High stocking density induces crowding stress and affects amino acid metabolism in Senegalese sole Solea senegalensis (Kaup 1858) juveniles

Fish held at high stocking densities are generally exposed to chronic stress situations that impose severe energy demands and may predispose the fish to infection. Senegalese sole Solea senegalensis (Kaup) juveniles (78.8±18.9 g body weight) were maintained at low (LSD; 4 kg m^?2 at the end of the experiment), medium (MSD; 9 kg m^?2) and high (HSD; 14 kg m^?2) stocking densities during a period of 63 days. Although disease outbreaks were observed in fish reared at HSD, growth and food consumption did not vary among different treatments. Results from plasma cortisol and free amino acids (FAAs) showed significant differences among different rearing densities pointing to HSD as stressful rearing condition. However, higher plasma glucose and osmolality levels indicated that fish held at MSD may also be under stress. The higher usage of FAAs from the HSD group may be due to the higher demand for energy production in order to cope with stressful rearing conditions, higher rate of protein synthesis or due to synthesis of other important metabolites related to stress response. Therefore, results from the present study point out to HSD as a stressful rearing condition and suggest that crowding stress may affect amino acid requirements.     

Physiological short-term response to sudden salinity change in the Senegalese sole (Solea senegalensis)

The physiological responses of Senegalese sole to a sudden salinity change were investigated. The fish were first acclimated to an initial salinity of 37.5?ppt for 4?h. Then, one group was subjected to increased salinity (55?ppt) while another group was subjected to decreased salinity (5?ppt). The third group (control group) remained at 37.5?ppt. We measured the oxygen consumption rate, osmoregulatory (plasma osmolality, gill and kidney Na⁺,K⁺-ATPase activities) and stress (plasma cortisol and metabolites) parameters 0.5 and 3?h after transfer. Oxygen consumption at both salinities was higher than for the control at both sampling times. Gill Na⁺,K⁺-ATPase activity was significantly higher for the 55?ppt salinity at 0.5?h. Plasma osmolality decreased in the fish exposed to 5?ppt at the two sampling times but no changes were detected for high salinities. Plasma cortisol levels significantly increased at both salinities, although these values declined in the low-salinity group 3?h after transfer. Plasma glucose at 5?ppt salinity did not vary significantly at 0.5?h but decreased at 3?h, while lactate increased for both treatments at the first sampling time and returned to the control levels at 3?h. Overall, the physiological response of S. senegalensis was immediate and involved a rise in oxygen consumption and plasma cortisol values as well as greater metabolite mobilization at both salinities.     

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.     

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.     

Influence of tank bottom surface on growth and welfare of Senegalese sole (Solea senegalensis)

Tank bottom surface is an important question to take into account in flatfish aquaculture considering the intimate contact that the fish have with it. The most usual materials in aquaculture tank surfaces are inert materials such as epoxy resins or plastics with null porosity, and cement that presents a high alkalinity, roughness and porosity. To improve the cement characteristics, silica fume admixture is usually added in construction industry. The aim of this study was to evaluate the effect of three types of tank bottom surfaces on growth and welfare over a Solea senegalensis population. Moreover, the degree of fin erosion, the appearance of skin lesions and malpigmentation patterns were also monitored. The bottom surfaces tested were plain cement (CE), the same plain cement blended with silica fume (10%) (SF) and epoxy coated surface (EP). Specific growth rate for each bottom surface were compared. The Image Processing Activity Index (IPAI) was used to evaluate the Senegalese sole welfare. Higher SGR and lower IPAI values were obtained in the surface of cement with silica fume, suggesting a higher welfare. The use of plain cement affected negatively the performance of fish, probably due to the high pH and alkalinity of the water in direct contact with fish skin. The null porosity of EP seems to be related to the main cause of higher activity level of fish kept on it outcoming in lower growth. Silica fume that is frequently use to improve the mechanical characteristics of plain cement, also provides benefits in terms of fish welfare.     

Effect of stocking density and protein/fat ratio of the diet on the growth of Dover sole (Solea solea)

Two hundred and fifty soles (30 g initial weight) were randomly stocked in 12 tanks (recirculation system) at two different stocking densities (2.3 and 1.3 kg m^?2) and fed on two different diets (50% and 54% of crude protein and 21% and 18% of total lipid, respectively, for diets A and B) with triplicate tanks for treatment. The trial lasted for 300 days. Fish stocked at low density showed a significantly higher weight gain than the fish reared at 2.3 kg m^?2 density (94.1±7.4 vs. 78.9±8.2 g, P<0.01) with a specific growth rate (SGR) of 0.46±0.11 and 0.43±0.16 (P<0.01) respectively. Feed conversion ratio (FCR) was more favourable for lower density groups (2.50 vs. 2.64, P<0.01). Diet B led to a higher final weight (124.1 vs. 110.2 g, P<0.01) and a better feed utilization (FCR: 2.49 vs. 2.65, P<0.01). Except for the lipid content, which was higher in the low‐density group (2.6% vs. 2.3%, P<0.05), proximate composition of sole's muscle was not influenced by treatments. Dover sole reared at low stocking density showed higher monounsaturated fatty acid (35.27% vs. 34.40%, P<0.01) and lower saturated fatty acid (24.36 vs. 26.13%, P<0.01) contents, and lower atherogenic (0.67 vs. 0.75, P<0.01) and thrombogenic (0.28 vs. 0.32, P<0.01) indexes.     

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.     

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.     

Effect of dietary protein level and source on digestive proteolytic enzyme activity in juvenile Senegalese sole, Solea senegalensis Kaup 1850

The effect of dietary protein level and protein source on growth and proteolytic activity of juvenile Solea senegalensis was studied. In Experiment 1, fish were fed on four experimental diets containing increased protein levels (36, 46, 56 and 67%). In Experiment 2, Senegalese soles were fed on five diets with partial substitution of fish meal by soybean meal, soybean protein concentrate, soybean protein isolate, wheat gluten meal or pea protein concentrate. Results prove that growth and proteolytic activity in the distal intestine of fish were affected by the quantitative increase in dietary protein. The origin of protein source used in the elaboration of experimental diets affected both the amount and composition of the alkaline proteases secreted into the intestinal lumen; however, it did not decrease animal growth. Juvenile Senegalese sole showed capability to modulate digestive protease secretion when the concentration and/or source of dietary protein were modified. Quantity and quality of dietary protein affected protein hydrolysis in Senegalese sole intestine. This study establishes that 30% fish meal protein can be replaced by soybean derivatives without affecting intestinal proteases. Replacement with wheat gluten meal or pea protein concentrate should be taken cautiously, but further research is needed to establish whether growth performance and digestive enzyme physiology of Senegalese sole are affected by plant protein-supplemented diets in a long-term trial.     

Replacement of fishmeal by increasing levels of plant protein blends in diets for Senegalese sole (Solea senegalensis) juveniles

A growth trial was conducted to evaluate the growth potential and nutrient utilization of Senegalese sole fed diets containing increasing replacement levels of dietary fishmeal (FM) by mixtures of plant protein (PP) sources. Six extruded isonitrogenous and isolipidic diets (55% crude protein and 8% crude fat on a dry matter basis) were fed to juvenile sole (mean initial body weight: 8 g ± 0.06) during 92 days. A reference diet containing FM as the main protein source (Control) was compared with five PP based-diets with increasing PP levels: 25% (PP25), 35% (PP35), 45% (PP45), 60% (PP60) and 75% (PP75). PP45, PP60 and PP75 diets were supplemented with crystalline amino acids to simulate the level present in the Control diet. The environmental impact of the tested diets was assessed through the determination of N and P budgets and calculating approximate Fish-in:Fish-out (Fi:Fo) ratios. Fish fed PP25 and PP75 reached a final body weight and daily growth index (DGI) similar to the Control group (P > 0.05). Diet PP45 displayed the lowest DGI (0.8 vs 1.3%), lowest protein efficiency ratio (1.03 vs 1.8) and highest FCR (1.7 vs 1.01). The dietary treatments with highest percentage of FM replacement (PP60 and PP75) displayed similar FCR values as the Control (1.2–1.0). Apparent digestibility coefficients (ADCs) of protein varied between 77 and 85% and were not significantly affected by the PP inclusion level. Whole body protein and energy content did not differ significantly among treatments, but protein gain was significantly higher in the Control group compared to PP35, PP45 and PP60 groups. The results indicate that Senegalese sole can effectively cope with plant protein-based diets, but growth rate and nutrient gain mainly depend on the selection of adequate plant protein blends, rather than on the plant protein incorporation level. Sole fed the highest PP level (PP75) showed good growth performance, efficient dietary nutrient utilization and a lower Fi:Fo ratio compared to the Control. The present results clearly show that increasing FM replacement level can have a positive environmental impact as reduces P fecal waste and the fishmeal used per kg of sole produced.     

Effect of temperature and dietary protein/lipid ratio on growth performance and nutrient utilization of juvenile Senegalese sole (Solea senegalensis)

A 74‐day trial was undertaken to evaluate the effects of temperature (16 and 22 °C) and dietary protein/lipid ratio on the performance of juvenile Senegalese sole (mean body weight: 6.4 g). Four experimental diets were formulated to contain two protein levels (550 g kg^?1 and 450 g kg^?1) combined with two lipid levels (80 g kg^?1 and 160 g kg^?1). Growth was higher at 22 °C and within each temperature in fish fed diets 55P8L and 45P16L. Feed efficiency, N retention (% NI) and energy retention (% EI) were higher at 22 and at both temperatures in fish fed diet 55P8L. Temperature affected whole‐body composition, with dry matter, protein, lipid and energy being higher and ash lower in fish kept at higher temperature. Independently of temperature, whole‐body lipid, energy and ash were higher and protein was lower in fish fed the high‐lipid diets. Visceral and hepatosomatic indices were not affected by diet composition but were higher in fish kept at 16 °C. Liver glycogen and lipid contents and activities of glutamate dehydrogenase, alanine and aspartate aminotransferases were not affected by diet or water temperature. Malic enzyme (ME) and glucose 6‐phosphate dehydrogenase activities were higher in fish fed the low‐lipid diets. ME activity was higher at lower temperature. In conclusion, increasing water temperature from 16 to 22 °C improves growth and feed efficiency of Senegalese sole juveniles; regardless of water temperature, the diet with 550 g kg^?1 protein and 80 g kg^?1 lipid promoted the best growth and feed efficiency.     

Feed deprivation in Senegalese sole (Solea senegalensis Kaup, 1858) juveniles: effects on blood plasma metabolites and free amino acid levels

The effects of prolonged feed deprivation were assessed on blood plasma metabolites and free amino acid levels in Solea senegalensis. Juvenile specimens were maintained at two experimental conditions (24 h fasted and 21 days feed-deprived). In feed-deprived fish, relative growth rate and hepatosomatic index as well as plasma triglycerides and proteins levels were significantly lower. However, plasma cortisol levels were higher in feed-deprived fish, while plasma glucose and lactate values were not significantly different between treatments. Furthermore, feed-deprived fish showed higher levels of total plasma free amino acids than 24 h fasted fish. In 21 days feed-deprived sole, an increase in plasma cortisol levels may suggest a functional role in mobilizing energy due to the catabolic action of this hormone in teleosts. Higher levels of glutamine, arginine and ornithine in 21 days feed-deprived fish may be indicative of a dual role for these amino acids: ammonia detoxification and carbon source for gluconeogenesis. The increased plasma glucogenic and branched-chain amino acid levels, together with the maintenance of plasma glucose in 21 days feed-deprived sole, suggest active liver gluconeogenic processes supported by tissue proteolysis.     

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.     

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma

Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non‐ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.     

Immune responses and gut morphology of Senegalese sole (Solea senegalensis,Kaup 1858) fed monospecies and multispecies probiotics

The current study aimed to determine the effects of dietary probiotic supplementation on growth, gut morphology and non‐specific immune parameters in Senegalese sole (Solea senegalensis) juveniles during a 1‐month trial. Fish were fed for 1‐month two diets with 1.0 or 4.6 × 10⁶ CFU kg^?1) of probiotic A (Bacillus sp., Pediococcus sp., Enterococcus sp. and Lactobacillus sp.) and two diets with 3.5 or 8.6 × 10⁵ CFU kg^?1 of probiotic B (Pediococcus acidilactici) and tested against an unsupplemented diet (control). Growth performance, as well as respiratory burst activity, nitric oxide (NO), alternative complement pathway (ACH50), lysozyme and peroxidase activities, was not affected by the dietary treatments. Probiotic supplementation tended to increased growth homogeneity between tanks having diet A₁ the best possible alternative to decrease costs associated to size grading. Villous length and number of goblet cells of the anterior intestine did not vary among treatments. Muscle duodenal layer was significantly thicker in fish fed probiotic A compared to probiotic B, when included at the lowest level (A₂ versus B₂). The current study indicate that the use of the multispecies probiotic at 1.0 × 10⁶ CFU kg^?1 might enhance protection against pathogen outbreak and increase nutrient absorption, whereas at the highest concentration could reduced size dispersion among tanks.     

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.     

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.