Rebeca Cerezuela

Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)), or B. subtilis + inulin (10(7) cfu g(-1) + 10 g kg(-1)) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

Impact of date palm fruits extracts and probiotic enriched diet on antioxidant status, innate immune response and immune-related gene expression of European seabass (Dicentrarchus labrax).

The application of additives in the diet as plants or extracts of plants as natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics and immunoprophylactics. The aim of the current study was to evaluate the potential effects of dietary supplementation of date palm fruit extracts alone or in combination with Pdp11 probiotic on serum antioxidant status, on the humoral and cellular innate immune status, as well as, on the expression levels of some immune-related genes in head-kidney and gut of European sea bass (Dicentrarchus labrax) after 2 and 4 weeks of administration. This study showed for the first time in European sea bass an immunostimulation in several of the parameters evaluated in fish fed with date palm fruits extracts enriched diet or fed with this substance in combination with Pdp 11 probiotic, mainly after 4 weeks of treatment. In the same way, dietary supplementation of mixture diet has positive effects on the expression levels of immune-related genes, chiefly in head-kidney of Dicentrarchus labrax. Therefore, the combination of both could be considered of great interest as potential additives for farmed fish.

Effects of inulin on gilthead seabream (Sparus aurata L.) innate immune parameters

Inulin, a fructooligossacharide, is a prebiotic that plays an important role in the immune function in mammals, but it has never been assayed in other vertebrate groups. Thus, we have studied the inulin effects on the gilthead seabream (Sparus aurata L.) innate immune response both in vitro and in vivo. For the in vitro study, head-kidney leucocytes were incubated with inulin (ranging from 0 to 1000 microg ml(-1)) for 30, 90, 180 and 300 min and 24h and any effect was observed on leucocyte viability or the main innate cellular immune responses (leucocyte peroxidase, phagocytic, respiratory burst and natural cytotoxic activities). For the in vivo study, seabream specimens were fed for 1 or 2 weeks with a commercial diet supplemented with inulin: 0 (control), 5 or 10 g inulin kg(-1) diet (0.5 and 1%, respectively). Inulin produced a significant inhibition in phagocytosis and respiratory burst in leucocytes from specimens fed diets containing 0.5% or 1% of inulin for 1 week. Based on the present results, inulin does not seem to be a good immunostimulant for seabream, though its effects in other species and combined with other immunostimulans (i.e. probiotics) might be of great interest.

Current Knowledge in Synbiotic Use for Fish Aquaculture: A Review

Synbiotics are nutritional supplements that combine probiotics and prebiotics, enhancing their beneficial effects. Nowadays, probiotics are also becoming an integral part of aquaculture practices to obtain high production, and, although considerably less information is available regarding probiotics applications for fish, they offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature to these feed ingredients, to date the use of synbiotics in fish farms has been poorly investigated and available data are still scarce. The parameters investigated in these studies have been the following: effect on survival and growth, feed conversion, body composition, haematological/biochemical parameters, digestive enzyme activity, immunological response and disease resistance. This review compiles the results from recent studies, aiming to highlight the properties of synbiotics evaluated for potential application in farming fish. Various factors as fish specie, time of feeding treatment and supplement dose as well as the type of prebiotics and probiotics can significantly affect the activity of synbiotics. Moreover, many aspects such as mechanism which mediate host benefits are poorly understood. Future studies are needed to provide a better knowledge and fully conclude on the effects of adding synbiotics in fish fed.

Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damselae.

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabreams immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.

Effects of dietary Bacillus subtilis, Tetraselmis chuii, and Phaeodactylum tricornutum, singularly or in combination, on the immune response and disease resistance of sea bream (Sparus aurata L.)

Combined or individual effects of two microalgae (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis on immune response, gene expression, and survival to challenge with Photobacterium damselae subsp. piscicida of gilthead sea bream were investigated. To test the capacity of B. subtilis to grow employing the microalgae polysaccharides as energy and carbon source, an in vitro assay was defined, and demonstrated that the digestion product of microalgae, mainly P. tricornutum, supported the growth of B. subtilis much better than glucose. For the in vivo study, fish were distributed in six equal groups (each of two replicates) and received one of the following experimental diets: C) control, non-supplemented diet; T) T. chuii 100 g kg(-1); P) P. tricornutum 100 g kg(-1); B) B. subtilis (10(7) cfu g(-1)); BT) B. subtilis (10(7) cfu g(-1))+T. chuii (100 g kg(-1)); and BP) B. subtilis (10(7) cfu g(-1))+P. tricornutum (100 g kg(-1)). The complement activity, serum IgM level, respiratory burst, phagocytic activity, and expression of seven selected immune-related genes in head-kidney were evaluated following two and four weeks of treatment. At the end of the feeding trial, fish were challenged by intraperitoneal injection of LD(50) concentration of P. damselae subsp. piscicida and mortality was recorded. This is the first study testing the immunomodulatory capacity of the microalgae used in the present work. The dietary applications of B. subtilis, T. chuii, and P. tricornutum, singly or in combination, may exhibit up-regulating effects on gilthead sea bream immune parameters. P. tricornutum demonstrated the highest immunostimulant activity. There were no significant differences between combination feeding and feeding ingredients separately. Our results demonstrated the potential of microalgae as immunostimulants for fish, although further studies regarding the implications and effects of a stimulated immune system against pathogens, especially the protective capacity against specific diseases, are necessary.

Effects of dietary vitamin D3 administration on innate immune parameters of seabream (Sparus aurata L.).

The present study assesses the in vivo effect of vitamin D(3) or cholecalciferol on some innate immune parameters of the gilthead seabream (Sparus aurata L.). Cholecalciferol was orally administered to seabream specimens in a commercial pellet food supplemented with 0 (control); 3750; 18,750 or 37,500 U kg(-1) and fish were sampled after 1, 2 and 4 weeks of treatment. Serum and head- kidney leucocytes were obtained and humoral (peroxidase and complement activity) and cellular (leucocyte peroxidase content, phagocytic, respiratory burst and natural cytotoxic activities) innate immune parameters were measured. Diet supplementation with 37,500 U kg(-1) cholecalciferol for 2 or 4 weeks resulted in a significant increase in phagocytic ability or serum peroxidase content, respectively, whereas the 3750 and 18,750 U kg(-1) supplemented diets led to significant increases in the phagocytic capacity of leucocytes at week 2 compared with the values found in control fish. Natural cytotoxic activity was increased in leucocytes from fish fed for 1 week with 3750 U kg(-1) cholecalciferol. No significant differences were observed in complement activity or in respiratory burst activity in the assayed conditions. These results suggested that dietary vitamin D(3) administration has an effect on the innate immune parameters of gilthead seabream. The immunostimulant effect was greater on the cellular innate immune parameters assayed, suggesting that similar receptors to those present in mammals are involved in the action of this vitamin in the fish immune system.

The treatment with the probiotic Shewanella putrefaciens Pdp11 of specimens of Solea senegalensis exposed to high stocking densities to enhance their resistance to disease.

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet.

In vivo actions of melatonin on the innate immune parameters in the teleost fish gilthead seabream

Abstract:  Melatonin, a molecule produced in the pineal gland and retina of vertebrates, plays a major role in day–night circadian rhythms and other physiological processes, including the immune responses. Because little is known in this respect in fish, we have evaluated the in vivo role of melatonin in the main innate immune activities and the expression of immune‐relevant genes in a teleost fish, the gilthead seabream (Sparus aurata L.). An intraperitoneal injection of 1 or 10 mg melatonin/kg of body weight produced a dose–time dependent increase in circulating melatonin serum levels. Several innate immune responses such as peroxidase, phagocytic, reactive oxygen intermediates and cell‐mediated cytotoxic activities were significantly enhanced by the administration of melatonin at different sampling times. The expression of immune‐relevant genes such as interleukin‐1β, major histocompatibility complex, virus‐related response (interferon‐regulatory factor‐1 and Mx) and lymphocyte markers (immunoglobulin M and T‐cell receptor for B and T lymphocytes, respectively), as analysed by semi‐quantitative reverse transcription‐polymerase chain reaction, was up‐regulated in the head–kidney of melatonin‐injected fish 1 and 3 days postinjection and decreased after 7 days. These data, together with our previous observations describing how photoperiod and in vitro melatonin treatment may affect seabream and sea bass immunology, confirm melatonin as a regulator of fish immunology. However, further studies are still needed to reveal the mechanisms underlying the direct or indirect interactions of melatonin with the fish immune system.

Effects of dietary administration of fenugreek seeds, alone or in combination with probiotics, on growth performance parameters, humoral immune response and gene expression of gilthead seabream (Sparus aurata L.)

ABSTRACT The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune‐relevant genes (igm, tcr‐&bgr;, csfr1 and bd) were measured in the head‐kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune‐associated genes in the head‐kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture. HIGHLIGHTSInnate immune response was enhanced by the fenugreek dietary supplementation.Humoral immune parameters were stimulates mainly by fenugreek + B. subtilis diet.Supplemented diets induced an important up‐regulation of igm gene expression.Fenugreek and probiotic tested could be considered as a good aqua feed supplements.The use of immunostimulants is a promising alternative to maintain animal welfare.