Investigating isotopic markers for hazelnut geographical authentication: Promising variables and potential applications.

Hazelnuts' features and price are influenced by their geographical origin, making them susceptible to fraud, especially counterfeit claims regarding their provenance. Stable isotope analysis is a recognised approach to establish the geographical origin of foods, yet its potential in hazelnut authentication remains unexplored. In this prospective study, we assessed multiple isotopic markers in hazelnuts from different origins and evaluated the most promising variables for geographical authentication by chemometric tools. Our findings indicate that bulk δ18O, along with δ2H and δ13C in the main fatty acid methyl esters, exhibit significant potential in discriminating geographical origins, and 87Sr/86Sr analysis could serve as a proficient confirmatory tool. Though no single marker alone can differentiate between all the studied origins, employing a multi-isotopic approach based on PLS-DA models achieved up to 92.5 % accuracy in leave-10 %-out cross-validation. These findings will probably lay the groundwork for developing robust models for hazelnut geographical authentication based on larger datasets.

Prospective exploration of hazelnut's unsaponifiable fraction for geographical and varietal authentication: A comparative study of advanced fingerprinting and untargeted profiling techniques.

This study compares two data processing techniques (fingerprinting and untargeted profiling) to authenticate hazelnut cultivar and provenance based on its unsaponifiable fraction by GC-MS. PLS-DA classification models were developed on a selected sample set (n = 176). As test cases, cultivar models were developed for "Tonda di Giffoni" vs other cultivars, whereas provenance models were developed for three origins (Chile, Italy or Spain). Both fingerprinting and untargeted profiling successfully classified hazelnuts by cultivar or provenance, revealing the potential of the unsaponifiable fraction. External validation provided over 90 % correct classification, with fingerprinting slightly outperforming. Analysing PLS-DA models' regression coefficients and tentatively identifying compounds corresponding to highly relevant variables showed consistent agreement in key discriminant compounds across both approaches. However, fingerprinting in selected ion mode extracted slightly more information from chromatographic data, including minor discriminant species. Conversely, untargeted profiling acquired in full scan mode, provided pure spectra, facilitating chemical interpretability.

Effect of feeding olive pomace acid oil on pork lipid composition, oxidative stability, colour, and sensory acceptance.

One of the targets of the meat industry is to reduce production costs and to increase the sustainability of the food chain, which has driven the attention towards the use of by-products as feed ingredients. Acid oils are fat by-products coming from the chemical refining process of edible oils, with a high energy value and that are approved as feed ingredients in the European Union. However, meat producers are hesitant to utilise them due to their varying composition and the limited understanding of their impact on animal performance and meat quality. The objective of this study was to evaluate the effects of using olive pomace acid oil (OPAO) instead of its corresponding crude olive pomace oil (OPO) or crude palm oil (PO) in pig diets on lipid composition, lipid oxidation and quality of pork loin (longissimus dorsi), fresh and after commercial refrigerated storage for 8 days. The experimental design consisted of feeding pigs with four diets supplemented with a 5% of PO, OPO, OPAO or a blend (B) of PO and OPAO (50:50, w/w). Fresh and refrigerated pork loin samples were assessed for fatty acid profile; tocopherol (T) and tocotrienol (T3) composition; lipid oxidative stability with the ferrous oxidation-xylenol orange method; 2-thiobarbituric acid (TBA) value; volatile compounds; colour; and sensory acceptance. Results showed that refrigeration reduced the total T + T3 levels and increased the TBA values and the volatile compound concentrations. The refrigerated storage also affected the instrumental colour parameters (L*, a* and b*) but not the overall acceptance of pork. Regarding the diet, pork from OPAO diet showed a higher unsaturated-to-saturated fatty acid ratio than pork from PO and B diets. The lowest T + T3 concentration was found in OPO and OPAO fresh pork and in OPAO refrigerated pork. The oxidative stability of fresh pork was lower for OPAO than for PO diet, but no significant effect of the diet was observed for this parameter in refrigerated pork. The TBA values and volatile compound concentrations of fresh pork were not affected by the diet. After refrigeration, OPAO pork had the highest TBA value and volatile compound concentrations. In any case, colour and consumer acceptance of pork were not affected by diet. In conclusion, in order to upcycle acid oils in pig diets, and considering results on the lipid oxidative stability of pork, it would be preferable to add the OPAO used in this study blended with PO.

Crude and acid oils from olive pomace as alternative fat sources in growing-finishing pigs.

The inclusion of crude and acid oils from olive pomace can lead to more unsaturated meat products and, especially in the case of olive pomace acid oil, achieve a more economically and environmentally sustainable swine production. The objective of this trial was to study the effect of dietary supplementation with crude and acid oils from olive pomace, which are rich in monounsaturated fatty acids (FAs) and have differing free FA content, on growth performance, digestibility, carcass parameters and FA profile of Longissimus muscle (LM) and backfat in growing-finishing pigs compared to the conventional crude palm oil. A total of 224 male and female pigs [(Landrace × Large White) × Duroc] were randomly distributed into 48 pens according to initial BW (58.7 ± 9.71 kg, mean ± SD) and sex. Four experimental treatments were randomly assigned (n = 12 pens/treatment; 4-5 pigs/pen) for the growing (0-42 days) and finishing (40-62 days) phases. Treatments consisted of a basal diet supplemented with 5% (as-fed basis) palm oil (PO), olive pomace oil (O), olive pomace acid oil (OA) or a mixture (M) of PO and OA at 50/50. No differences were found in the growth performance results between PO, O or M, but animals fed OA showed a lower gain to feed ratio than M (P = 0.008). No differences were found in apparent ileal digestibility among treatments, however, animals fed O and OA showed the highest values of total FA apparent total tract digestibility, while those fed PO had the lowest values, and M had intermediate values (P < 0.001). No differences were observed in carcass composition among treatments. In relation to backfat and the LM FA profile, O and OA treatments led to a higher unsaturated FA to saturated FA ratio and a lower content in saturated FA than PO. Moreover, O showed a higher intramuscular fat (IMF) content in LM than PO (P = 0.037). It is concluded that olive pomace oil is an interesting alternative fat source that can be included at 5% in growing-finishing pig diets, leading to meat products with more IMF, rich in monounsaturated FA, reaching high FA digestibility values and good pig performance parameters. Alternatively, olive pomace acid oil blended with conventional palm oil did not negatively impact fat utilisation nor performance. Including these fat by-products reduced feeding costs and led to a more efficient and environmentally sustainable production.

Effects of free-fatty-acid content and saturation degree of the dietary oil sources on lipid-class content and fatty-acid digestibility along the gastrointestinal tract in broilers from 22 to 37 days of age.

The aim of the present study is to assess the effect of the free-fatty-acid (FFA) content and saturation degree of dietary fat (added at 6%) on the fatty-acid (FA) digestibility and lipid-class content along the gastrointestinal tract and excreta in broilers from 22 to 37 d of age. This is essential to determine the potential use of acid oils (refining by-products rich in FFA) in broiler diets as an alternative to crude oils. The study consisted of a 2 × 4 factorial arrangement, which included 2 fat sources (soybean oils - unsaturated, or palm oils - saturated) and 4 levels of FFA (5, 15, 35, and 50%). Samples of digestive content of the gizzard, duodenum, jejunum, ileum and of the excreta were obtained at 37 d of age. Irrespective of the dietary fat source, more than 80% of total FA (TFA) was absorbed in the jejunum. Broilers fed with unsaturated diets had a higher absorption efficiency of FA than did those fed with saturated diets. This conclusion is supported by the lower FFA content and the higher TFA and polyunsaturated FA (PUFA) digestibility coefficients in the ileum (P < 0.001) observed in the former group. The dietary FFA level did not affect the FA absorption process as much as the dietary fat source did. This was supported by the lack of statistical differences among the diets with a similar saturation degree but rather different levels of FFA, for TFA, saturated FA, and PUFA digestibility coefficients both in the jejunum and ileum. However, the interactions reported in the ileum for triacylglycerol and diacylglycerol contents (P < 0.001), as well as for monounsaturated FA digestibility coefficients (P < 0.05) show that the dietary FFA content affects the FA absorption process. The present results show that the inclusion of acid oils in grower-finisher broiler diets with FFA levels up to 35% does not have a negative impact on the FA absorption process.

Effect of different dietary arachidonic, eicosapentaenoic, and docosahexaenoic acid content on selected immune parameters in gilthead sea bream (Sparus aurata).

The increasing replacement of fish oil (FO) by vegetable oils (VO) in aquafeeds for marine carnivorous species may lead to modifications of the dietary arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) levels and ratios that may compromise the fish immune status. Therefore, this study aimed to assess how different dietary ratios of ARA, EPA, and DHA modulate the immune response of gilthead sea bream juveniles. For that purpose, four isoproteic and isolipidic plant-based diets were formulated to include different dietary Long-Chain-Polyunsaturated Fatty Acids (LC-PUFA) ratios (%DM): Diet A (2.0% ARA: 0.2% EPA: 0.1% DHA); Diet B (1.0% ARA: 0.4% EPA: 0.4% DHA); Diet C (0% ARA: 0.6% EPA: 0.6% DHA); Diet D (0% ARA: 0.3% EPA: 1.5% DHA). Fish were fed the experimental diets for 56 days and then the haematological profile, humoral innate immune parameters, and expression of immune-related genes in the gut were determined. Our results showed that fish fed diet B exhibited an increase in the monocyte numbers, alternative complement activity, and bactericidal activity against Photobacterium damselae compared to the values found in fish fed other diets. Contrarily, peroxidase, antiproteases, and proteases activity and nitric oxide levels were not affected by the dietary treatments. Immune-related gene expression in the distal intestine did not show differences between dietary treatments. Overall, a diet with a balanced n-6 (ARA) and n-3 (EPA+DHA) LC-PUFA ratio seemed to better modulate the fish innate immune response, thus better preparing the fish innate immune system against potential immunological insults.

Mucosal immune responses in Senegalese sole (Solea senegalensis) juveniles after Tenacibaculum maritimum challenge: A comparative study between ocular and blind sides.

Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe.

Effects of dietary free fatty-acid content and saturation degree on lipid-class composition and fatty-acid digestibility along the gastrointestinal tract in broiler starter chickens.

The aim of the present study is to assess the effect of the dietary free fatty acid (FFA) content and dietary fat saturation degree on the fatty-acid (FA) digestibility and lipid-class content along the gastrointestinal tract and excreta in broiler chickens. The 8 experimental diets resulted from replacing crude soybean oil with soybean acid oil from chemical refining, or crude palm oil with palm FA distillate from physical refining. Thus, there were 4 soybean and 4 palm diets with 6% added fat varying in their FFA% (5, 15, 35, and 50%). Samples of digestive content (gizzard, duodenum, jejunum, and ileum) and excreta were collected at 14 D for the determination of the FA digestibility and lipid-class content. The total FA digestibility coefficients reported for the chickens fed S diets in the jejunum, ileum, and excreta were higher than for those fed P diets (P ≤ 0.02). The general greater digestibility of the unsaturated diets was mainly explained by a higher contribution of the ileum to the absorption of saturated FA. The dietary FFA content mainly affected the FA absorption process. The diets with 50% FFA presented lower saturated FA digestibility coefficients in the jejunum and ileum (P ≤ 0.03), and higher content of FFA in the ileum and excreta (P ≤ 0.014), in comparison to the diets with 5% FFA. The 15% FFA diets were not different from the 5% FFA diets, regarding the saturated FA digestibility in the jejunum and excreta, and the FFA content in the ileum and excreta. It was concluded that unsaturated diets with moderate content of dietary FFA (up to 15%) could be used in broiler-chicken starter diets, as they led to similar FA absorption and performance results to the diets with the lowest dietary FFA content. From the present study, it has also been concluded that dietary saturated FA content has a greater impact on FA absorption than the dietary FFA content has.

Mucosal and systemic immune responses in Senegalese sole (Solea senegalensis Kaup) bath challenged with Tenacibaculum maritimum: A time-course study.

Tenacibaculosis, caused by Tenacibaculum maritimum, continues to inflict substantial losses among cultured marine species, particularly in the Senegalese sole. However, the immune mechanisms in fish involved in fighting against this disease are still poorly understood. Thus, the present study aimed to investigate the skin mucus's terminal carbohydrate composition, several immune-related enzymes (i.e. lysozyme, peroxidase, proteases and antiproteases), the haemolytic activity of complement and the bactericidal activity in the skin mucus and plasma of the Senegalese sole in a time-course study following a bath challenge with T. maritimum. The haematological profile and the kinetics of cell migration post-infection were also considered. The bath challenge induced slight variations in the terminal carbohydrate composition of Senegalese sole skin mucus. In general, results from this study showed a delay in the mucosal immune response compared to that found at the systemic level (i.e. blood and plasma). For instance, a significant increase in the skin mucus's lysozyme, complement, protease and antiprotease activities were observed at the end of the experiment (14 d post-challenge). Interestingly, the higher activity of these enzymes could be related to the skin mucus's bactericidal capacity and haemolytic complement activity, suggesting that these enzymes play an important role in the defence against Gram-negative bacteria. The haematological profile revealed a significant increase in circulating neutrophils in challenged fish after 48 and 72 h, which was positively correlated to the increments observed in peroxidase and lysozyme activities, respectively, in the plasma of challenged fish at the same time. Although the route of entry and the survival strategy of T. maritimum are still not fully elucidated, results from the present study will contribute to this endeavour through the study of the mucosal immune responses of fish against this particular pathogen.

Evolution of lipid classes and fatty acid digestibility along the gastrointestinal tract of broiler chickens fed different fat sources at different ages.

The aim of the present study is to evaluate the effect of the dietary fat saturation degree and age on the lipid class (TAG, DAG, MAG, and FFA) composition and fatty acid digestibility along the gastrointestinal tract (GIT) and excreta in broiler chickens. A total of 120 one-day-old female broiler chickens were randomly distributed in 2 dietary treatments (6 cages/treatment), which resulted from the supplementation of a basal diet with 6% of soybean oil or palm oil. Two digestibility balances were carried out at 14 and 35 d and fatty acid digestibility and lipid class composition were determined in the gizzard, duodenum, jejunum, ileum, and excreta. Along de GIT, both fatty acid digestibility and lipid class composition were influenced by the dietary fat source and the age of the chickens. The absorption of the unsaturated fat was more efficient and faster than it was for the saturated fat. The ability of adult chickens to absorb fat was higher than for young chickens. The results show that the duodenum is the main place of fat digestion (hydrolysis), and the jejunum the main place of fat absorption. The role of the ileum on fat absorption is very important, as it is the last segment of the GIT where the absorption of fatty acids has been described. Thus, it was the contribution of the ileum that was responsible for the higher fat utilization observed for animals fed the unsaturated diet than for those fed the saturated diet at 14 d, and it was also responsible for the improvement on the utilization of the saturated diet between 14 and 35 d. All the results suggest that the absorption of fatty acids is more limiting than is hydrolysis, because the main differences were observed in the jejunum and ileum, where the absorption of fatty acids takes place.

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.

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.

Dietary administration effects of fenugreek seeds on skin mucosal antioxidant and immunity status of gilthead seabream (Sparus aurata L.).

Improving fish defense through the preventive administration of immunostimulants has an important role in controlling the outbreak of the disease in aquaculture. As a continuity of our previous studies, this paper describes the effects of dietary fenugreek (Trigonella foenum graecum) seeds on the skin mucosal antioxidant status and immune response of gilthead seabream (Sparus aurata L.). Fish were fed with four experimental diets: one a basal diet (control) and three diets with powdered fenugreek seeds incorporated in the fish feed at 1%, 5%, and 10%. After eight weeks of feeding, free radicals scavenging and antioxidant assays were assessed in skin mucus by measuring the peroxidation of phospholipid liposomes, hydroxyl radical and hydrogen peroxide scavenging, measurement of total antioxidant activity and the determination of antioxidant activity in a linoleic acid system. The skin mucosal immune response was evaluated by measuring the IgM levels and some enzymatic activities (peroxidase, antiprotease, protease, esterase, and ceruloplasmin). Our results demonstrated that fenugreek inclusion improved the hydroxyl radical scavenging capacity and conferred very high antioxidant activity. Besides, only the highest supplementation level (10%) was able to augment the peroxidase and protease activities confronted by a general decrement in the antiprotease activity in the experimental fed groups with 1% and 10%. These results suggest that the dietary administration of fenugreek at the higher inclusion dose enhances the skin mucosal immunity response and the antioxidant status of gilthead seabream a species with one of the highest rates of production in marine aquaculture.

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

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.

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

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.

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 in vitro exposure. AgNSs of 20, 50 or 100 nm in diameter were tested with the smallest ones (20 nm) clearly having the most deleterious effects, after an exposure period of 30 min, followed by the medium-sized ones; the NSs of 100 nm had no impact. The effective concentration was determined at 10 µg ml-1 while lower concentrations (1, 2.5 or 5 µg ml-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.

Effects of dietary supplementation with fenugreek seeds, alone or in combination with probiotics, on gilthead seabream (Sparus aurata L.) skin mucosal immunity.

Despite increasing interest in modulating the immune response of fish, providing a combination of probiotics and herbal immunostimulants in aquafeed has rarely has been studied. The effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds alone (FE), or combined with one of the following probiotic strains: Bacillus licheniformis (FEBL), Lactobacillus plantarum (FELP) or Bacillus subtilis (FEBS) were evaluated. Fish were fed a control or one of the supplemented diets for 3 weeks. After 2 and 3 weeks of the feeding trial, the abundance of terminal carbohydrates, IgM levels, enzymatic activities (proteases, alkaline phosphatase, esterase and ceruloplasmin) and bactericidal activity were determined in skin mucus. Our results demonstrated that the dietary administration of FE in combination with L. plantarum, particularly, increased carbohydrate abundance, the activity of certain enzymes such as ceruloplasmin, and bactericidal activity against the pathogenic bacterium Photobacterium damselae and the non-pathogenic bacterium B. subtilis in skin mucus at the end of the trial. The carbohydrates most affected by the FELP diet were mannose/glucose, N-acetyl-d-galactosamine and N-acetyl-ß-d-glucosamine. Interestingly, IgM levels were significantly higher in fish fed the FELP and FEBS diets whilst protease activity generally increased in all supplemented diets, which could suggests that the main effect in this activity was to the result of FE supplementation although that fact cannot be confirmed because the effects of probiotics addition alone were not studied. These results suggest that the combined dietary administration of fenugreek and L. plantarum will best enhance the skin mucosal immunity response of gilthead seabream.

Vitamin D3 affects innate immune status of European sea bass (Dicentrarchus labrax L.).

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500 U kg-1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2 weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets after 4 weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total IgM level. At gene level, fbl and rbl transcripts were up-regulated in HK in fish fed with the highest concentration of vD3-supplemented diets after 4 weeks, while in the gut, an up-regulation of hep gene was observed in fish fed with the different doses of vD3. These results suggest that vD3 may be of great interest for immunostimulatory purposes in fish farms.

The short-term effects of farmed fish food consumed by wild fish congregating outside the farms.

We simulated in the laboratory the possible effects on fatty acids and immune status of wild fish arriving for the first time in the vicinity of a sea-cage fish farm, shifting their natural diet to commercial feed consumption, rich in fatty acids of vegetable origin. The flesh fatty acid profile of golden mullet specimens was altered after 2weeks of commercial feed consumption, showing an increase in fatty acids of vegetable origin. The serum peroxidase and bactericidal activities, and head-kidney leucocyte phagocytic capacity, increased after eight weeks of the new diet, while the respiratory burst activity decreased. The extent of these changes cannot be considered large enough to regard them as compromising the health status of fish. More research is needed in order to elucidate whether the rapid assimilation of the dietary fatty acids could harm the immune status of fish when feeding for longer periods than two months.

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

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.

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.).

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-ß, 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.