Nucleotides supplementation (Nucleoforce fish™) in Caspian roach (Rutilus caspicus) diet: Growth performance, skin mucosal immune response, and resistance to salinity stress.

In this study, the growth, epidermal mucosal immunity, expression of growth-related genes, cross-protection, and resistance to salinity stress of Caspian roach were scrutinized in response to dietary levels of nucleotides (NT). Accordingly, 1200 fish (0.51 ± 0.01 g) were fed ad libitum with a basal diet (38.88 % crude protein and 10.04 % crude lipid in dry basis) containing incremental levels of NT at 0 (NT-0; control), 0.3 g kg-1 (NT-0.3), 0.6 g kg-1 (NT-0.6), and 1.2 g kg-1 (NT-1.2) for 8 weeks in triplicates. The growth performance was significantly increased in the fish fed with NT-0.6 and NT-1.2 diets compared to the control group (p < 0.05). A significant elevation in the growth hormone and insulin-like growth factor-I gene expression was recorded in NT-added groups at 0.6 and 1.2 g kg-1 compared to the control group (p < 0.05). In contrast to the control group, feeding on NT-0.6 and NT-1.2 diets had a remarkable effect on the skin mucus soluble protein and immunoglobulin levels (p < 0.05). After the feeding trial, we examined how salinity stress (15 g/l salinity) lonely and salinity stress under non-lethal thermal shock (+10 °C) affected heat shock protein (HSP70). Then, the mRNA expression of HSP70 gene from the gill was analyzed at 0, 2, 8, and 24 h post-challenge tests. The HSP70 gene expression level was approximately up-regulated more than 2-fold in NT-6 and NT-1.2 treatments compared to the control group under the salinity stress. Altogether, this research represents that the addition of NT at 0.6 and 1.2 g kg-1 in Caspian roach diet can improve overall performance and resistance to salinity stress.

Fractionation of the Caspian sand goby epidermal exudates using membrane ultrafiltration and reversed-phase chromatography: an investigation on bioactivities.

Bioactive peptide-based drugs have gained exceeding attention as promising treatments for infectious and oxidative-stress-related diseases, are exacerbated by the advent and spread of various multidrug-resistant bacteria and industrial lifestyles. Fish skin mucus has been recognized as a potential source of bioactive peptides, providing the first line of fish defense against invading pathogens which are targeted here to be explored as a new source of biopharmaceutics. Peptide fractions were isolated from the epidermal exudates of Caspian sand goby, Neogobius fluviatilis pallasi, by solid-phase extraction (SPE), ultrafiltration, and reversed-phase chromatography. The resulting fractions were characterized for their antibacterial and antioxidant properties, and results showed that the molecular weight fraction < 5 kDa represented the highest (p < 0.05) bacterial inhibition activity against Staphylococcus aureus and Bacillus subtilis as well as scavenging activity against DPPH and ABTS radicals. Overall, these results introduce the epidermal mucus of Caspian sand goby as a valuable source of bioactive compounds that can be considered new and efficient biopharmaceutics.

Sex and reproductive development impact skin mucosal epithelium immunity, antimicrobial capacity, and up-regulation of immune-related gene of goldfish (Carassius auratus).

This study evaluated the epidermis mucosal capacity of goldfish (Carassius auratus) during different stages of reproductive development in both females and males. In this regard, the activity of mucolytic immune enzymes, i.e., lysozyme, complement and peroxidase, as well as the activity of alkaline phosphatase (ALP) were evaluated. There were five stages for females i.e., immature (f1), cortical alveoli (f2), early and late-vitellogenesis (vtg) (f3 and f4) and ripe (f5); as well as two stages for males spermatogenesis (m1) and spermiation (m2). Some stages were also examined for the mucosal antimicrobial activity against specific pathogens. The results showed that the mucosal lysozyme activity increased significantly during vitellogenesis (P < 0.05), but no lysozyme activity was detected in plasma. On the contrary, the complement activity was only observed in female plasma, and it was significantly higher at f3 compared to the other developmental stages. Both the plasma and mucosal ALP and peroxidase activities showed a significant increase by female reproductive development with the highest amounts at f4. Contrary to the female, no significant changes were observed in plasma and mucosal immune agents and biochemistry of the male. The f5-staged goldfish showed the highest antimicrobial activities against Gram-positive bacteria, i.e., Streptococcus faecium, Staphylococcus aureus and Micrococcus luteus (P < 0.05). Our results also represented the up-regulation of lysozyme (c-lys) gene expression by effects of female maturational development in ovary, liver and skin, while male goldfish showed no significant changes in c-lys expression. Moreover, there were positive correlations between c-lys expression, mucosal lysozyme activity and calcium levels in females (P < 0.01). Overall, our findings revealed that vtg process improves mucosal innate immunity that leads to activate antimicrobial components at spawning season.

Instrumental and bioanalytical assessment of pharmaceuticals and hormone-like compounds in a major drinking water source-wastewater receiving Zayandeh Rood river, Iran.

Zayandeh Rood river is the most important river in central Iran supplying water for a variety of uses including drinking water for approximately three million inhabitants. The study aimed to investigate the quality of water concerning the presence of pharmaceutical active compounds (PhACs) and hormonelike compounds, which have been only poorly studied in this region. Sampling was performed at seven sites along the river (from headwater sites to downstream drinking water source, corresponding drinking water, and treated wastewater) affected by wastewater effluents, specific drought conditions, and high river-water demand. The targeted and nontargeted chemical analyses and in vitro bioassays were used to evaluate the presence of PhACs and hormonelike compounds in river water. In the samples, 57 PhACs and estrogens were detected with LC-MS/MS with the most common and abundant compounds valsartan, carbamazepine, and caffeine present in the highest concentrations in the treated wastewater in the concentrations of 8.4, 19, and 140 µg/L, respectively. A battery of in vitro bioassays detected high estrogenicity, androgenicity, and AhR-mediated activity (viz., in treated wastewater) in the concentrations 24.2 ng/L, 62.2 ng/L, and 0.98 ng/L of 17ß-estradiol, dihydrotestosterone and 2,3,7,8-TCDD equivalents, respectively. In surface water samples, estrogenicity was detected in the range of <0.42 (LOD) to 1.92 ng/L of 17ß-estradiol equivalents, and the drinking water source contained 0.74 ng/L of 17ß-estradiol equivalents. About 19% of the estrogenicity could be explained by target chemical analyses, and the remaining estrogenicity can be at least partially attributed to the potentiation effect of detected surfactant residues. Drinking water contained several PhACs and estrogens, but the overall assessment suggested minor human health risk according to the relevant effect-based trigger values. To our knowledge, this study provides some of the first comprehensive information on the levels of PhACs and hormones in Iranian waters.

Comparative study on accuracy of mucosal estradiol-17ß, testosterone and 11-ketotestosterone, for maturity, and cutaneous vitellogenin gene expression in goldfish (Carassius auratus).

Providing a non-invasive procedure to track fish maturity remains a priority in broodstocks' management. In the present study, the main goal was to assess reproduction status by measuring sex steroids and vitellogenin (VTG) in the skin mucosa, as a non-invasive method. For this purpose, the present study compared the levels of estradiol-17ß (E2 ), testosterone (T), 11-ketotestosterone (11-KT), VTG and calcium (Ca) in skin mucosa and blood plasma of goldfish (Carassius auratus). Skin mucosal and blood samples were collected, as well as gonad tissues, from goldfish, as a seasonal spawner. Histological analysis confirmed the gender and maturity status from females' ovaries (as primary-growth, cortical-alveoli, initial and late-vitellogenesis) and males' testes (as spermatogenesis and spermiation). Furthermore, vitellogenin (vtg) expression was observed in skin, liver and gonads. The results indicate that mucosal E2 concentrations were significantly higher during initial and late vitellogenesis than the other stages. Mucosal 11-KT concentrations significantly increased at spermiation (P < 0.05). E2 /T and 11-KT/E2 ratios significantly increased at early vitellogenesis and spermatogenesis, respectively (P < 0.05). Females' mucosal VTG levels were significantly fluctuated according to the maturity stage. Ca showed a similar trend, but Ca was more accurate for sex identification than the VTG. Although mucus showed high levels of VTG, ovarian vtg expression was strongest while liver and skin had the similar results. These results show that measuring the mucosal androgens could be considered as an accurate, non-invasive method to monitor fish maturity.

In vitro Selection of Synbiotics and in vivo Investigation of Growth Indices, Reproduction Performance, Survival, and Ovarian Cyp19α Gene Expression in Zebrafish Danio rerio.

In this study, we tested the compatibility of two extracts from the plant Jerusalem artichokes and button mushrooms with two different Lactobacillus probiotics (Lactobacillus acidophilus; La and Lactobacillus delbrueckii subsp. Bulgaricus; Lb) to develop a synbiotic formulation to improve the growth, survival, and reproductive performances of farmed fishes. Initially, we employed in vitro approach to monitor the growth of the probiotic lactobacilli in the presence of the different doses of the plant-based prebiotics, with the aim of selecting interesting combination(s) for further verification under in vivo conditions using zebrafish as a model. Results from the in vitro screening assay in the broth showed that both the probiotic species showed a preference for 50% mushroom extract as a source of prebiotic. A synbiotic formulation, developed with the selected combination of L. acidophilus, L. bulgaricus, and 50% mushroom extract, showed a positive influence on the growth and reproductive performances of the zebrafish. Our findings also imply that the improvement in the reproductive indices was associated with the upregulation of a cyp19a gene. Overall results suggest that a combination of L. acidophilus, L. bulgaricus, and mushroom extract can be considered as a potential synbiotic for the successful production of aquaculture species.

Stimulatory effect of dietary chasteberry (Vitex agnus-castus) extract on immunity, some immune-related gene expression, and resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus).

Chasteberry is highly recommended as an herbal medicine across the globe for treating of many gynaecological disorders. In this study, chasteberry extract (CBE) was supplemented in goldfish diet to evaluate the immunity responses at the cellular and molecular levels. Moreover, after the feeding trial, the fish were challenged with Aeromonas hydrophila. The fish (300 individuals, 2.4 ± 0.12 g initial weight) were randomly distributed in 12 tanks and were fed with 0 (control), 5, 10, and 15 g CBE per kg of feed for 8 weeks. Based on the results, lysozyme activity, alkaline phosphatase, and total immunoglobulin (Ig) in the skin mucus samples were significantly enhanced in the fish fed with 15 g/kg CBE (P < 0.05). Moreover, dietary CBE positively affected lysozyme activity, complement components, and IgM content of the serum samples compared to the control group. Also, the number of monocytes and lymphocytes were increased significantly with increasing CBE in the diet (P < 0.05). The highest mRNA levels of tumor necrosis factors (TNF-α, TNF-2α) and Lysozyme were observed in 15 g/kg CBE treatment. After the challenge test, the highest relative percentage survival value (60%) was observed in the fish fed with 15 g/kg CBE. We concluded that dietary CBE especially at 15 g/kg has an immunomodulatory effect in goldfish by stimulating the innate immunity and some inflammatory cytokines as well as disease resistance against A. hydrophila.

Immunity and growth improvement of rainbow trout (Oncorhynchus mykiss) fed dietary nettle (Urtica dioica) against experimental challenge with Saprolegnia parasitica.

In this study, effects of nettle (Urtica dioica) on growth, immunity, and gene expressions were examined in rainbow trout after an 8-week feeding period. A total of 264 juvenile rainbow trout (10.72 ± 0.55 g) were selected and stocked randomly in 12 aquaria. Nettle powder was added to the fish feed at three doses, 0.5,1 and 1.5% served as treatments. At the end of 8-week feeding period, the fish were exposed to Saprolegnia parasitica for 3 weeks. Results showed that all treatments fed with nettle diets exhibited significant increases in weight gain and SGR, and decreased FCR compared to the control. Feeding the fish with dietary nettle resulted in significant rises in blood indices and non-specific immunity in comparison with the control. Furthermore, fish fed 0.5% of dietary nettle showed significantly increased expressions of TNF-α, IL-1b, IL-6 and IL-8 genes following 8 weeks of feeding. A significant reduction in mortality rate was observed in the fish treated with 0.5% of nettle compared to the control following challenging with S. parasitica. Our observations indicate that the use of 0.5% nettle powder in rainbow trout diet can improve growth and immunity parameters as well as fish resistance against S. parasitica contamination.

The potential benefits of orange peels derived pectin on serum and skin mucus immune parameters, antioxidant defence and growth performance in common carp (Cyprinus carpio).

This study was performed to determine the effects of pectin derived from orange peel (PDOP) on growth performance, antioxidant enzyme activity and serum and skin mucus immune response of common carp (Cyprinus carpio). Common Carp (16.94 ± 0.03 g) were distributed into 12 tanks representing four treatments repeated in triplicates. Four diets were prepared to contain four levels of PDOP as follows: 0 (control), 0.5, 1, and 2% PDOP. Growth and immunological parameters as skin mucus lysozyme activity (SMLA) and total immunoglobulin (SMTIg), serum total immunoglobulin (STIg), serum peroxidase activities (SPA), Catalyse activity (CAT), DPPH radical scavenging activity, specific growth rate (SGR), weight gain (WG), final weight (FW), and feed conversion ratio (FCR) were assessed. Fish fed diets supplemented with PDOP showed an improvement of SGR, WG, FW, and FCR (P < 0.05). In terms of skin mucus immunological parameters, dietary inclusion of pectin significantly (P < 0.05) increased SMTIg. Likewise, carps fed either 1 or 2% PDOP showed notable enhancement of SMLA. In the case of serum immune parameters and antioxidant defence, carps in 1% PDOP treatment showed significantly (P < 0.05) higher SPA and CAT compared to fish fed either control diet or 0.5% OPDP. Additionally, no significant change (P > 0.05) was found in SPA and CAT of fish fed either 1% PDOP or 2% PDOP. Also, no significant (P > 0.05) difference was noticed between treated groups and control in the case of STIg. Furthermore, no significant differences were observed in DPPH radical activity among treatments (P > 0.05). Overall, these results suggested that inclusion of PDOP in common carp diet can beneficially affect growth and immune response.

Dietary nanoencapsulated quercetin homeostated transcription of redox-status orchestrating genes in zebrafish (Danio rerio) exposed to silver nanoparticles.

The present study assessed the protective effect of chitosan-nanoencapsulated quercetin (Qu-ChiNPs) against oxidative stress caused by silver nanoparticles (AgNPs). To this end, the transcription of prime genes regulating hepatic Keap1-Nrf2 pathway as well as downstream antioxidant enzymes were monitored prior to and after oxidative stress by AgNPs. Zebrafish (Danio rerio; n = 225) was assigned into five experimental groups based on feeding with diets supplemented with different additives as follows: negative and positive control groups, without additive; ChiNPs, 400 mg nanochitosan per kg diet; Quercetin, 400 mg free quercetin per kg diet; and Qu-ChiNPs, 400 mg Qu-ChiNPs per kg diet. At the end of the feeding trial (40 days), the experimental groups, except the negative control, were exposed to sublethal concentration of AgNPs (0.15 mg L-1) for 96h. Before exposure to AgNPs, free quercetin-treated diet significantly upregulated Keap1, Nrf2, Cat, SOD, GPx, and GST genes in the liver tissue when compared with the control diet, whereas Qu-Chi.NPs downregulated their transcription to the lowest levels. After exposure to AgNPs, all genes exhibited different responses in the AgNPs-exposed groups. The highest transcription of Nrf2, Cat, SOD, GPx, and GST was observed in the positive group, with being upregulated about 8, 10, 8, 8, and 7 times, respectively, when compared to the respective ones in the negative control. However, Keap1 showed a reverse response with being transcripted 12 times lower. The quercetin treatments, especially Qu-Chi.NPs, significantly reduced the transcription of Nrf2, Cat, SOD, GPx, and GST genes, yet enhanced Keap1 expression. Qu-Chi.NPs reduced the expression of Nrf2, SOD, Cat, GPx, and GST about 11, 10, 15, 10, and 10 times, respectively, yet increased that of Keap1 about 12 times. Taken together, nanoencapsulation can improve the antioxidant efficacy of quercetin against AgNPs toxicity and might reduce involvement of the cellular antioxidant system through tuning redox status. More broadly, it would be interesting to assess the effects of Qu-Chi.NPs against other metallic and organic oxidative stressors or pollutants.

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Biochemical biomarkers of skin mucus in Neogobius melanostomus for assessing lead pollution in the Gulf of Gorgan (Iran).

The present study was conducted to provide basic information on the physiological damages (mucosal indicators) of Round Goby under laboratory and environmental conditions of lead contamination. The other goal was to find mucosal biomarkers of lead contamination in the Gulf of Gorgan. In the laboratory study, 400 pieces of fish with an average the weight of 35 ± 7.16 g were caught alive and were exposed to lead concentrations of 0, 3.75, 7.5 and 15 mg / l for 14 days. Once the experiment was completed, the samples of mucus and liver were collected from the fish samples with the samples being surveyed by analyses of SDS-PAGE, mucus safety and toxicology. In the field test, the samples of fish, water, and sediment were prepared from four stations. In addition to measuring the lead in water, sediment and liver tissue, the samples of mucus from the caught fish were similarly analyzed under the laboratory conditions mentioned. According to the results under laboratory conditions, obvious differences were observed between the protein pattern of samples of treatment with the control group. Further, among innate immune parameters of the mucus, only lysozyme showed a significant correlation with the concentrations of lead accumulated in the liver of Round Goby (P < 0.05). Under environmental conditions, obvious differences were also observed between the protein pattern of samples of contaminated stations with the control station. Further, among innate immune parameters of the mucus, lysozyme indicated a positive correlation while alkaline phosphatase showed a negative correlation with lead contamination in the liver of Round Goby (P < 0.05). In total, in the pattern of mucus protein, the intensity of the band within the range of 25 kDa was enhanced in both conditions, suggesting the presence of proteases in this region. Also, among the immune parameters, lysozyme enzyme showed a significant and positive correlation with liver lead contamination in both conditions (P < 0.05). These can be considered as the biomarkers of protein pattern and safety in Round Goby mucus for assessing lead pollution in Gulf of Gorgan.

Can dietary jujube (Ziziphus jujuba Mill.) fruit extract alter cutaneous mucosal immunity, immune related genes expression in skin and growth performance of common carp (Cyprinus carpio)?

The present study investigates the effect of dietary Ziziphus jujube fruit extract [ZJFE] on skin mucus non-specific immune parameters as well as mRNA levels of immune related gene in the skin of common carp fingerlings. An eight-week feeding trial was performed using different levels of dietary ZJFE (0, 0.25, 0.5 and 1%). At the end of the trial skin mucus immune parameters (total Ig, lysozyme and protease activity), cytokines genes (il1b, il8, il10 and tnf-alpha) expression in skin and growth performance were studied. The result showed highest and lowest skin mucus total Ig were observed in fish fed diet containing 0.5% ZJFE and control group, respectively (P < 0.05). There were no significant difference among treatments regarding skin mucus lysozyme activity (P >0.05). It should be noted that, feeding on 0.5 and 1% ZJFE significantly increased skin mucus protease activity (P <0.05). Likewise, gene expression studies in skin showed significant increase of il1b expression in fish fed 0.5% ZJFE compared other treatments (P <0.05). Also, il8 gene was noticeably up-regulated in 0.5 and 1% treatments compared to the control group (P <0.05). While there were no significant difference between 0.25% JFE treatment and control in case of relative il10 gene expression (P >0.05), feeding on diets containing 0.5% or 1% ZJFE significantly down-regulated il10 gene (P <0.05). Our study indicated that relative expression of tnf-alpha gene significantly increased in treated groups (P <0.05). Also, feeding on ZJFE supplemented diet improved growth performance parameters. Overall, this experiment demonstrated the potentially useful effects of ZJFE on skin mucosal immunity and performance of common carp fingerlings.

Dietary sodium butyrate (Butirex® C4) supplementation modulates intestinal transcriptomic responses and augments disease resistance of rainbow trout (Oncorhynchus mykiss).

Intestine in fish is a complex multifunctional organ, not only plays roles in digestion and absorption of nutrient, but also has critical role in immunity. The present study evaluated the effects of different levels of dietary sodium butyrate [Butirex® C4 (Butirex)] on intestinal immune-,antioxidant-and tight junction-related gene expression injuvenile rainbow trout(Oncorhynchusmykiss). 240 healthy rainbow trout were dispensed in 12 fiberglass tanks appointed to four treatments [0 (control), 1.5 (B1.5), 2.5 (B2.5) and 5 (B5)g Butirex per kg diet]. After a 45-day feeding trial, the fish fed with the Butirex-supplemented diets showed higher intestinal lysozyme (LYZ), complement(ACH50) and bactericidal activities; the elevations in ACH50 and bactericidal activities depended on Butirex levels (P < 0.05). The Butirex-supplemented groups, particularly the B2.5 group, had significantly higher LYZ gene expression compared to the control group (P < 0.05). Butirex at 2.5 and 5 g/kg levels led to significantly higher IL-1ß gene expression. B2.5 and B5 had significantly lower and higher TNF-α gene expression compared to the control group (P < 0.05). The B2.5 group had significantly higher TGF-B, and significantly lower IL-8 compared to the control group (P < 0.05). The B1.5 and B2.5 group had significantly higher IL-10 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher SOD gene expression compared to the other groups; the highest expression was related to the B2.5 group (P < 0.05). Dietary Butirex supplementation significantly up-regulated CAT and GPx genes expression compared to the control group; the highest expression as related to the B2.5 and B5 groups (P < 0.05). The B2.5 group had significantly lower CLD12 gene expression compared to the control group (P < 0.05). The B2.5 and B5 groups had significantly higher CLD3, OCLD and ZO-1 gene expression compared to the control. The highest CLD3, ZO-1 gene expressions was related to the B2.5, and B5 groups respectively (P < 0.05). After challenge with Streptococcus iniae, B2.5 and B5 had significantly higher survival compared to the control group (55.6 ±â€¯7.70 and 68.9 ±â€¯10.2 vs. 33.3 ±â€¯6.67). In conclusion, Butirex is efficient immune stimulant and health booster in rainbow trout, which augments the fish resistance to disease. Modulation of immune components, cytokines, antioxidant system and intestinal integrity might involve in improving disease resistance in Butirex-treated fish. Although most of the examined genes were modulated by 2.5 g/kg Butirex under normal conditions, 5 g/kg level is recommended under pathogenic state to mitigate mortality.

Immunomodulatory effects of orally administrated florfenicol in rainbow trout (Oncorhynchus mykiss) following experimental challenge with streptococcosis/lactococcosis.

The purpose of the present work was to determine whether florfenicol (FFC) as the prominent broad-spectrum antibiotic could affect serum biochemical and immunological parameters, as well as immune-related genes expression in rainbow trout (55 ±â€¯7.6 g) challenged with the Lactococcus garvieae and Streptococcus iniae. In the first trial, the doses of the pathogens for challenge test were determined based on LD50. The therapeutic dosage of the drug (15 mg.kg-1 BW for 10 consecutive days) was administrated as medicated feed. After anesthesia, blood and kidney samples were collected from individual fish and were kept in deep freezing mode until the beginning of the measurements. Serum biochemical and enzymatic indices were measured using commercial kits. Immune parameters including total immunoglobulin level, lysozyme, ACH50, respiratory burst (RB), and phagocytic activities (PA) and the expression of immune genes namely TNF-α, IL-1ß, IL-8, and IgM was evaluated. The levels of lysozyme and RB activities, as well as the expression of TNF-α and IL-1ß genes, showed a significant increase in the FFC treated/infected fish compared to untreated diseased fish (P < 0.05). In contrast, serum total immunoglobulin and IgM-related genes expression were suppressed following drug administration represented by a significant reduction in untreated streptococcal infected fish compared to other treatments (P < 0.05). However, no significant effect of FFC was observed on serum ACH50 activity, PA values and IL-8-related gene expression (P > 0.05). These results demonstrated that FFC treatment could improve some physiological status including stress resistance and some liver function parameters, and much innate immunity was invigorated, but at the same time, the suppressive effects of FFC on acquainted immunity cannot be ignored.

Effects of dietary b-glucan, mannan oligosaccharide, Lactobacillus plantarum and their combinations on growth performance, immunity and immune related gene expression of Caspian trout, Salmo trutta caspius (Kessler, 1877).

This study investigated the effect of individual and combination of dietary pre- and probiotics (ß-glucan, 3 mg/g; mannan oligosaccharide (MOS), 4 mg/g; and Lactobacillus plantarum; 108 CFU/mg diet) on growth performance, blood immune parameters, expression of immune related genes, and intestinal microbial of Caspian trout (Salmo trutta caspius). On the basis of feeding with immunostimulant diets, the fish were assigned into eight groups denoted as: control (basal diet), bß (basal diet + ß-glucan), bM (basal diet + MOS), bLp (basal diet + L. plantarum), bßLp (basal diet + ß-glucan + L. plantarum), bMLp (basal diet + MOS + L. plantarum), bMß (basal diet + MOS + ß-glucan), and bMßLp (basal diet + MOS + ß-glucan + L. plantarum). All of the immunostimulant diets, in general, reduced feed intake (FI) and food conversion ratio (FCR) and increased WG, PER, and final weight. Condition factor (CF) demonstrated the lowest level in the experimental group received bMßLp. Total lipid increased in the fish received the additives, especially bM and bMß. Ash content demonstrated significant increase in the fish fed on bß and bMßLp, whereas moisture content was reduced in the group fed with L. plantarum-supplemented diet. All immunostimulant diets enhanced the activity and levels of lysozyme, Immunoglobulin M (IgM), and serum alternative complement activity (ACH50); the highest value for these indices was observed in the groups fed with bMß, bMßLp, and bßLp. bMß-treated fish group displayed the highest cortisol and glucose levels. bM diet induced the highest mRNA transcription of TNF-α1 in head kidney, whereas bLp, bMß, and bMßLp showed no effect. IL1ß exhibited the greatest up-regulation, about 8.75 fold change, in response to the diet supplemented only with ß-glucan. bßLp and bß significantly enhanced the relative IL-8 mRNA expression in the head kidney (about 2.75 and 1.9 folds, respectively), yet in response to bMßLp treatment it showed a decrease of about 5.7 times lower than the control group. In addition, intestinal population of L. plantarum showed the highest loads in the groups fed on the diets which were treated with the probiotic. Taken together, combinational use of these immunostimulants enhanced humoral innate immune system, whereas their individual and combinational application could increase and decrease the transcription of inflammation-related genes, respectively.

Cloning, characterization and tissue-specific expression of the antimicrobial peptide hepcidin from caspian trout (Salmo caspius) and the antibacterial activity of the synthetic peptide.

Antimicrobial peptides have a wide range of antimicrobial activity and widely occur in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine-rich antimicrobial peptides that are active against a variety of pathogens including gram-positive and gram-negative bacteria, as well as viruses. In this study, the hepcidin gene of Caspian trout (CtHep) was identified and characterized. Our results showed that CtHep cDNA has a 267-bp Open Reading Frame (ORF), which is translated to 88 amino acids. The CtHep was classified in the HAMP1 class of hepcidins. Comparison of DNA and cDNA sequences showed that CtHep has 3 exons and 2 introns. The signal, prodomain and mature part of CtHep have 24, 39 and 25 amino acids, respectively. The mature peptide has a molecular weight of 2881.43 Da and a theoretical isoelectric point of 8.53. The expression of CtHep mRNA was detected in different tissues of healthy and infected fish. CtHep expression in the liver, head kidney, spleen and skin was significantly enhanced after bacterial challenge. Expression of CtHep in different embryonic development stages was also substantial. Antibacterial activity of synthetic CtHep peptides was investigated against a number of Gram-positive and Gram-negative bacteria. CtHep inhibited some pathogenic bacteria such as Streptococcus iniae and Aeromonas hydrophila. In the in vivo experiment, CtHep upregulated the cytokines IL-6 and TNF-α in both kidney and spleen tissues after 24 h of the peptide injection. In conclusion, our study showed that CtHep plays an important role in the immune system of Caspian trout and also in the embryonic stages. Moreover, CtHep peptide has a potential to be used as an antimicrobial therapeutic agent as well as an immunostimulant in aquaculture.

Enhanced mucosal immune responses, immune related genes and growth performance in common carp (Cyprinus carpio) juveniles fed dietary Pediococcus acidilactici MA18/5M and raffinose.

The present study was conducted to evaluate the effects of dietary Pediococcus acidilactici (PA) and raffinose (RF) alone or in combination on growth performance, mucosal immune factors and immune related genes expression in common carp (Cyprinus carpio) juveniles. Fish with initial weight of 10.0 ±â€¯2.5 g were fed the following experimental diets for 60 days: control (without supplementation), prebiotic (2 g RF kg-1 diet), probiotic (6 × 108 CFU g-1PA) and synbiotic (2 g RF kg-1+ 6 × 108 CFU g-1PA). Carp fed synbiotic and probiotic diets had the highest (19.53 ±â€¯0.16) and the lowest (18.05 ±â€¯0.65) final body weight, respectively and the other experimental groups showed intermediate values. Singular administration of PA or in combination with 2 g RF kg-1 significantly increased skin mucus total immunoglobulin (Ig) and protein compared other groups, meanwhile, values of skin mucus protease activity enhanced by dietary immunostimulants administration in comparison with the control (P < 0.05). The expression of gene encoding lysozyme in skin pronouncedly increased by supplementing diets with singular or mixture of PA and RF; however, the expression of intestinal lysozyme gene as well as tumor necrosis factor-α genes expression in skin and intestine were not affected by administrating different immunostimulants (P > 0.05). The highest growth performance was noticed in fish fed synbiotic (P < 0.05). Overall, the combination of 2 g RF kg-1 with 6 × 108 CFU g-1PA is recommended for improving immunological responses of C. carpio juveniles.

Enrichment of common carp (Cyprinus carpio) fingerlings diet with Psidium guajava: The effects on cutaneous mucosal and serum immune parameters and immune related genes expression.

The present study aimed at evaluation of possible effects of Psidium guajava as immunostimulant on mucosal and serum immunity, as well as related genes expression in common carp (Cyprinus carpio) fingerlings. In an eight weeks feeding trial, fish were fed with experimental diets containing guava leaf powder [GLP] (0, 0.25, 0.5 and 1%). Then, skin mucus and serum immune parameters (total Ig, alkaline phosphatase activity, lysozyme activity), as well as immune related genes expression (TNF-alpha, IL1b, IL8) were measured. All levels of GLP inclusion in the diet brought about the increase in total Ig in the serum, while in the mucus only the highest level of GLP was effective. No significant differences were observed in mucus alkanine phosphatase activity, while fish fed 0.25% of GLP had noticeably higher serum lysozyme activity. The mucus total protein electrophoretic pattern revealed some differences between the control and the treated fish. Two high MW bands were present in the control group but not in the treated groups. Three bands with a low MW comprised between 35 and 17 mw were noticed in GLP fed carps and not in the control. Gene expression studies revealed no noticeable alterations in TNF-alpha gene between treated fish and control. However, the level of expression of IL-8 was strongly upregulated in fish fed 0.5 and 1% of GLP. Also, feeding on 0.5% of GLP significantly upregulated IL1b gene expression. Evaluation of growth performance revealed improved performance parameters in GLP treated groups. These results confirmed possible immunomodulatory and beneficial effects of GLP on common carp fingerlings.

Enrichment of rainbow trout (Oncorhynchus mykiss) fingerlings diet with microbial lysozyme: Effects on growth performance, serum and skin mucus immune parameters.

A two-month study was conducted to determine the influence of different levels of microbial lysozyme (LZ) contents (0, 0.5, 1.0, and 1.5 g kg-1 of diet) on growth performance, serum and skin mucus immune parameters as well as intestinal immune-related genes expression in rainbow trout Oncorhynchus mykiss fingerlings (5.5 ±â€¯0.1 g). Growth performance and feed utilization were not affected significantly by dietary LZ. Fish fed LZ-supplemented diets had higher serum total immunoglobulins concentration than the control group. In addition, fish fed 1.5 g LZ kg-1 diet had the highest skin mucosal total protein and immunoglobulin contents compared to other experimental groups. Furthermore, skin mucosal lysozyme and alkaline phosphatase activities as well as intestinal tumor necrosis factor-α and interlukine-1ß relative genes expression were higher in fish fed 1.0 and 1.5 g LZ kg-1 diets than the other groups. Overall, the present results clearly showed that LZ powder can be considered as a potential immunostimulant in O. mykiss fingerlings, but in the long term period it may result in negative effects on intestinal health as a consequence of inducing pro-inflammatory cytokines gene expression in the intestine.