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 At the start and end of the trial, whole‐body samples were withdrawn for proximate composition analysis.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.     

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.     

Rearing temperature affects Senegalese sole (Solea senegalensis) larvae protein metabolic capacity

The present work examined the short- and long-term effects of three rearing temperatures on protein metabolism and growth trajectories of Senegalese sole larvae using ¹⁴C-labelled Artemia protein as feed. A first feeding trial was performed on larvae reared at 15, 18 and 21 °C (at 26, 17 and 14 days post-hatching (dph), respectively) and a second trial conducted on post-larvae after transfer to the same rearing temperature (~20 °C) (49, 35 and 27 dph, in larvae initially reared at 15, 18 and 21 °C, respectively). Temperature greatly influenced larvae relative growth rate (RGR) and survival, since growth at 15 °C was severely depressed. Protein digestibility and retention was highest at 18 °C during the first trial (85.35 ± 1.16 and 86.34 ± 2.33 %, respectively). However, during the second trial, post-larvae from 15 °C had the highest feed intake and protein digestibility (3.58 ± 1.54 and 75.50 ± 1.35 %, respectively), although retention was similar between treatments. Furthermore, after transfer to 20 °C larvae from 15 °C experienced compensatory growth, which was observed until 121 dph, and confirmed by RGR values, which were significantly higher at 15 ºC than at 21 ºC or 18 ºC. Results from the present study show that Solea senegalensis larval development, survival and protein digestion and retention are highly affected by thermal history.     

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.     

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 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.     

Tissue composition,blood biochemistry and histology of digestive organs in Senegalese sole (Solea senegalensis) juveniles fed diets containing different plant protein ingredients

The aim of this study was to determine the impact of diets with different plant protein ingredients on proximate composition, tissue metabolites and tissue fatty acid composition, as well as hepatic and intestinal histological modifications in Senegalese sole (Solea senegalensis). Fish (21.5 ± 2.8 g body weight) were fed six isonitrogenous and isoenergetic diets during 11 weeks. A control diet containing fish meal as the main protein source was compared with five experimental diets replacing 30% fish meal protein by different plant protein sources: soybean meal (SBM), soybean protein concentrate (SPC), soybean protein isolate (SPI), wheat gluten meal (WGM) or pea protein concentrate (PPC). The inclusion of different plant protein did not significantly affect growth and proximate composition of fish. The hepatosomatic index was not significantly different to the control group; however, utilization of WGM significantly increased hepatocyte size. Plasma protein values and muscle triglycerides were influenced by the inclusion of SBM and WGM in the diets respectively. Feeding fish on SBM, WGM and PPC diets significantly enhanced n‐6 fatty acid levels in muscle, particularly linoleic acid. None of the plant protein ingredient used in the diets decreased arachidonic, eicosapentaenoic as well as docosahexaenoic acid values in liver and muscle. Overall, histological studies did not reveal the existence of any intestinal alterations induced by the inclusion of different plant proteins. Despite moderate changes produced by SBM, SPC and WGM, inclusion of dietary plant protein ingredients has no major impact on growth, tissue and blood biochemistry, fatty acid profile and gut integrity of Senegalese sole juveniles.     

Dietary protein/carbohydrate ratio in low‐lipid diets for Senegalese sole (Solea senegalensis,Kaup 1858) juveniles. Influence on growth performance,nutrient utilization and flesh quality

Four isoenergetic (21 kJ/g dry matter, DM) and isolipidic (65 g/kg DM) diets containing different crude protein/total carbohydrate (CHO) ratios: 60/26, 56/30, 52/34 and 48/38, were tested in 22 g Senegalese sole for 104 days. Apparent digestibility coefficients (ADC) were not affected by the treatments, and all groups presented extremely low starch ADC values (22.8%–36.5%). Replacement of dietary protein by CHO did not affect daily growth index (0.9–1.0), but significantly increased voluntary feed intake of fish. Regression analyses demonstrated that digestible protein content, rather than digestible energy, was the main dietary factor influencing such feeding activity (R² = .952). A significantly increased feed conversion ratio was observed in sole fed increasing CHO contents. The dietary protein/CHO ratio did not influence whole‐body composition. Sole fed the 48/38 diet showed the lowest efficiency in terms of N and energy utilization. PUFA were the most represented fatty acid fraction in fillet, regardless of the dietary protein/CHO ratio, mainly due to the high content of DHA. Senegalese sole increase feed intake under low dietary protein/CHO ratios to ensure an adequate N intake. Such compensatory mechanism seems to be triggered to satisfy a specific protein metabolic requirement for energy purposes as tissue accretion remained unchanged.     

Effective replacement of protein‐bound amino acids by crystalline amino acids in Senegalese sole (Solea senegalensis) juveniles

The objective of this study was to evaluate the potential of free amino acids to replace protein‐bound amino acids in diets for Solea senegalensis juveniles. For this purpose, fish with 4.26 g mean initial weight were fed during 55 days with diets containing 500 g kg^?1 protein and 100 g kg^?1 lipid, based on fish meal and fish oil as main protein and lipid sources (control diet), or diets where 20%, 35% and 50% of fish meal were replaced by crystalline amino acids, using L‐lysine HCl as lysine source. To access the efficiency of the crystalline lysine source, another diet was formulated with 35% fish meal replacement but using L‐lysine sulphate instead of L‐lysine HCl as lysine source. Overall, growth performance, feed utilization, nutrient retention, whole‐body and amino acid composition did not show significant differences among groups. In conclusion, up to 50% protein‐bound amino acids can be replaced by crystalline amino acids in diets for Senegalese sole juveniles without negative effects in fish performance. Additionally, it was shown that L‐lysine sulphate and L‐lysine‐HCl may be used interchangeably by Senegalese sole juveniles, with the potential economic benefits involved.     

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.     

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.     

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.     

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.     

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.     

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.     

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.