Iodine‐enriched rotifers and Artemia prevent goitre in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system

To study the effect of dietary supplementation of iodine in Solea senegalensis, larvae were randomly distributed in six tanks. Larvae in three tanks were given rotifers and Artemia enriched with iodine in addition to Rich Advance or Super Selco from 2 days after hatch (DAH) until 31 DAH. Larvae in a second set of three tanks were fed control rotifers and Artemia, enriched only with Rich Advance or Super Selco. Samples were collected at 2, 5, 10, 15 and 31 DAH to determine dry weight, total length, myotome height and thyroid status. Larvae fed the iodine‐enriched diet had significantly higher weight at 31 DAH and higher levels of whole body iodine concentration, compared to control larvae. At 31 DAH, larvae from the control treatment showed typical goitrous thyroid follicles. Thyroid cells of larvae from this treatment appeared columnar or afollicular, with the colloid partly or completely depleted, representative of hyperplasia (goitre). The lower growth rate in fish larvae from the control treatment was possibly a consequence of the hyperplasia, and the iodine enrichment prevented Senegalese sole larvae from developing goitre. This study demonstrates the importance of iodine enrichment of live feed for fish reared in a recirculation system.     

Intestinal microbiota variation in Senegalese sole (Solea senegalensis) under different feeding regimes

The aim of this study was to determine the influence of the feeding regimes in Senegalese sole (Solea senegalensis) cultured under extensive, semi‐extensive and intensive production systems. A total of 254 bacterial isolates from guts of fish cultured under different production systems and feeding regimes were tested. Biochemical tests and genetic analyses based on the 16S rDNA sequence analysis were conduced to identify bacterial strains. Vibrio species were the most represented taxonomic group in the culturable microbiota of S. senegalensis guts tested. Particularly, Vibrio ichthyoenteri was the most frequently isolated Vibrio species. Comparison among diets showed a significant reduction (P<0.05) in vibrio percentages and a higher occurrence of Shewanella species in Senegalese soles fed polychaeta. In addition, a major influence of environmental temperature on microbiota composition was detected. Cold temperatures brought about a change in the percentages of Vibrio species and a higher representation of α‐Proteobacteria in both outdoor systems (extensive and semi‐extensive). The significant differences between intestinal bacterial composition in Senegalese soles fed commercial diets and natural preys (polychaeta) reveal the necessity to develop specific optimized diets for the intensive rearing of this fish species.     

Effect of feeding time on dietary protein utilization and growth of juvenile Senegalese sole (Solea senegalensis)

The present study aimed to determine the effect of feeding time on growth and nitrogen excretion in juvenile sole. An 84‐day growth trial was conducted, in which food was supplied to three triplicate groups of juvenile Senegalese sole (3 g wet weight) at different schedules – diurnal, nocturnal and mixed. At the end of the growth trial, ammonia and urea excretion was assessed during a 24 h cycle. Improved growth (1.3% vs. 0.9% day^?1, specific growth rate), higher nitrogen retention (0.35 vs. 0.27 g N kg^?1 day^?1), lower ammonia excretion (209 vs. 272 mg N‐NH₄ kg^?1 day^?1) and lower total nitrogen excretion (278 vs. 352 mg N kg^?1 day^?1) were found in daytime‐fed fish compared with night‐fed fish. Fish in the mixed feeding regime showed intermediate values of ammonia and total nitrogen excretion, but did not differ from day‐fed fish regarding the other parameters. Results indicate that juvenile sole at a period of their life cycle appear to use more efficiently dietary protein for somatic growth under a diurnal than under a nocturnal feeding regime. This suggest that at least during a time‐window in the juvenile rearing a diurnal feeding regime might be more effective in the production of this species.     

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis,Kaup) at early stages of development

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. ‘Minor’ anomalies were predominant, and some of the detected disorders might be a result of non‐/low‐pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.     

Growth, stress response and free amino acid levels in Senegalese sole (Solea senegalensis Kaup 1858) chronically exposed to exogenous ammonia

Stressful husbandry conditions are likely to affect growth and amino acid metabolism in fish. In this study, chronic ammonia exposure was used to test the effects of a stressor on growth and amino acid metabolism of Senegalese sole juveniles. The fish were exposed for 52 days to 11.6 mg L⁻¹ [low‐TAN (L‐TAN)] or 23.2 mg L⁻¹ [high‐TAN (H‐TAN)] of total ammonia nitrogen (TAN), or to 0 mg L⁻¹ (Control). Growth in L‐TAN groups was slightly but significantly different from the Control groups [relative growth rate (RGR=0.35±0.13 and 0.52±0.23% day⁻¹ respectively)]. In H‐TAN groups, growth was severely affected (RGR=0.01±0.13% day⁻¹). Stress parameters (plasma cortisol and glucose) showed slight or no significant differences between treatments. Plasma free amino acid (FAA) concentrations were affected after H‐TAN treatment. Increases in glutamine and aspartate concentrations in H‐TAN fish suggest alterations in amino acid metabolism related to nitrogen excretion processes. Some of the changes in FAA concentrations also suggest mobilization to energy supply and synthesis of metabolites related to stress response. Therefore, Senegalese sole seem to adapt to the L‐TAN concentration tested, but the H‐TAN concentration reduced growth and affected amino acid metabolism.     

Effect of dietary administration of probiotics on growth and intestine functionality of juvenile Senegalese sole (Solea senegalensis, Kaup 1858)

The effects of the dietary administration of two bacterial probiotic strains (Ppd11 and Pdp13) from the Alteromonadaceae family for 60 days, were assessed by measuring growth and feed efficiency, activities of leucine aminopeptidase and alkaline phosphatase and structural changes in the intestine of juvenile Senegalese sole. In addition, the profile of intestinal microbiota was studied by Denaturing Gradient Gel Electrophoresis. Growth and nutrient utilization were significantly higher in fish receiving probiotics than in those fed the control diet. No differences were observed in proximal composition between treatments, though higher lipid muscle content was measured in fish receiving Pdp13. Those fish also exhibited higher activities of AP when compared to Ppd11 and control groups. The profile of intestinal microbiota clearly separated those fish receiving probiotics from those of the control group. Microscopical examination revealed accumulation of lipid droplets in the enterocytes of fish receiving the control diet, but not in those fed on probiotics. Interactions between those structural changes and growth performance are discussed.     

The effect of dietary oxidized lipid levels on growth performance,antioxidant enzyme activities,intestinal lipid deposition and skeletogenesis in Senegalese sole (Solea senegalensis) larvae

Fish tissues, particularly rich in n‐3 PUFA, are prone to lipid peroxidation that can damage cellular membranes, cause severe lesions and subsequently incidences of disease and mortality. However, fish possess antioxidant defences, such as vitamin E (VE) and antioxidant enzymes, to protect them against oxidative damage. This study investigated the effects of an increasing gradient of oxidized dietary lipid on the survival, growth performance, skeletogenesis and antioxidant defensive processes occurring in Senegalese sole (Solea senegalensis) larvae. Four groups of fish were fed live prey enriched with experimental emulsions containing an increasing gradient of oxidized oil: non‐oxidized, NO+VE, 34.5 nmol MDA g^?1 w.w.; mildly oxidized, MO+VE, 43.1 nmol MDA g^?1 w.w.; highly oxidized, HO+VE, 63.3 nmol MDA g^?1 w.w. and highly oxidized without VE, HO‐VE, 78.8 nmol MDA g^?1 w.w. The oxidation levels increased in enriched rotifers following the oxidation gradient of the emulsions, but were not affected in enriched Artemia metanauplii. The oxidation status of Senegalese sole larvae increased during development, but this was not related to the dietary treatments. The increasing dietary oxidation levels did not affect the fatty acid profile, survival, growth performance and metamorphosis processes of sole larvae. Senegalese sole seem to activate antioxidant defence mechanisms in response to the increasing amounts of dietary peroxidized lipids, in a manner efficiently enough to prevent detection of any alterations of these physiological processes. Antioxidant systems and detoxification mechanisms appeared to occur through the consumption of dietary α‐tocopherol, the activation of the antioxidant enzymes (catalase, superoxide dismutase, glutathione S‐transferase, glutathione reductase) and the retention of oxidized fat in the intestinal enterocytes for detoxification prior to their utilization. However, fish fed the highest oxidized diet presented a reduction in bone mineralization, but lower incidence of deformities in the vertebral and caudal regions than fish fed the other diets. This study exemplifies the importance of rearing Senegalese sole larvae on non‐oxidized diets during the early larval development to avoid detrimental consequences in older fish, most notably in the process of skeletogenesis.     

Iodine and selenium supplementation increased survival and changed thyroid hormone status in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system

To test how iodine and both iodine and selenium supplementation affected the thyroid status as well as growth and survival in Senegalese sole, larvae were reared in a recirculation system from 15 to 34 DAH. Sets of three tanks were assigned to each of the following three diets: control (C), iodine (I) and iodine and selenium (I?+?Se). Samples were collected at 15, 27 and 34 DAH to determine dry weight, iodine and selenium levels, GPx and ORD activities, thyroid hormone levels and thyroid follicles histology. At 34 DAH, fish from the control (C) treatment suffered from hyperplasia of the thyroid follicles (goitre), whereas iodine-treated larvae did not (I and I?+?Se). Lower survival rates in the C groups were probably a consequence of the hyperplasia. Moreover, there was an improvement in thyroid hormone status in I- and I?+?Se-treated larvae, showing that further supplementation of live feed with iodine can be crucial for fish at early life stages, as it seems to sustain normal larval development, when reared in a recirculation system. Selenium did not affect the results. Together with previous results, this indicates selenium supplement is more important at younger life stages.     

Soy protein concentrate as a protein source for Senegalese sole (Solea senegalensis Kaup 1858) diets: effects on growth and amino acid metabolism of postlarvae

The objective of this work was to evaluate the effect of a dietary amino acid imbalance, originating from the use of a soy protein concentrate (SPC) as the major protein source, on the growth performance and amino acid metabolism of Senegalese sole (Solea senegalensis) postlarvae. Senegalese sole (85.6±24.6 mg wet weight) were fed one of two experimental diets: one based on fish meal (FM) and another based on SPC. Diets were isonitrogenous (around 56% crude protein) and isoenergetic. Diet acceptability was very good and the growth rate was 6.9% day^?1 for sole eating the FM diet and 6.0% day^?1 for sole eating the SPC diet. Mass‐specific ammonia excretion and the activities of selected amino acid metabolic enzymes (ALAT, ASAT and GDH) did not present significant differences between treatments, although this may have been due to the high variability found for these parameters in the SPC treatment. This variability may suggest different capacities of individual fish to adapt to the possible methionine dietary deficiency. The utilization of amino acids as a substrate for lipogenesis does not seem to be affected by the dietary protein source, since NAPDH‐generating enzymes (G6PD and ME) had similar activities in both treatments. Amino acid metabolism in Senegalese sole postlarvae seems to be slightly affected by the dietary protein source. Nevertheless, the changes induced by the SPC diet do not seem to impair growth, at least at the high dietary protein level used in this experiment.     

A growth equation for sole postlarvae (Solea senegalensis Kaup, 1858) in earthen ponds in the region of Tagus and Sado Estuaries (Portugal)

The growth of 1-month-old juvenile sole, Solea senegalensis (Kaup, 1858), was monitored for 6 months in earthen ponds of commercial fish farms located at the Tagus and Sado Estuaries. The postlarvae were introduced into net cages at a density of 50 m⁻². Data obtained from different growth experiments carried out under similar environmental conditions were analysed. To describe the growth of juvenile sole, the comprehensive model proposed by Schnute was fitted to the data. This model provides a better fit than, for instance, the Gompertz model, to the length at age of S. senegalensis juveniles. Seven-month-old sole juveniles have 80% probability of attaining a length of 10.9–11.7 cm and a weight between 11.0 and 13.7 g. The growth equation can be used to predict fish growth under these environmental conditions and thus identify appropriate farm management options.     

Feed intake and growth performance of Senegalese sole (Solea senegalensis Kaup, 1858) fed diets with partial replacement of fish meal with plant proteins

To be able to study nutrient requirement and utilization in any species, a diet supporting normal feed intake and growth equally well as a traditional fish meal‐based diet is needed. Additionally the formulation of the diet should allow low levels of the nutrient under study. When studying the amino acid metabolism and requirements, one cannot rely on the fish meal‐based diets as fish meal are nicely balanced according to requirements. Therefore the current study aimed to develop a plant protein‐based diet (with low fish meal inclusion) to be used in the nutritional studies of Senegalese sole juveniles supporting feed intake and growth close to that obtained in a fish meal‐based control feed. Two experiments were conducted to evaluate whether Senegalese sole juveniles would accept and utilize diets containing high plant protein inclusion. For testing the acceptance of high plant protein inclusion, two diets were formulated: a reference diet that contained fish meal as the main protein source (450 g kg^?1 dry matter) whereas in the test diet, fish meal was substituted by a mixture of plant ingredients (soybean meal, corn and wheat gluten) with l ‐lysine supplementation. In order to improve the palatability, 50 g kg^?1 squid meal was added to both diets. The indispensable amino acids (IAA) profile of the test diet was made similar to the control diet by adding crystalline amino acids. Further, automatic feeders were used to improve the feed intake. Fish (24 g initial body weight) were fed the diets for a period of 4 weeks. As fish accepted both diets equally well, a second study was undertaken to test the growth performance. Fish (6 g initial BW) were fed the diets for a period of 12 weeks. The use of automatic feeders to deliver the feed and the addition of both squid and balancing the indispensable amino acids resulted in growth performance and accretion not differing from the fish meal fed control. It can be concluded that juvenile Senegalese sole are able to grow and utilize high plant‐protein diets when both diet composition and feeding regime are adequate for this species.     

Lipid enrichment for Senegalese sole (Solea senegalensis) larvae: effect on larval growth, survival and fatty acid profile

Results from three larval Senegalese sole (Solea senegalensis) feeding trials using non-enriched Artemia and Artemia enriched with Super HUFA®, Arasco®, sunflower oil and microalgae are presented and the effects on larval survival, growth and fatty acid (FA) composition are reported. The FA profile of Senegalese sole eggs was analysed to gather information about the nutritional requirements of the early larval stages and a quite high DHA/EPA ratio (4.3) was found. However, there was no evidence of a high dietary demand for DHA or EPA, given that no relationship was found between dietary HUFA concentration and larval growth and survival. When larvae were fed non-enriched Artemia a significantly better growth and comparable survival were obtained than with Artemia enriched with Super HUFA® (containing the highest HUFA level and DHA/EPA ratio). The FA profiles of the larvae generally reflected those of their diets. DHA was an exception, as it was present in high proportions, even in larvae fed DHA-deficient prey. Total FAME concentration decreased during larval development, with SFA, MUFA and PUFA being equally consumed; HUFA appeared to be less used, with its relative concentration being either kept constant (particularly EPA and ARA) or increased (DHA). A specific requirement for ARA in the first larval stages could not be confirmed but it was always present in considerable amounts, even in larvae fed an ARA poor diet.     

Effects of dietary eicosapentaenoic acid on growth, survival, pigmentation and fatty acid composition in Senegal sole (Solea senegalensis) larvae during the Artemia feeding period

We examined the effect of dietary eicosapentaenoic acid (EPA, 20:5n‐3) on growth, survival, pigmentation and fatty acid composition of Senegal sole larvae. From 3 to 40 days post‐hatch (dph), larvae were fed live food that had been enriched using one of four experimental emulsions containing graduated concentrations of EPA and constant docosahexaenoic acid (DHA, 22:6n‐3) and arachidonic acid (ARA, 20:4n‐6). Final proportions of EPA in the enriched Artemia nauplii were described as ‘nil’ (EPA‐N, 0.5% total fatty acids, TFA), ‘low’ (EPA‐L, 10.7% TFA), ‘medium’ (EPA‐M, 20.3% TFA) or ‘high’ (EPA‐H, 29.5% TFA). Significant differences among dietary treatments in larval length were observed at 25, 30 and 40 dph, and in dry weight at 30 and 40 dph, although no significant correlation could be found between dietary EPA content and growth. Eye migration at 17 and 25 dph was affected by dietary levels of EPA. Significantly lower survival was observed in fish fed EPA‐H diet. Lower percentage of fish fed EPA‐N (82.7%) and EPA‐L (82.9%) diets were normally pigmented compared with the fish fed EPA‐M (98.1%) and EPA‐H (99.4%) enriched nauplii. Tissue fatty acid concentrations reflected the corresponding dietary composition. ARA and DHA levels in all the tissues examined were inversely related to dietary EPA. This work concluded that Senegal sole larvae have a very low EPA requirement during the live feeding period.     

Use of probiotic bacteria in the rearing of Senegalese sole (Solea senegalensis) larvae

Three candidate probiotics, which had shown antimicrobial activity in vitro against two fish pathogens, were used in the rearing of Senegalese sole larvae and postlarvae. These probiotics improved the survival of starved sole yolk‐sac larvae. A feeding experiment of sole larvae and postlarvae comprised three treatments: A, B and C. Cultures of a Vibrio sp. (2J18) were added to treatment A, whereas a gram‐positive (J84) and a Shewanella sp. strain (2J27), were added to treatment B, while in a control treatment C no bacteria were added. Addition of bacteria in treatment B increased survival of larvae in the first phase of the experiment [0–20 days after hatching (DAH) (P<0.05)] and decreased the numbers of colony‐forming units (CFU) in larval gut 5 DAH compared with the control treatment (P<0.05). No differences were observed in survival (25–47%) during the second phase of the experiment (20–60 DAH). Nevertheless, the total numbers of CFU in fish gut 40 DAH were significantly lower in treatment B (P<0.05). The colonization rates of the added bacteria were the highest 5 and 11 DAH, and the highest average values reached were 65%, 82% and 17% of the total CFU count for the strains 2J18, 2J27 and J84 respectively.     

Interactions of microorganisms isolated from gilthead sea bream, Sparus aurata L., on Vibrio harveyi, a pathogen of farmed Senegalese sole, Solea senegalensis (Kaup)

Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole.     

Immune responses and gut morphology in Senegalese sole (Solea senegalensis) fed dietary probiotic supplementation and following exposure to Photobacterium damselae subsp. piscicida

The aim of this study was to determine the effect of a dietary multi‐species probiotic on growth, gut morphology and immune parameters in Senegalese sole (Solea senegalensis). Fish were fed with two experimental diets, a control diet and the same diet supplemented with the probiotic for 72 days. A sub‐lethal bath challenge with Photobacterium damselae subsp. piscicida was performed after the growth trial. Intestine and blood samples were collected to study gut structure and plasmatic immune parameters. No significant differences were found in growth performance. The analysis of gut morphology showed a significant increase in intestinal villi height of Senegalese sole fed the probiotic. Regarding circulating leucocytes, dietary probiotic supplementation increased thrombocytes levels whereas a decrease in the proportion of lymphocytes was observed. No significant differences were observed in humoural immune parameters. Bath challenge differentially affected leucocyte counts and increased peroxidase activity. This study presents the possibility of using dietary probiotic supplementation to increase Senegalese sole welfare since gut morphology was positively affected. Although the immune response after a bacterial challenge was not modulated by dietary treatment, further studies would be instrumental to unravel eventual dietary benefits on immune mechanisms.     

Arachidonic acid, arachidonic/eicosapentaenoic acid ratio, stearidonic acid and eicosanoids are involved in dietary-induced albinism in Senegal sole (Solea senegalensis)

Senegal sole larvae were fed live prey enriched with different amounts of arachidonic acid (ARA, 20:4n‐6) and eicosapentaenoic acid (EPA, 20:5n‐3) to re‐evaluate the effect of these two fatty acids on flatfish pigmentation. Echium oil, a plant‐derived oil rich in gamma‐linolenic acid (18:3n‐6) and stearidonic acid (18:4n‐3) was also used as a component of one of the enrichment emulsions. Although ARA content did not have any effect on growth there was a clear influence on pigmentation that correlated clearly with prostaglandin production. Inclusion of Echium oil, on the contrary, exerted a positive effect on pigmentation rate even though dietary ARA levels were as high as in the other emulsions. The relationships between dietary ARA levels and dietary ARA/EPA ratio, prostaglandin production and pigmentation are discussed.