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The study isolated two strains of intestinal autochthonous bacteria Lactiplantibacillus plantarum1 (MH155966.1) (L1) and Lactiplantibacillus plantarum2 (MH105076.1) (L2) from the Choobdeh Abadan region. The aim of this study was to investigate the effects of different strains of probiotic bacteria on the growth performance, digestive enzyme activity, histopathologic and histomorphometric characterization of the intestine, expression of immune and growth related genes, and evaluate Lates calcarifer resistance against Vibrio alginolyticus. To achieve this, for each treatment 60 L. calcarifer juveniles (75 ± 12 g) were randomly distributed in three fiberglass tanks (300 L) and fed for 45 days. The treatments were established as Diet 1 (control diet); L1 (diet with Lb. plantarum isolated 1); L2 (diet with Lb. plantarum isolated 2) with a bacterial concentration of 1 × 109 CFU/g. Nine fish from each treatment were sampled and examined, after euthanasia. The fish were placed 2 cm from the beginning of the intestine for microscopic sampling of villi height, villi width and thickness of the epithelium, with 3 treatments: The result showed differences in the mean values of total weight were found at the end of the experiment. After 45 days of culture, the fish fed with L1 had higher (P < 0.05) growth performance than the other treatment groups. But at the end of the trial, in L2, the digestive enzyme activities were higher (P < 0.05) than the other treatment groups. The fishes fed diets supplemented with the L2 group, like the digestive enzyme activities test, presented an increase in the thickness of the epithelium of the intestine, and villus height, and villus width were greatest in L2. Fish feeding with L1 and L2 probiotics induced higher transcription levels of interleukin-10 (IL-10), granulocyte-macrophage colony-forming cells (GMCFC), epidermal growth factor (EGF), and Transforming Growth Factor Beta (TGF-ß) genes in the gut, which may correlate with better immune and hematological parameters in these groups. The results of the challenge test revealed that the percentage of survival was significantly higher in L1 (76.2%) and L2 (80.95%) treatments than in the control (P < 0.05). These results indicate that host-derived probiotics (Lb. plantarum) have significant potential as important probiotics to enhance nutrient utilization, Digestive enzymes, and metabolism by increasing the gut surface area of Lates calcarifer juveniles at 45 days of culture.

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A 60-day study was carried out to determine the effect of dietary selenium nanoparticles (SeNP) on growth, digestive enzymes, and health status of Asian seabass (Lates calcarifer, 46.5 ± 0.2 g) juveniles reared in high saline water (48 ppt). Five levels of SeNP were added to a basal diet (45% protein, 15% lipid), including control (0), 0.5 (SeNP0.5), 1.0 (SeNP1), 2 (SeNP2), and 4 (SeNP4) mg SeNP kg-1 diet. Fish were stocked into fifteen 2,000 L tanks (50 fish tank-1) filled with 1,800 L sand-filtered seawater (26.5 ± 1.5°C, 48.0 ± 0.2 ppt) in a flow-through system. Each dietary treatment was performed in three replicates. The growth rate positively increased in both linear and quadratic trends with increasing dietary SeNP level (P < 0.05). The liver Se concentration increased with increasing SeNP in diet (P < 0.05). Gut total protease, trypsin, chymotrypsin, alkaline phosphatase (ALP), lipase, and α-amylase activities were significantly enhanced in the SeNP4 group compared to the other treatments (P < 0.05). Antioxidant capacity improved in fish-fed SeNP2 and SeNP4 diets regarding catalase and superoxide dismutase activities and the liver glutathione content. Serum lysozyme and hemolytic activities and white blood cells' respiratory burst activity in the control were lower than in fish-fed SeNP-supplemented diets. Serum total protein, globulin, and globulin/albumin ratio in fish-fed SeNP1, SeNP2, and SeNP4 diets were higher than the other groups (P < 0.05). The interleukin-10 and granulocyte-macrophage colony-stimulating factor genes' relative transcription levels in the gut of fish-fed SeNP4 were higher than the other groups. Serum cholesterol, triglycerides, ALP, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly decreased in fish with increasing SeNP content in the diet. In conclusion, supplementing the diet with 4 mg kg-1, SeNP was recommended to improve growth and health indices in L. calcarifer juveniles reared in high saline water (48 ppt).

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A 16-week feeding trial was done to examine the impacts of continuous feeding (CF) or pulse-feeding (PF) of multi-strain probiotics on Asian seabass (Lates calcarifer, 30.0 ± 0.1 g) juveniles. In this study, three different multi-strain probiotic mixtures were added to a basal diet, including (I) a mixture of different strains of Lactobacillus plantarum, (II) a mixture of the first probiotic (I) + L. delbrueckii sub bulgaricus, L. rhamnosus and L. acidophilus, and (III) a mixture of the second probiotic (II) + two quorum quenching (QQ) bacteria (Bacillus thuringiensis QQ1 and B. cereus QQ2). CF (every day) or PF (every two weeks) strategies were applied for using the abovementioned probiotics to design seven experimental groups including C (control, without probiotics), CF-I (continuous feeding of fish with the probiotic mixture I), CF-II (continuous feeding of fish with the probiotic mixture II), CF-III (continuous feeding of fish with the probiotic mixture III), PF-I (pulse-feeding of fish with the probiotic mixture I), PF-II (pulse-feeding of fish with the probiotic mixture II), and PF-III (pulse-feeding of fish with the probiotic mixture III). Four hundred and twenty fish were stocked into 21 circular polyethylene tanks with 220 L volume (20 fish/tank). Each dietary treatment had three replicates. Tanks were supplied with seawater (temperature = 30.5 °C, salinity = 45 g L-1) in a flow-throw system. Fish in CF-I, CF-II, and CF-III had higher growth rate (ca. 113-145%) and better feed conversion ratio than fish fed C and PF-I (P < 0.05). Fish in the CF-III group had the highest protease activity. Continuous feeding strategy resulted in a higher amount of glutathione and catalase activities in both the liver and plasma as well as higher superoxide dismutase activity in the liver of fish. Pulse-feeding strategy resulted in lower plasma lactate dehydrogenase and aspartate aminotransferase levels than the CF strategy. Regardless of feeding strategy, different probiotic mixtures significantly enhanced blood hemoglobin and hematocrit levels compared to the control. Continuous feeding with the multi-strain probiotics resulted in a higher survival rate against Vibrio harveyi than the PF method. Continuous feeding induced higher mRNA transcription levels of granulocyte-macrophage colony-forming cells and interleukin 10 genes in the gut of fish than PF strategy. In conclusion, continuous feeding with multi-strain probiotics is better than pulse-feeding on growth, feed utilization, antioxidant capacity, and the gut's immune-related genes and led to higher resistance of L. calcarifer in challenge with V. harveyi.

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The present study investigated the effects of combined and singular oral administration of Bio-Aqua® with different dosages of sodium diformate (NaDF) on biochemical indices, innate immune responses, antioxidant effects, and expressions of immunological related genes of Caspian brown trout (Salmo trutta caspius). Fingerlings Salmo trutta caspius (n = 1800; initial weight 15 ± 3 g) were randomly allocated into five groups (120 fish group-1 in triplicates). Control diet: without any addition, G1, G2, G3, and G4 received diets containing 0.2 g kg-1 commercial probiotic Bio-Aqua® combined with 0, 0.5, 1.0, and 1.5% NaDF to the basal diet for 60 days according to recommended dosages reported in previous studies. Results indicated that serum bactericidal activity (G3 on day 60 and G1 on day 30) and classic complement in all groups (on day 60) (G1 and G2 on day 30) were significantly elevated (P < 0.05). The serum lysozyme, glucose, globulin, and albumin levels showed no significant differences between all groups compared to the control group (P > 0.05). On days 30 and 60 of the sampling, no significant difference was observed in the amount of superoxide disotase (SOD) and catalase (CAT) between the treatments (P > 0.05) but activity of malondialdehyde (MDA) was lower in G1 than the control (P < 0.05). The expression of the immune-regulating genes IL-10, IL-1ß, GTP, FATP, and IGF was significantly improved in all probiotic + acidifier-treated groups (P < 0.05). The current findings showed that mixture of Bio-Aqua® and NaDF (1.5% + pro) is beneficial, as it effectively improves some immune parameters and expression of immunological and growth-related genes in Caspian brown trout.

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In this study, the effect of dietary supplementation of mannan oligosaccharide (MOS) + ß-glucan (Immunogen®) was investigated on growth performance, body composition, gut microflora, innate immune responses and gene expression of some proinflammatory cytokines in shabout (Tor grypus). Shabout fingerlings (35 ± 1.2 g) were fed with basal diet (control) or basal diet supplemented with Immunogen® at 0.5, 1 and 1.5% of feed for 90 days. According to the results, growth parameters were significantly improved in fish fed with prebiotic (1 and 1.5%) for 90 days (p < 0.05). The carcass protein content was significantly higher in fish nourished by prebiotic at 1.5% of feed for 90 days compared to fish received the basal diet (p < 0.05). Feeding with various levels of Immunogen® resulted in the significant promotion of the population of intestinal Lactobacillus spp. in the prebiotic-treated groups relative to the control group (p < 0.05). Serum total globulin was significantly higher in all prebiotic groups relative to the control group at day 60. Serum bactericidal and lysozyme activities were significantly (p < 0.05) elevated after feeding with dietary prebiotic at all intervals (days 30, 60 and 90). However, the highest serum bactericidal activities were recorded in fish fed with Immunogen® at 1.5% of diet (p < 0.05). The transcription levels of interleukin 1 beta (IL-1ß), interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) were significantly increased in the head kidney of fish treated with dietary prebiotic at all intervals. The results show that dietary supplementation with Immunogen®, particularly at the level of 1.5%, can positively alter growth parameters, carcass protein, intestinal microflora and immune responses of shabout.

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The present study was conducted to investigate the effects of probiotic and encapsulated Lactobacillus bulgaricus on hematological and immunological factors after lead toxicity in rainbow trout (Oncorhynchus mykiss). Two hundred and forty fish weighing about 16 ± 3.8 g were divided randomly in to four groups including two groups which were fed by a diet containing ~ 108 CFU g-1Lactobacillus bulgaricus and encapsulated Lactobacillus bulgaricus bacteria and also the third group diet without Lactobacillus bulgaricus. After 45 days, in addition to probiotic (~ 108 CFU g-1), 500 µg kg of lead nitrate was added to the food of the three groups for 21 days. The fourth group (control) was first fed to the normal diet for 45 days then exposed to Pb. Blood samples were collected at days 45, 52, 59, and 66, and hematological and some immunological parameters were assessed. Results showed that hemoglobin, red blood cells, white blood cells, and lysozyme activity in the two probiotics groups were increased significantly up to 45 day (P < 0.05), but followed by a decreasing trend by adding Pb. Complement and bactericidal activity were enhanced significantly in the bulgaricus group (P < 0.05). Respiratory burst activity at day 45 in group bulgaricus had significant increase (P < 0.05) and decreased in all groups particularly after Pb exposure (P < 0.05). The achieved data shows that microencapsulation of probiotics with alginate-chitosan may be a suitable method to improve the fish condition against heavy metal.

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For investigating the possible effect of Lactobacillus casei on the immune status of Shabot, Tor grypus, 480 fish weighing 40 ± 10 g were randomly divided into four groups in triplicate. The first group (control) was fed on a commercial diet without probiotic supplementation. Other groups (A, B, and C) were received diet supplemented by different levels of L. casei, i.e., 5 × 106, 5 × 107, and 5 × 108 CFU g-1, respectively. The feeding trial was performed for 60 continuous days followed by 15 days as withdraw period, i.e., day 75. Serum immune responses were determined on days 0, 30, 60, and 75 of feeding trial. Gene expression in the kidneys was analyzed on days 0, 60, and 75 as well as following bacterial challenge. The results showed that dietary L. casei supplementation significantly (P < 0.05) increased serum lysozyme, serum bactericidal, complement, and respiratory burst activities as compared with control group, while it was observed that treated fish did not show any significant differences in immune responses on day 75 (P < 0.05). Results of gene expression (TNF-α, IL-1ß, and IL-8) showed significant increases among different experimental times (P < 0.05). According to obtained results, it might be concluded that the feeding by L. casei could likely enhance the immune responses and gene expression in T. grypus. Treatments A and C were better than B, but the lower concentration is better as an economic viewpoints and biosecurity. Therefore, concentration A is suggested.

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Quorum quenching (QQ), the enzymatic degradation of N-acyl homoserine lactones (AHLs), has been suggested as a promising strategy to control bacterial diseases. In this study, 10 AHL-degrading bacteria isolated from the intestine of barramundi were identified by 16S rDNA sequencing. They were able to degrade both short and long-chain AHLs associated with several pathogenic Vibrio species (spp.) in fish, including N-[(RS)-3-Hydroxybutyryl]-l-homoserine lactone (3-oh-C4-HSL), N-Hexanoyl-l-homoserine lactone (C6-HSL), N-(ß-Ketocaproyl)-l-homoserine lactone (3-oxo-C6-HSL), N-(3-Oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL), N-(3-Oxotetradecanoyl)-l-homoserine lactone (3-oxo-C14-HSL). Five QQ isolates (QQIs) belonging to the Bacillus and Shewanella genera, showed high capacity to degrade both synthetic AHLs as well as natural AHLs produced by Vibrio harveyi and Vibrio alginolyticus using the well-diffusion method and thin-layer chromatography (TLC). The genes responsible for QQ activity, including aiiA, ytnP, and aaC were also detected. Analysis of the amino acid sequences from the predicted lactonases revealed the presence of the conserved motif HxHxDH. The selected isolates were further characterized in terms of their probiotic potentials in vitro. Based on our scoring system, Bacillus thuringiensis QQ1 and Bacillus cereus QQ2 exhibited suitable probiotic characteristics, including the production of spore and exoenzymes, resistance to bile salts and pH, high potential to adhere on mucus, appropriate growth abilities, safety to barramundi, and sensitivity to antibiotics. These isolates, therefore, constitute new QQ probiotics that could be used to control vibriosis in Lates calcalifer.

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The present study was tested how Oncorhynchus mykiss can respond to dietary supplementations of autochthonous probiotics, including Lactobacillus delbrukei subsp. bulgaricus and Lactobacillus acidophilus and Citrobacter farmeri by measuring different parameters. To address that, 300 fish weighing 19.08-32.9 g were fed by probiotics-enriched diets, containing 5 × 107 CFU g-1 for 60 days. Our results indicated that probiotics, especially L. acidophilus and L. bulgaricus are involved in enhancing the growth performance of this species as compared with the control group. Blood profile (Hemoglobin and Hematocrit) showed significant (P < 0.05) increases in probiotic fed groups compared with the control. Serum lysozyme and complement activities were higher in probiotic-fed fish while similar changes were not observed in the case of bactericidal activity and Nitroblue Tetrazolium (NBT) reduction. Better colonization of lactic acid bacteria in fish intestine was observed following L. acidophilus and L. bulgaricus administrations (P < 0.001). Digestive enzyme activities of intestine, including amylase, trypsin, lipase and alkaline phosphatase were elevated either significant or insignificant while protease activity did not act the same. All probiotic treatments led to mild or strong (P < 0.001) up-regulation of cytokine and growth gene expressions of intestine in comparison with the control group. Higher in vitro antagonist activities of L. acidophilus and L. bulgaricus against the Lactococcus garvieae were coincident with in vivo challenge test. The relative percentage of survival (RPS) was obtained 63.71 and 51.56 for L. bulgaricus and L. acidophilus, respectively, which were higher in those treated fish as compared to control fish. Our results may suggest that the probiotics, applied here, can promote growth performance by improving digestive enzyme activity, gut micro flora and growth gene expression. Up-regulation of immune regulatory proteins may increase the non-specific immune responses and bacterial resistance in this species as well.

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In this study, the effects of orally administrated two native probiotics (Lactobacillus plantarum and Lactobacillus delbrueckii ssp. bulguricus), isolated from the intestine of Shabot fish, Tor grypus, on some immune response parameters and immune-related genes expression against Aeromonas hydrophila in T. grypus were evaluated. Four hundred and eighty juveniles weighing 45 ± 10 g were randomly divided into four groups (with three replications) and fed with the experimental diet containing 5 × 107 cfu g-1 of L. plantarum (G1), Lactobacillus bulgaricus (G2), Lactobacillus casei (G3), and a control diet (without probiotics) for 60 continuous days. At the end of the dietary treatments, fish were challenged with a lethal concentration of A. hydrophila (5 × 108 CFU ml-1) via intra peritoneal (i.p) injection. Blood and head kidney samples were taken from six fish in each treatment before challenging and 6, 12, 24, and 48 h and also 7 days after injection. The results showed that lysozyme, complement, bactericidal, and NBT activity of probiotic-treated groups were significantly elevated (P < 0.05). The IL-8, IL-1ß, and TNF-α gene expressions were significantly higher in all probiotic-treated groups (P < 0.05). Meanwhile, a high direct correlation was observed between serum immune parameters and expression of immune-related genes (P < 0.0001); furthermore, the highest correlation (R 2 = 0.634, P < 0.0001) was recorded between IL-1ß expression and NBT activity. It can be concluded that not only two native probiotics strains stimulate serum immune responses parameters and immune-related gene expression in T. grypus, but also a high correlation was seen among these indices. The study suggests that gastrointestinal colonization is preferred for host specificity as the strain previously derived from shabot fish displayed better colonization than the non-indigenous bacteria strain such as L. casei. Therefore, these native probiotics bacteria can be accounted as suitable candidates to immune stimulation in fish.

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Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae which causes significant losses in the aquaculture industry. To develop a recombinant vaccine for IPNV, a cDNA construct of IPNV VP2-VP3 fusion gene was prepared and cloned into an Escherichia coli (E. coli) expression vector (pET-26b) to obtain recombinant protein products. A study was conducted to determine the antibody responses and protective capacity of this recombinant vaccine expressing VP2-VP3 fusion protein. Subsequently, juvenile rainbow trout were inoculated by injecting purified recombinant IPNV VP2-VP3 proteins, followed by challenge with virulent IPNV in rainbow trout. Our results demonstrate that recombinant E. coli derived VP2-VP3 fusion protein induced a strong and significantly (P < 0.05) higher IgM antibody response in serum samples compared to control groups. Following intraperitoneal challenge, the relative percent survival (RPS) rate of survivors was 83% for the vaccinated group. Statistical analysis of IgM levels indicated that immunogenicity of recombinant VP2-VP3 protein, combined with adjuvant, was much higher than any other groups of rainbow trout challenged with virulent IPNV. This result was confirmed by measuring the viral loads of IPNV in immunized rainbow trout which was drastically reduced, as analyzed by real-time RT-PCR. In summary, we demonstrate that E. coli-expressed IPNV VP2-VP3 injectable vaccine is highly immunogenic and protective against IPNV infection.

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The question of whether, as hormone therapies, spawning agents differ from each other to induce physiological pathways of gametogenesis and oocyte maturation in fish remains important, because it could modify undesirable changes, regulated by endocrine systems of individual fish. A series of experimental treatments were applied to investigate the underlying mechanism(s) in which female bunnei (Barbus sharpeyi) fish respond differently to hormone therapies. Female broodstocks were injected twice (with 12 h interval) by three different treatments namely A, B and C. The treatment A received carp pituitary extract (CPE) + luteinizing hormone-releasing hormone analogs (LHRHα2) (0.5 mg CPE kg(-1) BW for first injection and 2 mg CPE kg(-1) BW + 10 µg LHRHα2 kg(-1) for second injection), treatment B received CPE (0.5 and 3.5 mg kg(-1) BW), and treatment C received ovaprim (0.1 and 0.15 ml kg(-1) BW). Blood samples were collected at four different time intervals, including prior to injections, 6 h after first injection, 6 h after second injection and at the time of spawning, and serum steroid hormones, including testosterone, progesterone and estradiol-17ß as well as cortisol, were measured. Results showed significant increases in serum estradiol-17ß following all treatments, but the most profound response was found in treatments A and B. Testosterone was higher in larger broodfish than in small-sized broodfish (>1.5 vs. <1.5 kg) in all treatments. CPE led to higher concentration of testosterone rather than two other treatments. CPE also increases the progesterone following first injection and approximately remains unchanged till the end of experiment. Change in progesterone level was only significant after second injection of ovaprim as well as after spawning compared with previous time. Linear regression analyses indicated that cortisol had adverse effects on progesterone and testosterone levels of weight group <1.5 kg. These results suggest that among inducing agents, applied here, CPE can provide more reasonable response in reproduction of female B. sharpeyi.

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