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.     

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.     

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0 mg (control), 25, 50 or 100 mg kg(-1) chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.     

In vitro effect of chitin particles on the innate cellular immune system of gilthead seabream (Sparus aurata L.)

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.     

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.     

High dietary intake of alpha-tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of three high levels of dietary intake of alpha-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg alpha-tocopherol kg-1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg alpha-tocopherol acetate kg-1) for 15, 30 or 45 days. Growth, serum alpha-tocopherol levels, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between alpha-tocopherol acetate intake and serum alpha-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg-1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg-1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head-kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg-1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800 mg kg-1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg-1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.     

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 D₃ 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 D₃ 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.     

Effects of 2-deoxy-D-glucose on the immune system of seabream (Sparus aurata L.)

Stressful situations are a major problem in aquaculture because they affect the immune system. 2-Deoxy-d-glucose (2-DG) is a derivative of a glucose analogue that reduces the availability of energy, thereby inhibiting cell metabolism so that it is unable to enter the glycolysis pathway. In this paper, 2-DG has been administered in order to study if the immune function is compromised during metabolic stress. Blood glucose level was measured as an indicator of the inhibition of glycolysis, and the effects of intraperitoneal administration of 2-DG on the main parameters of the humoral (complement, IgM levels and peroxidase activity in blood plasma) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis activity) immune parameters of gilthead seabream (Sparus aurata, L) were evaluated. Furthermore, the expression levels of immune-associated genes (CSF-1R, NCCRP-1, Hep, TCR-β, IgM_H, MHC-IIα, C3 and IL-1β) were analyzed by real-time PCR in head-kidney. A total of 5 intraperitoneal injections were performed at 48 h intervals. Three experimental groups were established: a control group injected with phosphate buffer saline, group 2-DG 500 and group 2-DG 750 injected with 500 mg kg^?1 and 750 mg kg^?1 2-DG, respectively (N = 15). After the third and fourth injection, some specimens of both DG-treated groups died. Following the first and third injection, the blood glucose levels of both 2-DG treated groups increased to a statistically significant extent with respect to the control group. While the humoral immune parameters were not significantly affected as a consequence of 2-DG administration, the cellular activities of leucocytes were. The injection of 500 mg kg^?1 2-DG provoked up- or down-regulation of the immune-relevant genes analyzed, while the injection of 750 mg kg^?1 always caused down-regulation of these genes. The results suggest that 2-DG provokes metabolic stress, which reduces the activities carried out by immune cells (leucocytes) and induces down-regulation of the immune-relevant genes analyzed when the energy available to the cell decreases.     

Effects of lactoferrin on non-specific immune responses of gilthead seabream (Sparus auratus L.)

The main innate cellular immune responses of gilthead seabream (Sparus auratus L.) leucocytes were evaluated after in vitro incubation with human lactoferrin (Lf). Isolated head-kidney leucocytes were incubated with 0 (control) to 1 mg ml(-1) Lf-supplemented culture medium for 30, 120, 240 or 360 min and assayed for viability, peroxidase content, and respiratory burst, phagocytic and cytotoxic activities. Only respiratory burst activity was found to increase when using the highest Lf concentration (1 mg ml(-1)) and long incubation times (more than 120 min). Seabream were fed Lf-supplemented diets (0, control, 50, 100 or 200 mg kg(-1) diet). After 1 or 2 weeks of administration the leucocyte peroxidase content, respiratory burst, phagocytic and cytotoxic activities as a measure of cellular immune responses, as well as serum peroxidase and complement activity as a measure of humoral immune responses were evaluated. The results showed that Lf feeding at 100 mg kg(-1) diet for 1 week enhanced the cellular innate immune responses although only the cytotoxic activity did so significantly. The humoral immune response was not influenced by Lf feeding. In conclusion, Lf seems to affect innate immune cellular activity, mainly respiratory burst and natural cytotoxic activity. The possible use of Lf as an immunostimulant for farmed gilthead seabream is discussed.     

Quantitative trait loci affecting morphology traits in gilthead seabream (Sparus aurata L.)

We report a quantitative trait loci (QTL) mapping study on 18 morphometric characters in gilthead seabream based on a total of 74 informative microsatellite markers genotyped in 409 offspring coming from 10 paternal half‐sib families. Statistical analysis was carried out using a linear regression approach, and various suggestive and significant morphology QTL were detected in three (9, 21 and 25) of nine linkage groups examined. Fitting body weight as a covariate reduced the significance of some QTL but revealed three new QTL in other linkage groups (LG6 and LG10). Current results combined with those obtained from previous studies underline highly significant loci affecting overall growth and morphology in S. aurata.     

17Beta-estradiol triggers postspawning in spermatogenically active gilthead seabream (Sparus aurata L.) males

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6-14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.     

Cytochemical characterization of leucocytes from the seawater teleost,gilthead seabream (Sparus aurata L.)

The cytochemical characterization of head-kidney and peripheral blood leucocytes of gilthead seabream (Sparus aurata L.) was studied by light and electron microscopy. Neutrophilic granulocytes show some cytoplasmic granules, which are positive for alkaline phosphatase and peroxidase but acid phosphatase negative. The scarce granules found in the cytoplasm of the circulating neutrophils and their cytochemical features seem to be indicative of an immature stage. Acidophils are also alkaline phosphatase and peroxidase positive at pH 11.0. They are strongly positive for acid phosphatase and acid phosphatase activity may thus be considered a cytochemical marker to characterize and differentiate neutrophilic from acidophilic granulocytes in this fish species. Three granule populations are characterized in the cytoplasm of the gilthead seabream acidophils: the first is positive only for peroxidase and the second contains a dense core with acid and alkaline phosphatase activities, surrounded by a thin peroxidase positive electron-dense halo. The third granule type contains an eccentric core, which is strongly positive for acid and alkaline phosphatase and peroxidase. As regards their cytochemical features, the first and second granule types seem to correspond respectively to the azurophilic and specific granules found in acidophils of mammals and could be involved in phagocytic processes, thus playing an important microbicidal role in this species. The monocytes, monocyte-macrophages and macrophages show different cytochemical features. The first have scarce acid phosphatase-positive lysosomes, while blood monocyte-macrophages and macrophages are positive for acid and alkaline phosphatases and for peroxidase; the monocyte-macrophages show scarce lysosomes.     

Modulatory effects of deltamethrin-exposure on the immune status,metabolism and oxidative stress in gilthead seabream (Sparus aurata L.)

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.     

Retinal development in the gilthead seabream Sparus aurata

The retinal development of the gilthead seabream Sparus aurata has been analysed from late embryonic development to juvenile stages using classical histological and immunohistological methods. Five significant phases were established. Phases 1 and 2 comprise the late embryonic and hatching stages, respectively. The results indicate that during these early stages the retina is composed of a single neuroblastic layer that consists of undifferentiated retinal progenitor cells. Phase 3 (late prolarval stage) is characterized by the emergence of the retinal layers and the appearance of neurochemical profiles in differentiating photoreceptors, amacrine and ganglion cells. Phases 4 and 5 comprise the late larval and juvenile stages. In these stages, all the retinal cell types can be detected immunohistochemically. All the maturational events described are first detected in the central retina and, as development progresses, spread to the rest of the retina following a central‐to‐peripheral gradient. The results of this study suggest that S. aurata is an altricial teleost species that hatches with a morphologically undifferentiated retina. The most relevant processes involved in retinogenesis occur during the late prolarval stage (phase 3).     

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.)

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0.1g vitamin C and 0.1g vitamin E kg(-1) acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg(-1), 1.2g vitamin E kg(-1) or both 3g vitamin C and 1.2g vitamin E kg(-1). After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.     

Effects of dietary inulin,Bacillus subtilis and microalgae on intestinal gene expression in gilthead seabream (Sparus aurata L.)

The present work describes effects of dietary inulin, two microalgae (Tetraselmis chuii and Phaeodactylum tricornutum) and Bacillus subtilis (solely or combined with inulin or microalgae) on the expression of different genes in the intestine of the gilthead seabream (Sparus aurata L.) following four weeks of a feeding trial. Selected genes were grouped into five categories: genes involved in inflammation (genes encoding proinflammatory proteins), genes related to the cytoskeleton, genes encoding proteins of junction complexes, genes implicated in digestion processes and genes related to transport proteins. Regarding proinflammatory genes, interleukin-8 (IL-8) expression showed a significant increase in the fish fed all the assayed diets, except the B. subtilis + inulin diet, whereas the expression of caspase-1 (CASP-1) was also increased by the B. subtilis and B. subtilis + T. chuii diets. Cyclooxygenase-2 (COX-2) gene expression only increased in fish fed the B. subtilis diet. Among cytoskeletal and junctional genes, only β-actin and occludin were significantly affected by the assayed diets. β-actin expression was up-regulated by the inulin-containing diets (inulin and B. subtilis + inulin diets), whereas occludin expression increased in the fish fed all the assayed diets, except the P. tricornutum diet. Finally, the expression of transport protein genes demonstrated that the inulin diet and all the experimental diets containing B. subtilis significantly increased transferrin expression, whereas digestive enzymes were not affected by the experimental diets. In conclusion, our results demonstrated that inulin, B. subtilis and microalgae can modulate intestinal gene expression in the gilthead seabream. To our knowledge, this is the first study of the effects of some food additives on the intestinal expression of different genes in this species. More studies are needed to understand the role of these genes in maintaining the integrity and functionality of the intestine.     

Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate

The effects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial non-supplemented diet containing 1.75 mg of vitamin A kg(-1) (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg(-1) (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were unaffected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg(-1) diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three different groups of seabream were i.p. injected with 1 ml of phosphate buffer containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0.05 or 0.30 mg 100 g(-1) biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic effects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection.     

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.)

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref‐sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit‐SMsa1 and kit‐SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.