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The present study evaluated the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on intestinal histomorphology, microbiota profile, and digestive and oxidative stress-related enzyme activities of gilthead seabream (Sparus aurata). To this purpose, two practical diets were formulated: one with 50% P and 10% CH (P50/CH10) and other with 40% P and 20% CH (P40/CH20). Triplicate groups of fish with9.1±0.01 gwere fed these diets for 60 days until visual satiation at a FF of 1, 2, or 3 meals per day. Distal intestine histomorphology was not affected by diet composition or FF. However, the pyloric caeca (PC) of fish fed 1 meal per day presented more gut fold height alterations than the other groups, except in fish fed diet P50/CH10 3 meals per day, where no changes was observed. Fish fed diet P40/CH20 3 meals per day also presented higher PC submucosa cellularity than the other groups. Fish fed diet P40/CH20 presented a higher number of operational taxonomic units, microbial richness, and diversity indices than fish fed diet P50/CH10. Amylase was the only measured digestive enzyme affected by the experimental conditions, presenting higher activity in fish fed diet P50/CH10 once per day. Glucose-6-phosphate dehydrogenase activity was lower in fish fed 2 meals per day than only 1. While catalase activity was lower in fish fed 2 than 3 meals per day. Glutathione reductase activity was the only measured parameter affected both by dietary P/CH ratio and FF, being inferior in fish fed once per day the P50/CH10 diet than the P40/CH20 diet and, also in the P50/CH10 diet, to fish fed 1 than those fed 3 meals per day. Overall, no major interactions was observed between dietary P/CH ratio and FF; however, a P40/CH20 diet fed 2 meals per day might be recommended for gilthead seabream juveniles.

The effect of partially replacing fishmeal (FM) by black soldier fly larvae meal (Hermetia meal--HM) in meagre (Argyrosomus regius) diet was evaluated for nutrient digestibility and digestive enzyme activity. For that purpose, triplicate groups of fish (18.0 ± 0.02 g) were fed during 48 days either a control diet (CTR), without HM, or one of three diets including 100, 200 and 300 g/kg of HM, replacing 17, 35 and 52% of FM, respectively. Apparent digestibility coefficients (ADCs) of dry matter, energy, protein, lysine, isoleucine, leucine, phenylalanine, alanine, glutamate, glycine and serine presented a decreasing response with increased HM inclusion. Chitin ADC was null, independent of dietary HM inclusion. Total alkaline protease activity increased while trypsin activity decreased with dietary HM inclusion. No intestinal chitinolytic activity was detected. Intestinal alkaline protease zymogram revealed nine bands with proteolytic activity against casein, with molecular weights ranging between 15 and 75 kDa. Anti-protease activity in the intestine was not affected by dietary inclusion of HM compared to the CTR diet. Overall, it is concluded that replacement of up to 17% FM with HM (100 g/kg HM inclusion level) in meagre diets has no major adverse effects in diet digestibility and digestive enzyme activity.

Nannochloropsis gaditana was tested as functional ingredient in low fishmeal diets for gilthead seabream juveniles, and its short‐term effects were evaluated in terms of intestinal morphology, digestive function, intestinal immunological and oxidative status, and intestinal microbiota. A diet with 850 g/kg of the protein provided by plant feedstuffs and 150 g/kg provided by fishmeal was used as control, and three other diets identical to the control were supplemented with 5, 7.5 and 15 g/kg N. gaditana meal and fed to 56.6 g fish for 37 days. At the end of this period, intestinal mucosa integrity and digestive capacity (luminal enzyme activity and absorption) were not altered by dietary microalgae supplementation. Intestinal transcript levels of key markers of inflammation (IL‐1β and TNF‐α) and of key enzymes involved in redox homeostasis (CuZnSOD, MnSOD and catalase) were also similar across experimental groups. However, microbiota richness increased with dietary supplementation of microalgae, and such modulation requires further investigation, particularly regarding its potential effects on disease resistance at short and long term.

This study aimed at evaluating the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on growth performance, hepatic metabolism, gut microbiota and digestive enzymes activities of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to control but supplemented with 10 g of scFOS, XOS or GOS per kilo diet, which were fed to fish during 12 weeks. Prebiotics had no effect on growth, feed efficiency or gut microbiota. Plasmatic triglycerides were lower in fish fed XOS than FOS and GOS diets. Malic enzyme activity was lower in fish fed XOS than FOS diet. Fish fed XOS diet had lower fatty acid synthetase (FAS), a key lipogenic enzyme and higher alanine aminotransferase activities. Fifteen days after the start of the trial, an enhancement of total alkaline protease, trypsin and lipase activities was observed in fish fed prebiotics, but such effect disappeared at 12 weeks. In conclusion, scFOS, XOS or GOS seem to have limited applicability in white sea bream feed.

The gut microbiomes of fish play important roles in host development, digestion and health. Evidence suggests that abiotic factors, such as diet and rearing temperature, could affect fish gut microbiota. In this study, the effect of dietary short-chain fructooligosaccharides (scFOS) on turbot intestinal health, microbiota and digestive enzymes was investigated at two rearing temperatures: 15 and 20 °C. Four practical diets were supplemented with scFOS at 0, 5, 10 and 20 g kg−1. scFOS did not affect fish performance. PCR-DGGE did not show differences in bacterial profiles between dietary treatments; however, the number of operational taxonomic units, richness and diversity were higher at 20 °C. Enzyme activities in the foregut were not affected by rearing temperature, while in the hindgut, enzyme activities were higher at 15 °C. Total alkaline protease, α-amylase and lipase activities in the foregut were higher in fish fed 20 g kg−1 scFOS. Prebiotic supplementation had no effect on hindgut α-amylase activity, while lipase activity of fish reared at 20 °C was higher in fish fed diet with 5 g kg−1 scFOS. No differences were observed in intestinal morphology. This was the first study to simultaneously evaluate the effect of dietary prebiotic level and rearing temperature on fish intestinal microbiota and health.

To assess the effect of feeding frequency on the utilization of dietary starch by white sea bream juveniles, triplicate groups of fish were fed an experimental diet (400 g kg−1 protein, 140 g kg−1 lipids and 350 g kg−1 pregelatinized maize starch) to apparent visual satiation 2, 3 or 4 times a day for 63 days. Growth performance, feed utilization efficiency, glycaemia, cholesterolaemia, plasma triacylglycerides, liver lipids and glycogen content were unaffected by feeding frequency. α-Amylase activity increased from the pyloric caeca to the posterior intestine and was higher in fish fed twice a day than in fish fed 3 or 4 times a day. Hepatic glucokinase (GK) activity decreased with the increase in feeding frequencies, whereas fructose-1,6-bisphosphatase (FBPase) activity increased. Hepatic glucose-6-phosphate dehydrogenase and glutamate dehydrogenase activities were unaffected by feeding frequency. Overall, feeding frequency did not improve white sea bream dietary starch utilization. α-Amylase, GK and FBPase activities responded to dietary starch consumed at each meal, denoting a good metabolic adaptation of the fish to the feeding conditions.

This study was the first to investigate the effect of soy antinutrients, saponins and phytosterols, in on-growing sea bass. Seven diets were formulated: a control diet (fishmeal and fish oil based) and six diets containing 1 or 2 g kg−1 levels of soya saponins, 5 or 10 g kg−1 levels of phytosterols or a combination of 1 g kg−1 saponins + 5 g kg−1 phytosterols or 2 g kg−1 saponins + 10 g kg−1 phytosterols. After a 59 days feeding trial fish were weighed, faeces collected for digestibility studies and plasma sampled for cholesterol quantification. At day 15 and 59, distal intestine (DI) samples were collected for histological evaluation and quantification of maltase (Malt), alkaline phosphatase (AlP) and leucine aminopeptidase (LAP) activities. At day 59, DI was collected for gene expression of AlP, Malt and immune related genes. Antinutrients had no effect on fish performance, digestibility, plasma cholesterol levels and digestive enzymes activities and gene expression. Only mild morphological changes were present in groups fed high levels of saponins + phytosterols. Expression of immune related genes showed no differences between groups. Overall, on-growing sea bass showed high tolerance to dietary antinutrients up to the levels tested.

A 14 weeks growth trial was performed to estimate the protein requirement for growth and maintenance of zebra sea bream (Diplodus cervinus) juveniles. For that purpose, nine isolipidic diets were formulated to contain increasing protein levels (from 50 to 550 g kg−1) at the expense of carbohydrate. Each diet was assigned to duplicate groups of 20 fish, with an average body weight of 7.7 g. Feed efficiency improved with dietary protein up to 400 g kg−1, no further differences being noticed at higher protein levels. Fish fed the 50 g kg−1 protein diet lost weight during the trial. In the other groups, weight gain improved as dietary protein increased up to 350–400 g kg−1. Fish fed diets with 250 g kg−1 protein or lower had lower whole-body protein content than the other groups. A curvilinear-plateau model was used to adjust weight gain and protein gain (g kg ABW−1 day−1) to dietary protein levels. Based on that model, the optimum dietary protein requirement for maximum weight gain was estimated to be 437.6 g kg−1 and for maximum protein gain 461.9 g kg−1, corresponding to a protein intake of 7.63 g kg ABW−1 day−1. Protein requirement for maintenance was estimated to be 1.01 g kg ABW−1 day−1.

The effect of short-chain fructooligosaccharides (scFOS) incorporation on growth, feed utilization, body composition, plasmatic metabolites and selected liver enzyme activities of turbot juveniles reared at winter (15 °C) and summer (20 °C) temperatures was studied. Four comparable diets were formulated to contain circa 50 : 50 fish meal and plant ingredients as protein sources. Experimental diets included increasing levels of scFOS (0, 5, 10 and 20 g kg−1). Final weight was higher at 20 °C, but thermal growth unit, feed efficiency, nitrogen and energy retention were better at 15 °C. scFOS supplementation did not affect fish growth performance. Fish reared at 15 °C had higher liver glycogen, visceral and hepatosomatic indices. Liver lipids, plasma triglycerides, total lipids, cholesterol HDL and LDL were higher in turbot reared at 20 °C. Malic enzyme, fatty acid synthetase, alanine aminotransferase and glutamate dehydrogenase activities were higher in fish reared at 15 °C. Malic enzyme was lower in turbot fed with 20 g kg−1 scFOS compared to control diet; however, fatty acid synthetase presented an increasing trend as dietary scFOS increased up to 10 g kg−1. Glutamate dehydrogenase activity was higher in fish fed the control diet. Results seem to indicate no benefits of scFOS incorporation to diets on growth performance of turbot.

This study aimed to elucidate the effects of two soy antinutrients, saponins and phytosterols, on growth, intestinal health and function of European sea bass juveniles. Seven fishmeal-based diets were formulated: a control diet without antinutrients and six experimental diets containing low or high levels of soyasaponins (SapL and SapH, respectively), low or high levels of phytosterols (PhytL and PhytH, respectively) and low or high levels of soyasaponins + phytosterols (SapPhytL and SapPhytH, respectively). A feeding trial was conducted for 59 days after which blood was collected for plasmatic cholesterol quantification and intestinal samples were collected for histology, digestive enzymes activity and gene expression analysis. Histology was also performed at day 15. PhytL resulted in high distal intestine (DI) relative weight and decreased plasma cholesterol, while PhytH caused inflammatory changes in the DI. SapH depressed maltase and alkaline phosphatase activity in DI and the histological evaluation indicated some inflammatory changes. The SapPhytH resulted in decreased maltase activity in the DI and indications of inflammatory changes that were supported by the results on gene expression profiles. The antinutrients tested did not compromise growth, however, caused some gastrointestinal disturbance that may affect fish in the long term.

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.

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.

The effects of various dietary protein levels on growth performance, whole body composition and nutrient utilization were studied in two-banded sea bream (Diplodus vulgaris), a candidate species for aquaculture. Fish (initial weight 6.1 g) were fed to satiety six iso-energetic diets, containing 5%, 12.5%, 25%, 35%, 45% or 55% of crude protein during 72 days. Fish fed 35% and 45% protein attained better growth and feed utilization than the other groups (P < 0.05). Daily growth index and feed conversion ratio were the poorest for fish fed 5% and 12.5% protein (P < 0.001), while the 25% and 55% protein groups had intermediate performance. Lipid retention increased significantly from 13.7% to 30.1% (P < 0.0001) and protein retention decreased from 35.5% to 21.3% (P < 0.01) with increasing protein levels from 12.5% to 45%. Muscle protein, lipid and energy concentrations were not significantly affected by dietary protein level. The estimated protein requirement for maintenance and maximum growth of two-banded seabream growing from 6 to 20 g were 7.5% and 35.7%, respectively. Protein requirements as calculated from body protein gain were 2.3 and 6.5 g of protein intake per kilogram body weight per day.

A trial was undertaken to estimate the protein requirement of white sea bream (Diplodus sargus). Five fish meal-based diets were formulated to contain graded levels of protein (from 60 to 490 g kg⁻¹). Each diet was assigned to triplicate groups of 25 fish with a mean individual body weight of 22 g. Fish fed the 60 g kg⁻¹ protein diet lost weight during the trial, while growth improved in the other groups as dietary protein level increased up to 270-370 g kg⁻¹. Feed efficiency improved as dietary protein level increased. Maximum protein efficiency ratio (PER) was observed with the 17% protein diet. N retention (NR) (% N intake) was not different among groups fed diets with 17% protein and above. Ammonia excretion (g kg⁻¹ABW day⁻¹) increased as dietary protein level increased, while no differences in urea excretion were noted. An exponential model was used to adjust specific growth rate and NR (g kg⁻¹ day⁻¹) to dietary protein level. Based on that model, dietary protein required for maximum retention was 330 g kg⁻¹, while for maximum growth it was 270 g kg⁻¹. On a wet weight basis, there were no differences in whole body composition of fish-fed diets with 170 g kg⁻¹ protein and above, except for the protein content, which was lower in group fed the 170 g kg⁻¹ protein diet than the 490 g kg⁻¹ protein diet. Specific activities of hepatic amino acid catabolism enzymes (glutamate dehydrogenase, alanine aminotransferase and aspartate aminotransferase) increased as dietary protein levels increased. There were no differences among groups in fatty acid synthetase and malyc enzyme but 6-phosphate dehydrogenase (G6PDH) was significantly lower in fish fed the 60 g kg⁻¹ protein diet than the 170 and 490 g kg⁻¹ protein diets.