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.     

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.     

Selenium supplementation changes glutathione peroxidase activity and thyroid hormone production in Senegalese sole (Solea senegalensis) larvae

Dietary selenium (Se) influences the glutathione peroxidase (GPx) activity and the thyroid hormone system in vertebrates, but these relations are poorly described in fish. The present study aimed to evaluate the effect of Se supplementation on Senegalese sole larvae, focusing on GPx activity and thyroid status. Two groups of larvae were fed on either control (C) or Se‐enriched live feed, from 2 days after hatch (DAH) until 34 DAH. Senegalese sole larvae fed Se‐enriched live feed significantly increased their whole‐body Se, compared to larvae from C group. The activity of GPx was higher in larvae from Se group, suggesting that the levels of this mineral were suboptimal in the control diet. TH levels were also higher in larvae fed Se‐enriched live feed, suggesting that Se may have a positive influence on the TH production in early life stages of marine fish larvae.     

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.     

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.     

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.     

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.     

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.     

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.     

Optimal dietary protein levels in juvenile Senegalese sole (Solea senegalensis)

A study was undertaken to determine the dietary protein level for optimal growth performance and body composition of juvenile Senegalese sole. Five experimental extruded diets were formulated to contain increasing levels of protein [430, 480, 530, 570 and 600 g kg^?1 dry matter (DM)] and a constant lipid level, ranging from 100 to 130 g kg^?1 DM. Triplicate groups of 35 sole (initial body weight: 11.9 ± 0.5 g) were grown over 84 days in 60‐L tanks supplied with recirculated seawater. Fish were fed by means of automatic feeders in eight meals per day. At the start and end of the trial, whole‐body samples were withdrawn for proximate composition analysis. At the end of 84 days of experimental feeding, daily weight gain and specific growth rate in fish fed diets P43 and P48 were significantly lower than those found in fish fed higher protein level diets (P53, P57 and P59). Similarly, feed efficiency was also significantly lower in fish fed diet P43 than in fish fed all other dietary treatments. Sole juveniles fed lower protein level diets (P43 and P48) showed a significantly lower protein content than fish fed the higher dietary protein level treatments (P53, P57 and P60). Changes within the tested dietary protein levels did not affect significantly protein productive value or total nitrogen (N) losses in fish. However, daily N gain was significantly higher (P < 0.05) in fish fed diets P53 and P60 than in fish fed the lowest protein level diet (P43). Data from the present study indicate that diets for juvenile Senegalese sole should include at least 53% crude protein to maintain a good overall growth performance. Based on a second‐order polynomial regression model, the daily crude protein requirement for maximum whole‐body N gain as estimated here for Senegalese sole juveniles was 6.43 g kg^?1 body weight day^?1 which corresponds to a value of 1.03 g N intake kg^?1 body weight day^?1. If the present data are expressed on a dietary crude protein concentration basis, the allowance for maximum protein accretion (N gain) would be met by a diet containing a crude protein level of 600 g kg^?1.     

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.     

Evaluation of protein hydrolysis in raw sources by digestive proteases of Senegalese sole (Solea senegalensis,Kaup 1858) using a combination of an in vitro assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of products

The hydrolysis of protein in different animal and plant sources by the intestinal proteases of Senegalese sole (Solea senegalensis) was studied using a combination of an in vitro digestibility assay and the evaluation of the protein fractionation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). In vitro hydrolysis was performed for 90 min in a closed reactor maintained at constant pH and temperature. Samples of the reaction mixture at different time intervals were developed in SDS‐PAGE gels to evaluate the progressive hydrolysis of the different protein fractions in each protein source. A numerical value [coefficient of protein degradation (CPD)], which integrates the information obtained after image analysis of the gels, is proposed for comparison among proteins, according to the intensity of the hydrolysis produced by sole proteases. Additionally, the total free amino acid released from proteins was measured during the in vitro assay. Casein, squid meal and soybean concentrate (SBC) proteins showed very similar degradation patterns, with a quick and almost complete proteolysis within the first minutes of the enzymatic reaction. Fish and krill meals were hydrolysed more progressively. On the contrary, pea meal (PM) and corn gluten meal (CGM) showed scarce changes in their protein profile after 90 min of reaction. For animal protein sources, the final CPD values ranged from 77.6% to 87.0%, showing not significant differences. By contrast, PM (30.5%) and CGM (32.3%) presented significantly lower CPD values (P<0.05) as compared with SBC (90.6%). In general, a linear fitting was found between CPD and the release of free amino acids during in vitro protein hydrolysis. The present study provides detailed information, which, combined with the conventional in vitro digestibility studies, may help in the evaluation of different raw sources according to their protein degradation patterns. This information can be applied directly to estimate the protein nutritional quality of ingredients for Senegalese sole feeds.