sturgeon,Acipenser persicus (Borodin, 1897) semen cryopreservation by 2‐hydroxypropyl‐beta‐cyclodextrin (HβCD)

In this study, cryopreservation feasibility of Persian sturgeon (Acipenser persicus) and the effect of different doses of 2‐hydroxypropyl‐beta‐cyclodextrin on thawed spermatozoa quality (motility duration and motility percentage) were investigated. For freezing, semen of seven male individuals was pooled in equal volumes and diluted with 4°C [Tris‐HCl (100 mM), pH = 8, DMSO 10%] extenders containing 0, 5, 10, 15 mM of HβCD in a ratio of 1:1(semen/extenders). Then semen was filled into 0.5‐mL straws, and was frozen with vapour of liquid nitrogen at 4‐cm above surface of liquid nitrogen. After 3 min, straws were plunged in to liquid nitrogen. Thawing was performed at 40°C water baths for 15 s. Motility duration of the 10 mM HβCD treated spermatozoa at days 14 (228.98 ± 16.39) and 56 (199.66 ±21.78) were longer than other treatments. In day 56, the motility percentage in treatment with 10 mM was significantly higher (16.14 ± 2.54) (P < 0.05) compared with 5 mM treatment (8.75 ± 2.47) (P < 0.05). Therefore, it is recommended that 10 mM of HβCD can be used as an additive cryoprotectant for increasing cryopreserved spermatozoa quality in this species.     

Effects of glutamate in low‐phosphorus diets on growth performance,antioxidant enzyme activity,immune‐related gene expression and resistance to Aeromonas hydrophila of juvenile mirror carp (Cyprinus carpio)

This study investigated the effects of glutamate (Glu) in low‐phosphorus diets on growth performance, haematological indices, antioxidant enzyme activity, immune‐related gene expression and resistance to Aeromonas hydrophila in juvenile mirror carp (Cyprinus carpio) (5.07 ± 0.02 g). Fish were fed either graded levels of Glu (0 g/kg, 5 g/kg,  10 g/kg and 20 g/kg, named G0, G0.5, G1 and G2, respectively) in a low‐phosphorus diet (15 g/kg NaH₂PO_4, 0.49), or a normal phosphorus diet ( 20 g/kg NaH₂PO_4, 0.61) without added Glu (C), for 8 weeks. At the end of the feeding trial, the fish were challenged with A. hydrophila. Compared with G0 group, 10 g/kg and 20 g/kg Glu supplementation of the low‐phosphorus diet significantly improved the final weight, WGR, SGR and PER, and decreased FCR (p < .05). Glu supplementation of the low‐phosphorus diet significantly enhanced the T‐AOC, SOD activity and GSH content in intestine (p < .05). Glu supplementation significantly reduced MDA content in foregut and midgut and increased CAT activity in midgut and hindgut (p < .05). Regarding immune‐related gene expression, Glu supplementation significantly diminished the up‐regulation of intestinal TNF‐α, IL‐1β and IL‐8 mRNA levels induced by phosphorus deficiency (p < .05). The survival rate of the G1 group was significantly higher than that of the G0 group (p < .05). In conclusion, 10 g/kg Glu supplementation in low‐phosphorus diets can improve the growth performance, enhance the activity of intestinal antioxidant enzymes and strengthen the immune function of juvenile mirror carp.     

Roles of zinc and selenium in protecting sea cucumber Apostichopus japonicus coelomocytes against lipopolysaccharide‐induced oxidative stress in vitro

This study investigated the attenuate effects of zinc (Zn) and selenium (Se) on lipopolysaccharide (LPS)‐induced oxidative stress in sea cucumber Apostichopus japonicus coelomocytes in vitro. Coelomocytes were first treated with different concentrations of Zn (0.12, 0.48 and 1.2 mM) and Se (0.06, 0.24 and 0.6 mM) for 12 h and the optimal concentrations of Zn and Se as antioxidants for A. japonicus coelomocytes were selected based on antioxidant parameters including total antioxidant capacity (T‐AOC) and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione‐S‐transferase (GST) and concentration of reduced glutathione (GSH). Next, coelomocytes were pretreated with 0 (control), 0.12 mM Zn or 0.24 mM Se for 12 h, and then were treated with LPS (100 μg mL^?1) for 6 h. Se completely inhibited LPS‐induced increase in superoxide anion () production and lipid peroxidation (LPO) and effectively prevented the LPS‐induced decreases of T‐AOC, activities of SOD, CAT, GPx and GST and concentration of GSH. Zn alleviated the LPS‐induced oxidative stress but the protective effects were not as effective as Se. The present work also proved that suboptimal amount of Zn and Se could impair the antioxidant system of A. japonicus coelomoytes. In conclusion, the present work demonstrated that both Zn and Se, especially Se, have the potentials to be the effective antioxidants for A. japonicus. Further work will be conducted for their optimal administration concentrations in vivo.     

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.     

Effect of short‐term fasting and re‐feeding on growth,digestive enzyme activities and antioxidant defence in yellowfin seabream,Acanthopagrus latus (Houttuyn, 1782)

An 80‐day feeding trial was conducted to evaluate the influence of different short‐term fasting and re‐feeding strategies on growth and physiological responses in yellowfin seabream, Acanthopagrus latus (2.4 ± 0.2 g) fingerlings. The fish were subjected to four different feeding regimes, and the control group fed four times daily to apparent satiation throughout the whole feeding period, while the other three groups were deprived for 2, 4 and 8 days followed by 8, 16 or 32 days of re‐feeding (F₂R₈, F₄R₁₆ and F₈R₃₂, respectively) in repeated cycles for 80 days. The fish in the control and F₂R₈ groups had the highest and the lowest total length, respectively (p < .05). Moreover, fish exposed to F₄R₁₆ had the highest hepatosomatic indices, while control fish had the lowest hepatosomatic indices (p < .05). Fish in the F₂R₈ group relatively had higher catalase and glutathione‐S‐transferase activities than other groups (p < .05). Furthermore, total protease, α‐amylase and alkaline phosphatase activities in the F₄R₁₆ and F₈R₃₂ were higher than the F₂R₄ and control groups (p < .05). Overall, this study showed that compensatory growth in weight and length and digestive enzyme activities were observed in the F₄R₁₆ and F₈R₃₂; however, the increase in the activity of antioxidant enzymes in the F₈R₃₂ group indicated that oxidative stress remained after 80 days of re‐feeding in the liver.     

Antioxidant and anti‐inflammatory activities of Pinus radiata bark extract in salmonid cell lines

A fish meal supply shortage is limiting aquaculture development. Currently, plant‐based proteins, such as soya bean meal, are being used as an alternative protein source, despite that such a diet can adversely affect fish, such as by inducing an inflammatory response. A possible solution is to include dietary additives in farm diets to counteract negative effects. One such solution originates from pine bark extracts, which present bioactive properties. In this study, the antioxidant and anti‐inflammatory properties of Pinus radiata bark extracts were evaluated for the first time in a salmonid cell line. This extract chemically demonstrated antioxidant activity through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH = 58.4 ± 1.1%) and ferric ion reducing antioxidant power (FRAP = 575 ± 17 mgEqFe(II)·g extract^?1) assays. Additionally, the extract showed high flavonoid and phenolic compound contents. Up to 100 mg mL^?1, the P. radiata extract showed no cytotoxicity in the CHSE‐214 salmonid embryo cell line. Moreover, the antioxidant activity of the extract (50 μg mL^?1) was evaluated by a dichlorofluorescein (DCFH) assay in the SHK‐1 salmon cell line challenged with an oxidant stimulus (H₂O₂), showing 58.9% activity. The extract also protected DNA from oxidative damage, as observed through a comet assay. When assessing anti‐inflammatory properties in an in vitro inflammation model, the extract significantly reduced the relative expression of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) and of the inducible cyclooxygenase‐2 (COX‐2) enzyme. These results suggest a potential application of P. radiata bark extract in functional foods in aquaculture.     

Does Ralstonia eutropha,rich in poly‐β hydroxybutyrate (PHB), protect blue mussel larvae against pathogenic vibrios?

The natural amorphous polymer poly‐β‐hydroxybutyrate (PHB‐A: lyophilized Ralstonia eutropha containing 75% PHB) was used as a biological agent to control bacterial pathogens of blue mussel (Mytilus edulis) larvae. The larvae were supplied with PHB‐A at a concentration of 1 or 10 mg/L for 6 or 24 hr, followed by exposure to either the rifampicin‐resistant pathogen Vibrio splendidus or Vibrio coralliilyticus at a concentration of 10⁵ CFU/ml. Larvae pretreated 6 hr with PHB‐A (1 mg/L) survived a Vibrio challenge better relative to 24 hr pretreatment. After 96 hr of pathogen exposure, the survival of PHB‐A‐treated mussel larvae was 1.41‐ and 1.76‐fold higher than the non‐treated larvae when challenged with V. splendidus and V. coralliilyticus, respectively. Growth inhibition of the two pathogens at four concentrations of the monomer β‐HB (1, 5, 25 and 125 mM) was tested in vitro in LB₃₅ medium, buffered at two different pH values (pH 7 and pH 8). The highest concentration of 125 mM significantly inhibited the pathogen growth in comparison to the lower levels. The effect of β‐HB on the production of virulence factors in the tested pathogenic Vibrios revealed a variable pattern of responses.