Synbiotic feed supplementation significantly improves lipid utilization and shows discrete effects on disease resistance in rainbow trout (Oncorhynchus mykiss).

Enteric redmouth disease caused by the bacterial pathogen Yersinia ruckeri is the main reason for antimicrobial prescription, and a cause of substantial economic losses and decreased animal welfare in aquaculture. Given the importance of the intestinal microbiota in digestion and disease, our aim was to investigate whether synbiotic feed supplementation strategies could improve feed performance and disease resistance. Four experimental synbiotic feeds formulated with pre- and probiotics were tested against a commercially available probiotic control feed. Each experimental feed was evaluated for feed performance, effects on gross as well as intestinal morphometrics, and finally their effect on resistance against a waterborne experimental infection with Yersinia ruckeri serotype O1, biotype 2. While co-supplementing Pediococcus acidilactici with citrus flavonoids or bacterial paraprobiotics significantly improved utilization of feed lipid content relative to the control group, a decrease in lipid utilization was observed for feeds that combined P. acidilactici with yeast paraprobiotics. No significant improvements on disease resistance were observed. Still, synbiotic formulations including P. acidilactici led to reduced risks relative to that of the control group, while an increased relative risk was observed for a Bacillus-based formulation. In conclusion, two of the synbiotic supplements significantly improved lipid utilization and contributed to minor increases in disease resistance.

Single or Combined Dietary Supply of Psidium guajava and Phyllanthus amarus Extracts Differentially Modulate Immune Responses and Liver Proteome in Striped Catfish (Pangasianodon hyphophthalmus).

Guava Psidium guajava L (Pg) and bhumi amla Phyllanthus amarus Schum. et Thonn (Pa) are well-known plants in traditional medicine. However, the capacity of these plants for improving the immune system of aquatic species has received less attention so far. This study aimed to investigate the effects of single supply or mixture of Pg and Pa extracts on immune responses, disease resistance and liver proteome profiles in striped catfish Pangasianodon hypophthalmus. Fish were fed diets including basal diet 0% or one of three doses of each plant extract, either alone or in mixture, 0.08, 0.2, or 0.5% Pg, Pa or mixture (Pg:Pa, v/v) for 6 weeks. The immune parameters (respiratory burst activity (RBA); nitric oxide synthase (NOS), total immunoglobulin, lysozyme, and complement activities) were examined at W3, W6 post-feeding, and after challenge test. The growth parameters and the challenge test with Edwardsiella ictaluri were done at W6. The liver proteome profiles were analyzed in W6 at 0.08 and 0.5% of each extract. The results showed that extract-based diets significantly improved growth parameters in the Pg0.2 group compared to control. The cellular immune responses in spleen and the humoral immune responses in plasma were significantly improved in a dose and time-dependent manner. Diets supplemented with single Pg and Pa extracts, and to lesser extent to combined extracts, could significantly decrease the mortality of striped catfish following bacterial infection compared to control. The proteomic results indicated that some pathways related to immune responses, antioxidant and lipid metabolism were enriched in liver at W6. Several proteins (i.e., CD8B, HSP90AA1, HSP90AB1, PDIA3, CASP8, TUBA1C, CCKAR, GNAS, GRIN2D, PLCG1, PRKCA, SLC25A5, VDAC2, ACTN4, GNAI2, LCK, CARD9, NLRP12, and NLRP3) were synergistically upregulated in mixture of Pg and Pa-based diets compared to control and single dietary treatments. Taken together, the results revealed that single Pg and Pa extracts at 0.2 and 0.5% and their mixture at 0.08 and 0.5% have the potential to modulate the immune mechanisms and disease resistance of striped catfish. Moreover, the combination of Pg and Pa in diets suggested positive synergistic effects liver proteome profile related to immune system processes.

Bactericidal effect on skin mucosa of dietary guava (Psidium guajava L.) leaves in hybrid tilapia (Oreochromis niloticus × O. mossambicus).

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the intensification practices in global aquaculture, fish are often confined in small volumes, which can results in outbreak diseases. In this context, the use of antibiotics is very usual. Thus, looking for natural substance able to reduce the use of the antibiotics is imperative. Among them, there is a great interest at present in the study of medicinal plants such as guava (Psidium guajava L.). These plants could help to develop a more sustainable aquaculture all over the world. The application of guava in traditional medicine dates for centuries and it is widely used in tropical countries for the treatment of diseases in human and animals. AIM OF THE STUDY: The purpose of this work was to study the effects of the dietary administration of dried leaves of Psidium guajava on the skin mucosal immunity of hybrid tilapia (Oreochromis niloticus × O. mossambicus). Furthermore, the ability of this plant to inhibit the bacterial load in different tissues after an experimental infection with Vibrio harveyi was studied. MATERIALS AND METHODS: P. guajava leaves collection and the experimentation was carried out in Dominican Republic. Fish were fed with a commercial diet supplemented with guava leaf at different concentrations (0%, 1.5% and 3%) for 21 days before being intraperitoneally injected with V. harveyi (1 × 104 cells mL-1). Thereafter, several immune activities were measured in fish skin mucus and after 48 h of injection, the skin, spleen and liver were collected to analyse the bactericidal activity of guava leaf and the gene expression of some immune related genes. RESULTS: The administration of P. guajava leaves significantly modulated some immune-related enzymes (protease, antiprotease and peroxidase) in the skin mucus of hybrid tilapia. In addition, the bacterial load after V. harveyi infection in skin, spleen and liver significantly reduced in fish supplemented with guava leaves. Finally, the expression profile of hepcidin gene in skin and liver was modulated in fish feed with control diet after V. harveyi infection. CONCLUSION: These results demonstrate that the dietary intake of guava leaves increases the skin mucosal barrier defences of hybrid tilapia and confers protection against V. harveyi colonization.

Physiological activity of Aloe vera in pacu (Piaractus mesopotamicus) inoculated with Aeromonas hydrophila.

Aloe vera is a traditional medicinal plant; however, its use in fish is fairly recent. We evaluated the effects of dietary A. vera on stress, innate immunity, and energy metabolism in pacu inoculated with Aeromonas hydrophila. For 7 days, 192 fish were fed with diets supplemented with 0% (control), 0.5%, 1.0%, and 2.0% of the plant extract and then inoculated with bacteria and sampled 3, 6, and 24 h later. All concentrations of A. vera reduced basal levels of cortisol, and 1.0% reduced cortisol levels more intensely 3 h after inoculation. A. vera increased the basal respiratory activity of leukocytes/RAL (0.5 and 1.0%), increased the serum levels of lysozyme (1.0 and 2.0%) 6 h after inoculation, and increased the activity of the complement system after 3 h. Spleen somatic index/SSI increased with 1.0 and 2.0% A. vera. A. vera also promoted metabolic effects. It increased basal levels of lipids in the liver and muscle, as well as hepatosomatic index (1.0%) and, 3 h after inoculation, prevented the reduction of serum triglyceride (1.0%) and reduced the mesenteric fat (1.0%). Bacterial inoculation increased RAL from 3 to 24 h and lysozyme levels at 24 h, increased serum cholesterol at 24 h, and decreased serum triglyceride from 3 to 24 h, regardless of A. vera. We concluded that A. vera offered for only 7 days had stress-reducing effects, stimulated innate immunity, protected triglyceride levels in blood, lipid depots in the liver and muscle, and directed the energy mobilization to visceral depots.

Choline supplementation prevents diet induced gut mucosa lipid accumulation in post-smolt Atlantic salmon (Salmo salar L.).

BACKGROUND: Various intestinal morphological alterations have been reported in cultured fish fed diets with high contents of plant ingredients. Since 2000, salmon farmers have reported symptoms indicating an intestinal problem, which we suggest calling lipid malabsorption syndrome (LMS), characterized by pale and foamy appearance of the enterocytes of the pyloric caeca, the result of lipid accumulation. The objective of the present study was to investigate if insufficient dietary choline may be a key component in development of the LMS. RESULTS: The results showed that Atlantic salmon (Salmo salar), average weight 362 g, fed a plant based diet for 79 days developed signs of LMS. In fish fed a similar diet supplemented with 0.4% choline chloride no signs of LMS were seen. The relative weight of the pyloric caeca was 40% lower, reflecting 65% less triacylglycerol content and histologically normal gut mucosa. Choline supplementation further increased specific fish growth by 18%. The concomitant alterations in intestinal gene expression related to phosphatidylcholine synthesis (chk and pcyt1a), cholesterol transport (abcg5 and npc1l1), lipid metabolism and transport (mgat2a and fabp2) and lipoprotein formation (apoA1 and apoAIV) confirmed the importance of choline in lipid turnover in the intestine and its ability to prevent LMS. Another important observation was the apparent correlation between plin2 expression and degree of enterocyte hyper-vacuolation observed in the current study, which suggests that plin2 may serve as a marker for intestinal lipid accumulation and steatosis in fish. Future research should be conducted to strengthen the knowledge of choline's critical role in lipid transport, phospholipid synthesis and lipoprotein secretion to improve formulations of plant based diets for larger fish and to prevent LMS. CONCLUSIONS: Choline prevents excessive lipid accumulation in the proximal intestine and is essential for Atlantic salmon in seawater.

Protective effects of seaweed supplemented diet on antioxidant and immune responses in European seabass (Dicentrarchus labrax) subjected to bacterial infection.

European seabass (Dicentrarchus labrax) production is often hampered by bacterial infections such as photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp). Since diet can impact fish immunity, this work investigated the effect of dietary supplementation of 5% Gracilaria sp. aqueous extract (GRA) on seabass antioxidant capacity and resistance against Phdp. After infection, mortality was delayed in fish fed GRA, which also revealed increased lysozyme activity levels, as well as decreased lipid peroxidation, suggesting higher antioxidant capacity than in fish fed a control diet. Dietary GRA induced a down-regulation of hepatic stress-responsive heat shock proteins (grp-78, grp-170, grp-94, grp-75), while bacterial infection caused a down-regulation in antioxidant genes (prdx4 and mn-sod). Diet and infection interaction down-regulated the transcription levels of genes associated with oxidative stress response (prdx5 and gpx4) in liver. In head-kidney, GRA led to an up-regulation of genes associated with inflammation (il34, ccr9, cd33) and a down-regulation of genes related to cytokine signalling (mif, il1b, defb, a2m, myd88). Additionally, bacterial infection up-regulated immunoglobulins production (IgMs) and down-regulated the transcription of the antimicrobial peptide leap2 in head kidney. Overall, we found that GRA supplementation modulated seabass resistance to Phdp infection.

Dietary Supplementation With ω6 LC-PUFA-Rich Algae Modulates Zebrafish Immune Function and Improves Resistance to Streptococcal Infection.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Dietary supplementation of Spirulina ameliorates iron-induced oxidative stress in Indian knife fish Notopterus Notopterus.

Iron though an essential cofactor for many proteins including haemoglobin and cytochromes, when in excess (>1 ppm in water and 100 ppm in fish tissue) elicits toxicity via Fenton reaction inducing oxidative stress. The present study aimed to evaluate the efficacy of dietary Spirulina (Arthrospira platensis) supplementation on waterborne-iron induced oxidative stress in the tissues of Notopterus notopterus. Juvenile fishes were divided randomly into 4 groups, namely, Group-I: control fed with commercial diet only, Group-II, III and IV treated with 0.75 ppm FeCl3 where Group-II fed with commercial diet only, Group-III with 10% (w/w) Spirulina supplemented commercial diet and Group-IV with 100% (w/w) Spirulina diet only; for 7 and 28 days (n = 6 per group). Tissue oxidative stress biomarkers like lipid peroxidation (LPx), protein carbonylation (PC) and protein thionylation (protein and nonprotein-SH content); antioxidant defence (superoxide dismutase: SOD; catalase; CAT; glutathione peroxidase/reductase: GPx/GR; glutathione s-transferase: GST; metalothionine: MT and reduced glutathione: GSH) and iron accumulation in the gill, liver and muscles tissue were analysed. The augmented oxidative predominance in the tissues with respect to LPx and PC along with decline in antioxidant defence (SOD, CAT, GPx, GR, GST, MT, PSH, NPSH and GSH) by iron was neutralized by Spirulina supplementation in the diet in a dose and duration dependent manner where 100% Spirulina diet for 28 days completely ameliorated iron-induced oxidative stress in fish tissues. Thus, Spirulina can be used as a dietary supplement for fishes cultured in water bodies with iron overload.

Effects of silver nanoparticles on stress biomarkers of Channa striatus: immuno-protective or toxic?

Nanotechnology is a novel arena with promising applications in the field of medicine, industry, and agriculture including fisheries. Cross-disciplinary interactions and the application of this technology in biological systems have led to the innovation of novel nanoparticle antioxidants, which are the subject of our study. In context with above background, we designed an experiment on nano-silver to elucidate its role for mitigation of abiotic and biotic stress. Three diets were formulated viz. silver nanoparticles (Ag-NPs) incorporated at 0.5 and 1 mg/kg diet and control diet (Ag-NPs at 0 mg/kg). Fish were exposed to sublethal level of 1/25th of LC50 (4 ppm) of lead (Pb) and temperature at 34 °C. The effect of Ag-NPs on productive performance (weight gain %, feed conversion ratio, protein efficiency ratio, and specific growth rate), stress biomarkers (catalase, super oxide dismutase, glutathione-s-transferase, acetylcholine esterase, cortisol, heat shock protein), biochemical and immunological response (protein and carbohydrate metabolic enzymes, phagocytic activity, serum total protein and albumin: globulin ratio), histopathology alterations in the liver and gill as well as survival of Channa striatus, following challenge with pathogenic bacteria were evaluated. Dietary Ag-NPs at 0.5-mg supplementation improved growth performance, immunity, survival, and reduced stress biomarker such as HSP 70, cortisol, and blood glucose in various fish tissues. Exposure to Pb and high temperature and group fed with Ag-NPs (1 mg/kg diet) demonstrated remarkable changes in the histo-architect of liver such as pyknotic nuclei, pyknosis, leucocyte infiltration, hemorrhage and karyokinesis, blood vessels with nucleated, lipid vacuoles in the liver tissue. Histology of gill displayed hyperplasia, aneurism, blood congestion, severe telengiectiasis, epithelial lifting, curling of secondary lamella, hyperplasia of epithelial cell of secondary lamella in the group exposed to lead and high temperature and supplemented with Ag-NPs at 1 mg/kg diet. In addition to histopathology, feeding with Ag-NPs at 1 mg/kg diet deteriorated and altered all studied parameters including reduced growth performance. Results obtained in the present study suggest that supplementation of Ag-NPs at 0.5 mg/kg diet has a definitive role to play in the mitigation of abiotic and biotic stress in C. striatus.

Leaves from banana (Musa nana) and maize (Zea mays) have no phyto-prophylactic effects on the susceptibility of grass carp (Ctenopharyngodon idella) to Aeromonas hydrophila infection.

BACKGROUND: The ubiquitous and opportunistic bacterial pathogen Aeromonas hydrophila has been associated with ulcerative dermatitis in fish, especially under stressful conditions. It can cause severe losses in fresh water aquaculture and is particularly prevalent in tropical and subtropical regions. Fresh leaves from maize and bananas have been used as feed supplement by fish farmers in Vietnam and it has been reported that they may have phyto-prophylactic benefits. In the present study, a feeding trial was conducted to investigate the benefits of providing maize and banana leaves as feed supplement: to determine if they were taken up and digested by grass carp (Ctenopharyngodon idella), if this uptake resulted in improved growth performance, and if leaf supplementation protected fish when challenged with A. hydrophila by intramuscular injection. RESULTS: All fish were fed an identical ratio of commercial pelleted feed relative to biomass. However, in 12/18 tanks, this diet was supplemented with either fresh banana leaves or fresh maize leaves; offered ad libitum. Addition of leaves increased the overall feed conversion ratio (FCR) significantly. However, if only the pellet were taken into account, then no difference was found between treatments. Changes to the isotopic composition of the fish showed leaf nutrient uptake occurred. No prophylactic effects of feeding banana or maize leaves were detected against infection with A. hydrophila, and the diet did not induce changes in the fish haematocrit. However, addition of the maize leaves was associated with significantly reduced severity of the skin lesions, which could improve the market value of the fish. CONCLUSIONS: Addition of the leaf supplement did not result in significantly improved growth performance. Similarly, the effect of the supplement on the fish survival to infection was not significant.

Activity of Brazilian propolis against Aeromonas hydrophila and its effect on Nile tilapia growth, hematological and non-specific immune response under bacterial infection

ABSTRACT The effect of the ethanolic extract of propolis (EEP) on Aeromonas hydrophila was analyzed by determination of minimum inhibitory concentration (MIC). Then, the effects of crude propolis powder (CPP) on growth, hemato-immune parameters of the Nile tilapia, as well as its effects on resistance to A. hydrophila challenge were investigated. The CPP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) was added to the diet of 280 Nile tilapia (50.0 ± 5.7 g fish-1). Hemato-immune parameters were analyzed before and after the bacterial challenge. Red blood cell, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and hydrogen peroxide (H2O2) and nitric oxide (NO) were evaluated. The MIC of the EEP was 13% (v/v) with a bactericidal effect after 24 hours. Growth performance was significantly lower for those fish fed diets containing 2.5 and 3% of CPP compared to the control diet. Differences in CPP levels affected fish hemoglobin, neutrophils number and NO following the bacterial challenge. For others parameters no significant differences were observed. Our results show that although propolis has bactericidal properties in vitro, the addition of crude propolis powder to Nile tilapia extruded diets does not necessarily lead to an improvement of fish health.

Activity of Brazilian propolis against Aeromonas hydrophila and its effect on Nile tilapia growth, hematological and non-specific immune response under bacterial infection.

The effect of the ethanolic extract of propolis (EEP) on Aeromonas hydrophila was analyzed by determination of minimum inhibitory concentration (MIC). Then, the effects of crude propolis powder (CPP) on growth, hemato-immune parameters of the Nile tilapia, as well as its effects on resistance to A. hydrophila challenge were investigated. The CPP (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0%) was added to the diet of 280 Nile tilapia (50.0 ± 5.7 g fish-1). Hemato-immune parameters were analyzed before and after the bacterial challenge. Red blood cell, hematocrit, hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and hydrogen peroxide (H2O2) and nitric oxide (NO) were evaluated. The MIC of the EEP was 13% (v/v) with a bactericidal effect after 24 hours. Growth performance was significantly lower for those fish fed diets containing 2.5 and 3% of CPP compared to the control diet. Differences in CPP levels affected fish hemoglobin, neutrophils number and NO following the bacterial challenge. For others parameters no significant differences were observed. Our results show that although propolis has bactericidal properties in vitro, the addition of crude propolis powder to Nile tilapia extruded diets does not necessarily lead to an improvement of fish health.

Bile components and lecithin supplemented to plant based diets do not diminish diet related intestinal inflammation in Atlantic salmon.

BACKGROUND: The present study was undertaken to gain knowledge on the role of bile components and lecithin on development of aberrations in digestive functions which seemingly have increased in Atlantic salmon in parallel with the increased use of plant ingredients in fish feed. Post smolt Atlantic salmon were fed for 77 days one of three basal diets: a high fish meal diet (HFM), a low fishmeal diet (LFM), or a diet with high protein soybean meal (HPS). Five additional diets were made from the LFM diet by supplementing with: purified taurocholate (1.8 %), bovine bile salt (1.8 %), taurine (0.4 %), lecithin (1.5 %), or a mix of supplements (suppl mix) containing taurocholate (1.8 %), cholesterol (1.5 %) and lecithin (0.4 %). Two additional diets were made from the HPS diet by supplementing with: bovine bile salt (1.8 %) or the suppl mix. Body and intestinal weights were recorded, and blood, bile, intestinal tissues and digesta were sampled for evaluation of growth, nutrient metabolism and intestinal structure and function. RESULTS: In comparison with fish fed the HFM diet fish fed the LFM and HPS diets grew less and showed reduced plasma bile salt and cholesterol levels. Histological examination of the distal intestine showed signs of enteritis in both LFM and HPS diet groups, though more pronounced in the HPS diet group. The HPS diet reduced digesta dry matter and capacity of leucine amino peptidase in the distal intestine. None of the dietary supplements improved endpoints regarding fish performance, gut function or inflammation in the distal intestine. Some endpoints rather indicated negative effects. CONCLUSIONS: Dietary supplementation with bile components or lecithin in general did not improve endpoints regarding performance or gut health in Atlantic salmon, in clear contrast to what has been previously reported for rainbow trout. Follow-up studies are needed to clarify if lower levels of bile salts and cholesterol may give different and beneficial effects, or if other supplements, and other combinations of supplements might prevent or ameliorate inflammation in the distal intestine.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, ß-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) ß1, TGF-ß2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1ß, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Dietary camu camu, Myrciaria dubia, enhances immunological response in Nile tilapia.

Camu camu, Myrciaria dubia, is an Amazon plant that presents high levels of vitamin C in its composition. Several studies in animals and humans have demonstrated their efficiency in the prevention and treatment of various diseases. However, there are no reports of its properties in fish. The aim of this study was to evaluate the effect of the oral administration of the extract of this plant in the immune parameters in Nile tilapia, Oreochromis niloticus. 400 Nile tilapia (80 ± 5 g) were randomly distributed into 20 tanks with 1500 L capacity each (20 fish/tank). After a week of adaptation to environmental conditions, it was provided a diet for 5 weeks, using different levels of inclusion of camu camu extract: 0, 50, 100, 250, and 500 mg/kg of feed. Each treatment consisted of four replicates. It was obtained 40.5 mg of vitamin C/g of camu camu pulp powder by high-performance liquid chromatography. At the end of the trial period, fish were inoculated with Aeromonas hydrophila in the swim bladder. Samples were taken after 6; 24 and 48 h of the challenge. Results revealed that fish supplemented with this herb showed significant increase (P < 0.05) in white blood cells counts in blood and exudate, burst respiratory activity, lysozyme activity, serum bactericidal activity, direct agglutination, and melanomacrophage centers count. Red blood cells count, hemoglobin, hematocrit, and biochemical profile of fish supplemented with the herb presented no statistical differences compared to control group (P > 0.05). No histopathological lesions were observed in intestine, kidney, spleen, and gills. It can be concluded that the addition of Myrciaria dubia in tilapia feed improves the immune response and the growth after 5 weeks, especially, at a dose of 500 mg/kg.

Hemato - Immunological and biochemical parameters, skin antibacterial activity, and survival in rainbow trout (Oncorhynchus mykiss) following the diet supplemented with Mentha piperita against Yersinia ruckeri.

This study was aimed to assess the potential effects of Mentha piperita on the hemato - immunological and biochemical parameters, skin antibacterial activity and protection against Yersinia ruckeri infection in rainbow trout Oncorhynchus mykiss. Fish were divided into 4 groups before being fed diets supplemented with 0, 1, 2 and 3% of Mentha piperita (MP) plant extract for 8 weeks. Dose-dependent increases immune (both in skin mucus and blood serum) and hematological parameters (number of red and white cells, hematocrit and hemoglobin contents), as well as in respiratory burst activity, total protein, albumin, and neutrophil levels in fish fed supplemented diets compared to the control fish. Furthermore, dietary MP plant extract supplements have no significant effect on blood biochemical parameters and enzymatic activities of liver determined in serum of rainbow trout. After 8 weeks the cessation of feeding with MP plant extract, survival rates of 54.4%, 63.6% and 75.2% were recorded in groups which received 1, 2 and 3% of MP plant extract of feed, respectively, compared to 34.6% survivals in the control. This study underlying several positive effects of dietary administration of MP plant extract to farmed fish.

Chlorophytum borivilianum Polysaccharide Fraction Provokes the Immune Function and Disease Resistance of Labeo rohita against Aeromonas hydrophila.

The present study aimed to investigate the effects of Chlorophytum borivilianum polysaccharide (CBP), as a dietary supplement administered at varying concentrations with feed (basal diet), on various cytokine-related responses in Labeo rohita fingerlings. Immune parameters and immune-related gene expressions were measured at 3rd, 4th, and 5th week after feeding. The results revealed that dietary administration of CBP at 0.2% and 0.4% for 4 weeks significantly upregulated serum lysozyme and phagocytic activity. Complement C3 and respiratory burst activity (RBA) were significantly higher after 4 weeks of CBP feeding. The immune related genes IL-8, IL-1ß, TNF-α, and iNOS were downregulated (P < 0.05) in groups with 0.2% and 0.4% CBP supplemented diets at week 4. Expression of anti-inflammatory cytokines (IL-10 and TGF-ß) was also downregulated (P < 0.5) after 4 weeks of feeding with 0.2% to 0.8% CBP. However, five weeks of CBP administration had no significant effect on immune gene expression, except TNF-α and IL-8. Fish fed with 0.4% CBP for 4 weeks showed maximum resistance against Aeromonas hydrophila (73.3% survival) compared to control. From these results, we recommend that CBP administration at 0.4% for 4 weeks could effectively improve immune response and disease resistance in L. rohita.

Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus.

The effect of Avicennia marina aqueous leaf extract on innate immune mechanisms such as total white blood cell counts (WBC), serum lysozyme activity, respiratory burst assay, alternative complement (ACH50) assay, phagocytic activity assay, disease resistance, gut bacteria, and survival rate of clownfish (Amphiprion sebae) against Vibrio alginolyticus is reported. Healthy fish challenged with V. alginolyticus (1 × 10(7) cells ml(-1)) were fed with diets supplemented (0, 1, 2, and 4%) with A. marina extract. The survival rate was 85% and 80% in infected fish fed with 4% and 8% supplementation diet; with 1% diet it was 70% while in the infected untreated group it was only 10%. The total gut bacteria flora was high in 8% and 4% supplementation diet groups with 2.8 × 10(5) and 4.7 × 10(4) cfu/g while it was 8.9 × 10(3) cfu/g in 1% diet group. The immunological parameters significantly increased on weeks 6 and 8 when infected fish were fed with 1% or 4% supplementation diet. This study reports that in clownfish challenged with V. alginolyticus, dietary administration of the 1% or 4% of A. marina extract improved the immune status and survival rate.

Effect of dietary supplementation with Suaeda maritima on blood physiology, innate immune response, and disease resistance in olive flounder against Miamiensis avidus.

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.

Effects of dietary biotin and avidin on growth, survival, feed conversion, biotin status and gene expression of zebrafish Danio rerio.

A study was conducted to investigate the effects of dietary avidin on growth, survival, food conversion, biotin status and gene expression of zebrafish (Danio rerio Hamilton-Buchanan) juveniles (average wet mass 0.178 g) fed 7 purified diets for 12 weeks. Experimental diets were formulated to provide 0×, 1×, 15×, 30×, 60× and 120× excess avidin versus biotin kg(-1) diet, on a molar basis; a control diet contained neither supplemental biotin nor avidin. Fish fed the control diet had the lowest percentage weight gain and the highest mortality, while the highest percentage weight gain and the lowest mortality was observed with the 0× diet (P<0.05). A linear relationship was observed between feed conversion ratio (FCR) and dietary avidin (r=0.876; P<0.0001). Fish fed diets with 120× more avidin than biotin had the highest whole-body biotin content, while the lowest value was obtained with the control and avidin-free diets (P<0.05). Elevated levels of acetyl CoA carboxylase-A (acca), methylcrotonyl CoA carboxylase (mcc) and propionyl CoA carboxylase-A (pcca) transcripts were recorded in fish fed the control diet, in comparison to the other diets. A broken-line analysis indicated that feeding zebrafish a diet with 60 times more avidin than the dietary biotin requirement level will cause biotin deficiency signs.