Francisco A. Guardiola

Quality and antioxidant response of gilthead seabream (Sparus aurata L.) to dietary supplements of fenugreek (Trigonella foenum graecum) alone or combined with probiotic strains

Abstract The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream. HighlightsAntioxidant status of seabream after dietary fenugreek + probiotics was evaluated.Supplemented diets did not affect the fatty acid profiles of the muscle.Experimental diets showed antioxidant properties (decreased the TBARS in muscle).SOD and CAT were enhanced after fed experimental diets for 3 weeks.Fenugreek + probiotics up‐regulated liver antioxidant‐related gene expression.

Healing and mucosal immunity in the skin of experimentally wounded gilthead seabream (Sparus aurata L)

Abstract Skin lesions are very common in fisheries, increasing the risk of pathogens entering through the wounded skin of the fish. In the present assay, the progression of wound healing was studied over a 7 day period in gilthead seabream (Sparus aurata L.) after making experimental wounds in two different locations: above (group A) or below (group B) the lateral line. Macroscopic observation confirmed faster wound healing of the wounds of fish from group B. Furthermore, several immune‐related components were studied in the skin mucus of wounded fish to ascertain whether wounding altered the mucus composition compared with the values obtained from non‐wounded fish (group C, control). Significant variations were detected depending on both the site of the wound and the studied parameter. At the same time, the gene expression profile of several immune‐relevant genes, including pro‐inflammatory (il1b,il6, tnfa), anti‐inflamamtory (tgfb, il10), immunoglobulins (ighm, ight), involved in oxidative stress (sod, cat) and in skin regeneration (krt1 and grhl1) were studied in the three groups of fish (A, B and C). The results throw further light on the complex process of skin wound healing in fish, since substantial changes in the skin mucus and in the skin gene expression originated by the presence of wounds were observed. This work underline some important differences depending on the place of the fish body where the wound is located. Of particular note was the fact that such changes depended on the site of the wound. Graphical abstract Figure. No Caption available. HighlightsSkin healing was studied in gilthead seabream wounded above or below the lateral line.The healing process is faster for wounds below the lateral line.Fish skin cells are more sensitive to physical aggression in the area below the lateral line.The gene expression of some immune‐related genes increased to a greater extent in the skin located above the lateral line.

Sterigmatomyces halophilus β-glucan improves the immune response and bacterial resistance in Pacific red snapper (Lutjanus peru) peripheral blood leucocytes: In vitro study

ABSTRACT &bgr;‐Glucans are naturally occurring polysaccharides that are produced by bacteria, fungi and yeast. They are considered immunostimulants in fish acting on non‐specific defense mechanism. Yeast‐derived glucans from cell wall (Sterigmatomyces halophilus, &bgr;‐Gluc/Sh) have been used for this purpose in this study. Therefore, an in vitro assay using peripheral blood leucocytes (PBLs) from Pacific red snapper was performed to evaluate the stimulant effects of &bgr;‐Gluc/Sh and zymosan A (positive control) for 12 and 24h and after bacterial challenge with Aeromonas hydrophila at 24h. In addition, structural characterization of this marine yeast glucan was performed by proton nuclear magnetic resonance (NMR) revealing structures containing (1–6)‐branched (1–3)‐&bgr;‐D‐glucan. PBLs responded positively to &bgr;‐Gluc/Sh where cell viability was higher than 80%. After challenge, &bgr;‐Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with &bgr;‐Gluc/Sh significantly increased the non‐specific immune response, such as phagocytic activity, respiratory burst, nitric oxide and peroxidase activities followed by an increase in superoxide dismutase and catalase activities after 12 and 24h post‐stimulation and after challenge with the pathogen. Regarding induction of antioxidant gene expression, it was more pronounced in stimulated &bgr;‐Gluc/Sh leucocytes compared to other groups at all experimental times of the trial and after bacterial challenge. Indeed, our results clearly showed the ability of leucocytes to strongly react to &bgr;‐Gluc/Sh with an increase in cytokine gene expression, particularly the IL‐1&bgr;, IL‐10 and IL‐17 genes. These results confirm that S. halophilus yeast‐derived &bgr;‐glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro. HIGHLIGHTSSterigmatomyces halophilus &bgr;‐glucan enhances immune parameters in fish leukocytes.Structural characterization by (NMR) revealing (1–6)‐branched (1–3)‐&bgr;‐D‐glucan.&bgr;‐Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila in PBLs.Antioxidant SOD and CAT activity was increased in &bgr;‐Gluc/Sh‐stimulated PBLs.Cytokine gene expression confirms the immunoestimulatory effects of &bgr;‐Gluc/Sh.

Dietary dehydrated lemon peel improves the immune but not the antioxidant status of gilthead seabream (Sparus aurata L.)

ABSTRACT Lemon (Citrus limon) is the third most important species of citrus in the world, while Spain is the major producer in Europe. Numerous beneficial effects of lemon are known, which explains their use in traditional medicine. The paper describes the effect of dietary dehydrated lemon peel (a sub‐product of the lemon industry) on the growth, immune and antioxidant status of gilthead seabream (Sparus aurata L.) over a period of 30 days. Fish fed diets enriched with dehydrated lemon peel (1.5% and 3%) for 15 days showed improved growth and both humoral (seric immunoglobulin M) and cellular (peroxidase activity and phagocytic ability of head kidney leucocytes) immunity, as well as the expression of some immune‐related genes (nkefa, il1&bgr;, igth and csfr1). However, decreases growth promotion was observed after thirty days of trial. Neither the anti‐oxidant enzymes activity nor the expression of several anti‐oxidants and anti‐stress genes in liver was improved by the diet. The possible inclusion of dehydrated lemon peel in fish diets for its immunostimulant effects is discussed. HIGHLIGHTSDietary dehydrated lemon peel (DLP) for 15 days improved gilthead seabream immunity.Supplemented DLP diets improved leucocyte, serum and skin mucus immune activities.Immune‐related gene expression was stimulated by the supplemented diets.More concentrated diet (3% DLP) decreased weight and growth promotion at 30 days.Dehydrated lemon peel could be considered as a good dietary supplement.

In vitro effects of Origanum vulgare leaf extracts on gilthead seabream (Sparus aurata L.) leucocytes, cytotoxic, bactericidal and antioxidant activities

ABSTRACT Origanum vulgare is a well‐known medicinal plant that has been used since ancient times as an additive in foods and cosmetic preparations. The possible application of O. vulgare extracts in fish was assessed by using gilthead seabream (Sparus aurata) as a marine fish model due to its importance in aquaculture. The in vitro effects of aqueous and ethanolic leaf extracts of O. vulgare were tested in order to observe any immunostimulant, cytotoxic, bactericidal or antioxidant properties. The results showed that medium or high concentration of aqueous extracts and low concentrations of ethanolic extract, increased head kidney leucocyte activities as well as the number of SAF‐1cells. However, moderate to high concentrations of ethanolic extracts decreased both leucocyte activities and the number of viable SAF‐1cells, suggesting some possible toxic effect towards them. Only the highest concentration of the aqueous extract and medium to high concentrations of the ethanolic extracts showed cytotoxic activity against the tumor PLHC‐1cell line. Bactericidal activity was only detected against Vibrio harveyi, V. anguillarum and Photobacterium damselae when using the highest concentration of aqueous extract and moderate to high concentrations of ethanolic extract. Finally, both plant extracts presented antioxidant activity particularly the aqueous extract. Overall, the results suggest that both extracts (when used at the appropriate concentration) have immunostimulant, cytotoxic, bactericidal and antioxidant properties, making O. vulgare an interesting candidate for incorporation as additive in functional diets for farmed fish. HIGHLIGHTSOregano aqueous and ethanolic extracts had immunostimulant activities on gilthead seabream HK leucocytes.Aqueous and ethanolic extracts had cytotoxic activities on SAF‐1 and PHLC‐1cell lines.Ethanolic extracts had more bactericidal activity than aqueous ones.Aqueous extracts had more antioxidant activity than ethanolic extracts.

Effects of dietary administration of fenugreek seeds on metabolic parameters and immune status of gilthead seabream (Sparus aurata L.)

ABSTRACT Medical plants could be used as a prophylactic method in aquaculture because they are considered safe and so very promising alternatives to the use of chemicals. The aim of the present work was to examine the effects of dietary fenugreek (Trigonella foenum graecum) seeds administered for 8 weeks on the metabolic and immune status of gilthead seabream (Sparus aurata L.). Four experimental groups were designated: one receiving a basal diet (control) and three fed powdered fenugreek seeds incorporated in the fish feed at 1%, 5% and 10%. The results show that significant decreases in aspartate aminotransferase, creatine kinase, potassium and the albumin/globulin ratio were detected in the serum of fish fed 10% fenugreek compared with the values recorded in control fish. As regards the immune status, fish fed the 5% supplemented diet had higher haemolytic complement and peroxidase activities than the control fish whilst antiprotease activity was higher in fish fed the 1% fenugreek level respect to control fish and the fish fed the highest fenugreek supplementation rate. Interestingly, the results also revealed a significant enhancement of most of the cellular immune parameters studied, especially in fish fed the highest level of fenugreek (10%). However, the bacteriostatic activity of serum against fish pathogenic and non‐pathogenic bacteria was non‐affected to any significant extent in fish fed the supplemented diets. Overall, the results suggest that the high level of dietary fenugreek tested in this work (10%) did not negatively affect any of the metabolic parameters measured in serum but increased some of them. In addition, the inclusion of fenugreek seeds in the gilthead seabream diet at 5% or 10% improved the humoral and cellular immune activities, respectively. Further studies are needed to better understand the effects of this natural product, which may be suitable for use as a feed additive in fish aquaculture. HIGHLIGHTSDietary supplements of fenugreek seeds for 8 weeks has beneficial effects in gilthead seabream.Fenugreek incorporation (10%) do not impact negatively to any of the 10 metabolic parameters tested.Fenugreek administration improved some of the seabream metabolism parameters evaluated.Seric complement and peroxidase activities were increased in fish fed 5% supplemented diet.The highest inclusion of fenugreek tested (10%) enhance the cellular innate immunity.

A time course study of glucose levels and innate immune response in gilthead seabream (Sparus aurata L.) after exposure to clove oil-eugenol derived anaesthetic

ABSTRACT Clove oil is used as an anaesthetic for many species of fish worldwide; however, relatively few studies have assessed its effects on the innate immune response on these species. The present work aimed to investigate the effects of clove oil‐eugenol derived anaesthetic on some humoral and cellular immune response in gilthead seabream (Sparus aurata L.). To compare with an unexposed control group, fish were exposed to 55 ppm clove oil for 5 min, before being sampled at 1, 24 and 48 h post‐exposure. Serum glucose level was also measured to obtain information on the fish physiological response after clove oil anaesthesia. One hour after exposure the haemolytic complement activity of fish was lower than in the unexposed group. By contrast, the leucocyte peroxidase activity in head‐kidney was significantly stimulated 24 h after exposure to clove oil‐eugenol. The rest of innate immune parameters evaluated and the glucose levels not were affected by clove oil exposure at any sampling point. Overall, the use of clove oil at 55 ppm as anaesthetic did not seem to alter the innate immune response and neither did it trigger a stress response. The use of clove oil‐eugenol derived had become common practice in aquaculture, and its use with gilthead seabream can be considered safe as it does not cause immunodepression in anesthetized fish. HighlightsFew studies have assessed the clove oil effects on the immune status.Glucose levels and immunity were evaluated in gilthead seabream after clove oil anaesthetic exposure.Glucose levels and the main immune parameters measured were not altered by 55 ppm clove oil exposure.Only the haemolytic complement activity was reduced in fish sampled after 1 h of clove oil exposure.Clove oil seems not to compromise the physiological and immune status in gilthead seabream.

Molecular oxidative stress markers in olive ridley turtles (Lepidochelys olivacea) and their relation to metal concentrations in wild populations

Due to their longevity and extensive migration areas, marine turtles are able to accumulate diverse contaminants over many years and as a consequence they represent an interesting bioindicator species for marine ecosystem pollution. Metals provoke toxicological effects in many aquatic animal species, but marine turtles have been under-investigated in this area. Thus, we have determined the presence of certain inorganic elements (As, Cd, Cu, Ni, Pb, Se and Zn) in olive ridley turtles (Lepidochelys olivacea) and related them to metallothionein (MT), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) transcription and/or enzymatic activities. Gene expression of sod, cat and gr was found to be higher in blood than liver or kidney but most of the significant relationships were found in liver, not only for gene expression but also for enzyme activities. This must be related to the role the liver has as the first filter organ. Several positive relationships of sod, cat and gr gene expression in the different tissues were found in this population, as well as very high Cd concentrations. This could mean that these turtles are adapting to the metals-production of ROS and damage through a high transcription of these antioxidants. Multiple positive relationships with GR seem to be part of its compensatory effect due to the decrease of SOD production against the high and chronic exposure to certain xenobiotics. CAT, on the other hand, seems not to be used much, and glutathione detoxification of H2O2 may be more important in this species. Finally, despite the very high Cd concentrations found in this population, no significant relationship was found in any tissue with metallothionein gene expression. These results, along with very high Cd concentrations and a negative relationship with Cu, lead us to consider some kind of disruption in mt gene expression in these turtles.

Evaluation of silver nanospheres on viability and innate cellular parameters of gilthead seabream (Sparus aurata L.) head-kidney leucocytes

The increasing use of nanomaterials, e.g. nanosilver, has lead to concerns about environmental contamination and possible toxic effects on aquatic organisms. Here, we present evidence for the impact of silver nanospheres (AgNSs) on fish innate immune cells after inxa0vitro exposure. AgNSs of 20, 50 or 100xa0nm in diameter were tested with the smallest ones (20xa0nm) clearly having the most deleterious effects, after an exposure period of 30xa0min, followed by the medium-sized ones; the NSs of 100xa0nm had no impact. The effective concentration was determined at 10xa0μgxa0ml-1 while lower concentrations (1, 2.5 or 5xa0μgxa0ml-1) were ineffective. Head-kidney mixed leucocyte population showed significant viability reduction which was attributable to diminished viability of macrophages/monocytes and lymphocytes only whereas granulocytes viability was not affected at the above exposure regime. Furthermore, cellular respiratory burst activity, phagocytic capacity and phagocytic ability were all reduced, with the first two parameters exhibiting the sharper reductions. Finally, transmission electron microscopy revealed that the AgNSs internalization was brought about via phagocytosis, pinocytosis, receptor-mediated endocytosis and macropinocytosis; also, that cell death could be effected in either an apoptotic or a necrotic manner. It is concluded that AgNSs are potentially very noxious for the teleost fish immune system as they can adversely affect the function and viability of the head-kidney leucocytes.

Local immune response of two mucosal surfaces of the European seabass, Dicentrarchus labrax, fed tryptophan- or methionine-supplemented diets

ABSTRACT Immune responses relies on an adequate provision of multiple nutrients that sustain the synthesis of key effector molecules. These needs are depicted in the already reported increase of circulating free amino acids in fish under stressful conditions. Since aquaculture and the inherent fish welfare are an emergent call, the immunomodulatory effects of amino acids on gut‐ and skin‐associated lymphoid tissues of the European seabass (Dicentrarchus labrax) were studied under unstressed conditions and after an inflammatory insult. To achieve this goal, fish were distributed in duplicate tanks (fifteen fish per tank) and were fed for 14 days with methionine or tryptophan‐supplemented diets at 2× dietary requirement level (MET and TRP, respectively) or a control diet meeting the amino acids requirement levels (CTRL). Afterwards, samples of skin and posterior gut were collected from 6 fish per dietary treatment for the assessment of the immune status while the remaining animals were intraperitoneally‐injected with inactivated Photobacterium damselae subsp. piscicida and subsequently sampled either 4 or 24 h post‐injection. The immune status of both mucosal surfaces was poorly affected, although a tryptophan effect was denoted after bacterial inoculation, with several immune‐related genes up‐regulated in the gut at 4 h post‐injection, which seems to suggest a neuroendocrine‐immune systems interaction. In contrast, skin mucosal immunity was inhibited by tryptophan dietary supplementation. Regarding methionine, results were often statistically non‐significant, though increasing trends were denoted in a few parameters. Overall, dietary methionine did not significantly affect neither gut nor skin immunity, whereas tryptophan supplementation seems to induce modulatory mechanisms that might be tissue‐specific. HighlightsMethionine and tryptophan potential as dietary immunomodulators was assessed.Dietary methionine surplus did not significantly affect local immune responses.Tryptophan induced immune‐related gene expression in the posterior gut.Skin mucus immune response was attenuated in fish fed tryptophan‐supplemented diets.