Cottonseed oil as a complementary lipid source in diets for gilthead seabream Sparus aurata juveniles

The efficacy of using cottonseed oil (CSO) as a fish oil (FO) substitute in gilthead seabream (Sparus aurata) juveniles feed was evaluated. Fish (BW_i 4.0 ± 2.9 g) were fed one of four isoproteic (~48% CP) and isolipidic (~18% L) diets for 9 weeks. Added oil was either FO (control diet, CTRL) or CSO, replacing 50% (CSO50 diet), 60% (CSO60 diet) and 70% (CSO70 diet) of dietary FO. Results indicated that FO replacement by CSO up to 60% level had no detrimental effects on growth or nutritive utilization and composition in fish muscles. Higher CSO intake (CSO70 diet, 56 g kg^?1) led to a 16% reduction in weight gain, 14% in feed utilization (FCR) and 57% in muscle n‐3 long‐chain polyunsaturated fatty acids (lc PUFA) as compared with CTRL and to abundant accumulation of lipid within the hepatocytes. Use of CSO altered fatty acid (FA) profiles of muscle and liver. Data suggested utilization of linoleic acid (LOA) by fish and retain of docosahexaenoic acid (DHA) in muscles. Therefore, limits of CSO inclusion as the main source of supplementary dietary lipid, with no negative effects on fish performance or nutritive composition and utilization in muscles, are: 40–48 g kg^?1 feed for gilthead seabream juveniles.     

Characterization of digestive enzyme activity during larval development of gilthead seabream (Sparus aurata)

The evolution of the digestive enzyme equipment in seabream from hatching to 30 days old larvae was studied; there was a progressive increase in the activity of protease, amylase and acid and alkaline phosphatase from day 15 onwards. The use of specific inhibitors, and SDS-PAGE provided evidence to suggest that most of the proteases belonged to the serine group. A high -amylase activity was also denoted. Zymograms of larval extracts indicated that exogenous food has more a qualitative than a quantitative role in the secretion of digestive enzymes in this species.     

Interference of Listonella anguillarum with potential probiotic microorganisms isolated from farmed gilthead seabream (Sparus aurata, L.)

Four isolates obtained from gilthead seabream have been tested for their adhesion to the skin, gill and intestinal mucus of gilthead seabream, and for their ability to interfere with Listonella anguillarum, an important pathogen of farmed gilthead seabream. The ability to adhere to mucus was higher than 7% for all isolates. Three isolates showed an antagonistic effect against some of the pathogenic strains tested. They were assayed to interfere with the attachment of L. anguillarum to the mucus of gilthead seabream. Only two isolates significantly reduced the adhesion of L. anguillarum to all of the mucus assayed under exclusion, competition and displacement conditions. According to the criteria applied, the isolate Pdp11 was selected and its in vivo probiotic potential was assessed by oral administration followed by challenge with the pathogen L. anguillarum. For the feeding trial, a group of 50 gilthead seabreams received a commercial diet supplemented with lyophilized 10⁸ CFU g^?1 of this isolate for 15 days. An other group of similar characteristics received the non‐supplemented commercial diet. After the challenge, the mortality of the fish receiving the diet supplemented with the potential probiotic was significantly lower than that observed in the groups of fish receiving the non‐supplemented commercial diet.     

Effects of dietary inclusion of peas,chickpeas and faba beans on growth,feed utilization and health of gilthead seabream (Sparus aurata)

Three legumes [field peas (P), chickpeas (CP) and faba beans (B)] at two inclusion levels [170 g kg^?1 (L) and 350 g kg^?1 (H)] were evaluated in a 13‐week experiment with triplicate groups of 92.6 ± 5.0 g gilthead seabream (Sparus aurata L.). A control diet included wheat meal, fishmeal (FM) and a mixture of plant ingredients as protein sources. Diets were formulated to be isonitrogenous and isoenergetic and processed in a twin‐screw extruder. Restricted feeding was applied (15 g kg^?1 of body weight) and growth, haematology and histology parameters were evaluated. Decreased, but not significant, growth values were observed for all diets including legumes compared to the control. Poorer feed conversion ratio values were observed for both P diets and for high level B diet. Liver glycogen increased with increasing starch level, but hepatosomatic index did not differ significantly for any of the diet treatments. Histological examination of internal organs showed no pathological abnormalities that could be related to nutritional treatment. The study indicated that the tested legumes are ingredients that could be used in farmed seabream diets up to 350 g kg^?1 without negative effects replacing other carbohydrate sources and part of FM.     

Virulence factors and pathogenicity of Hafnia alvei for gilthead seabream, Sparus aurata L

Virulence factors (eae gene, haemolytic capacity, fimbriae, resistance to the bactericidal effect of serum, siderophore production) and pathogenicity for gilthead seabream, Sparus aurata L., were analysed for 23 Hafnia alvei strains. None of the strains used in LD50 studies were lethal for seabream at doses as high as >10(8) cfu mL(-1). In chronic challenge studies differences in severity of the inflammatory response were observed between strains. On the basis of correlation of the inflammatory response to different strains of H. alvei in seabream with those virulence factors studied, it was only possible to establish a positive correlation between pathogenicity and resistance to the bactericidal effect of fish serum. Gilthead seabream is thus a species with considerable resistance to experimental infection with H. alvei. The bacterium does, however, have the capacity to remain viable in seabream for up to 3 months, without any clinical signs. Hafnia alvei is a well-recognized human and animal pathogen. Thus, as the pathogen can coexist with aquaculture operations, cultured gilthead seabream could represent a risk to human health as a carrier in some circumstances.     

Effects of the dietary protein : lipid ratio on growth and nutrient utilization in gilthead seabream (Sparus aurata L.)

Gilthead seabream Sparus aurata L. (initial mean body weight: 42.5 g) were fed four experimental diets containing either 47 or 51% of dry matter (DM) as protein and either 15 or 21% as lipid for 12 weeks. Each diet was hand-distributed to triplicate groups of 60 fish, three times a day until satiation. The digestibility coefficients of the dietary components were determined using chromic oxide as a marker. The levels of protein or lipid in the diets did not affect the digestibility. Fish regulated their feed intake and attained the same weight at the end of the experiment. However, feed efficiency varied between diets, with best values obtained with both diets containing 21% lipid. When diets contained only 15% lipid, feed efficiency increased with dietary protein level. Nitrogen retention was significantly higher with high fat diets regardless of dietary protein level. Neutral lipid deposition was significantly higher in liver for diets rich in lipids. It was elevated in muscle only in fish fed the diet containing 47% protein and 21% lipid and this deposition in muscle contributed to a significant increase in body fat content. Phosphorus load to the environment, measured as percentage retention of ingested or digestible phosphorus, was significantly lower with both diets higher in lipids.     

Evaluation of micronized lupin seed meal as an alternative protein source in diets for gilthead sea bream Sparus aurata L. juveniles

A 12‐week growth trial was performed to evaluate the effect of lupin seed meal as a protein source in diets for gilthead sea bream (Sparus aurata) juveniles. Six experimental diets were formulated to be isonitrogenous and isoenergetic and to contain 10%, 20% and 30% of raw lupin (Lupinus angustifolius) seed meal protein or 20% and 30% lupin (L. angustifolius) seed meal processed by infrared radiation (micronized) in place of fish meal protein, the only protein source of the control diet. Fish accepted all diets well and no significant differences in feed utilization among groups were noticed during the trial. Final weight of fish fed the experimental diets was identical or higher than the control group. Final weight of fish fed diets including 20% micronized lupin protein was even significantly higher than that of fish fed the fish meal‐based control diet. Moreover, at the same dietary lupin seed meal protein inclusion levels, final weight of fish fed diets including micronized lupin was significantly higher than with raw lupin. A trend was also noticed for a decrease of final weight with the increase in lupin seed meal in the diets. At the end of the trial no significant differences in proximate whole‐body composition, hepatosomatic and visceral indices were observed among groups. It is concluded that lupin seed meal can replace up to 30% fish meal protein in diets for gilthead sea bream juveniles with no negative effects on growth performance. Furthermore, micronization of lupin seeds improves its dietary value for gilthead sea bream juveniles. At the same dietary lupin inclusion levels, diets including micronized lupin seeds promote significantly higher growth rates than raw lupin seeds.     

Effects of dietary dehydrated lemon peel on some biochemical markers related to general metabolism,welfare and stress in gilthead seabream (Sparus aurata L.)

Dehydrated lemon peel (DLP) at two levels (1.5% and 3%) was included for 30 days in the diet of gilthead seabream (Sparus aurata L.) and after 15 and 30 days the serum and skin mucus of fish were analysed. In serum, both experimental diets led to an initial decrease in glucose and lactate after which the levels became comparable to the control. Dietary DLP modulated the activity of transaminases, alanine aminotransferase and aspartate aminotransferase. The total antioxidant status showed a progressive increase in relation to levels of inclusion of DLP and time of administration (p < .05). In skin mucus, some biomolecular markers related to general stress, oxidative stress and apoptosis exhibited variations that suggest an adaptive response of fish to the new metabolic situation, resulting from the experimental diet. The inclusion of DLP in the diet seems to positively modulate the central metabolism and welfare of gilthead seabream.     

Debaryomyces hansenii L2‐enriched diet enhances the immunity status,gene expression and intestine functionality in gilthead seabream (Sparus aurata L.)

This study evaluates the effects of the dietary administration of the live yeast Debaryomyces hansenii strain L2 on the immune responses of gilthead seabream for 4 weeks. Cellular immune parameters were measured from serum and head‐kidney leucocytes respectively. The expression levels of immune‐associated genes were quantified by real‐time polymerase chain reaction. In addition, the profile of intestinal microbiota was studied by denaturing gradient gel electrophoresis. The results showed that seabream fed a diet containing D. hansenii had significantly increased cellular immune parameters. The yeast‐supplemented diet up‐regulated the expression of most seabream genes at week 2 and down‐regulated all of them at week 4, except in the head‐kidney. Finally, a reduction in the diversity of the intestinal microbiota was detected in those specimens receiving the yeast‐supplemented diet. These results support the idea that the live yeast D. hansenii strain L2 stimulates the immune system of gilthead seabream.     

Effect of fishmeal replacement by poultry by‐product meal on growth performance,proximate composition,digestive enzyme activity,haematological parameters and gene expression of gilthead seabream (Sparus aurata)

Two feeding trials examined the replacement of fishmeal (FM) with poultry by‐product meal (PBM) in the diet of juvenile Sparus aurata. In Feeding trial I (100 days), three diets were formulated, where FM protein was replaced by 50% (PBM50) and 100% (PBM100) PBM, while in Feeding trial II (110 days), four diets were formulated using the same FM control diet, but FM was replaced at lower levels: 25% (PBM25), and 25% (PBM25 + ) and 50% (PBM50 + ) with the supplementation of lysine and methionine amino acids. PBM protein can successfully replace 50% of FM protein in the diet of S. aurata without adverse effects on survival, feed intake, growth performance and feed utilization, given that the diet is balanced with lysine and methionine. The proximate composition of body and muscle was unaffected by the diet, but the total FM replacement resulted in reduced lipid and energy contents in fish. A fifty per cent FM replacement by PBM did not affect haematological parameters indicating a good fish health. Similarities in trypsin and chymotrypsin activities with FM‐fed fish suggest a high digestibility of PBM. High dietary levels of PBM reduced the liver gene expression of GH/IGF axis and of cathepsin D suppressing fish growth and modulating the protein turnover.     

Fish oil replacement by different microalgal products in microdiets for early weaning of gilthead sea bream (Sparus aurata,L.)

The aim of this study was to determine if algal products rich in DHA or ARA are able to completely replace fish oil in microdiets for marine fish larvae, gilthead seabream and if extra supplementation with EPA may further enhance larval performance. For that purpose, 20 day‐old gilthead seabream larvae of 5.97 ± 0.4 mm mean total length and 0.12 ± 0.001 mg mean dry body weight were fed with five microdiets tested by triplicate: a control diet based on sardine oil; a diet containing AquaGrow^® DHA (diet DHA) to completely substitute the sardine oil; a diet containing AquaGrow^® ARA (diet ARA); a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA to completely substitute the fish oil; and, a diet containing both products, AquaGrow^® DHA and AquaGrow^® ARA, together with an EPA source. Temperature, air and salinity activity tests were also performed to detect larval resistance to stress. At the end of the experiment, final survivals did not differ among groups. The microorganism produced DHA was able to completely replace fish oil in weaning diets for gilthead seabream without affecting survival, growth or stress resistance, whereas the inclusion of microorganism produced ARA did not improve larval performance. Moreover, addition of EPA to diets with total replacement of fish oil by microorganism produced DHA and ARA, significantly improved growth in terms of body weight and total length. The results of this study denoted the good nutritional value of microorganisms produced DHA as a replacement of fish oil in weaning diets for gilthead seabream, without a complementary addition of ARA. However, dietary supplementation of EPA seems to be necessary to further promote larval performance.     

Influence of dietary levels of soybean meal on the performance and gut histology of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

The present study was performed to determine the effect of soybean meal (SBM) on the performance and gut histology of gilthead sea bream and European sea bass. Three isonitrogenous and isolipidic extruded diets (crude protein, 470 g kg⁻¹ diet; crude fat, 200 g kg⁻¹ diet) were formulated containing 0 (0 SBM), 180 (180 SBM) and 300 (300 SBM) g kg⁻¹ diet SBM and tested on both species in two separate experiments. Fish at an initial average weight of around 18 g were randomly allocated to 800 L square tanks connected to a closed recirculating system. The trials lasted 80 days for sea bream and 89 days for sea bass. Fish were hand-fed to apparent satiation. Increasing the level of SBM had no significant effects on the specific growth rate, feed intake and feed conversion rate in both the species. In the sea bream distal intestine, lamina propria was moderately and diffusely expanded in some fish due to an increase in cell infiltration represented by mononuclear cells, this finding being more frequent in animals fed diet 300 SBM. No other morphological alterations in intestinal folds, enterocytes or other inflammation signs were noticed in the sea bream distal intestine of any group. No histological differences were found in sea bass in any experimental group.     

Pigmentation of gilthead seabream, Sparus aurata (L. 1875), using Chlorella vulgaris (Chlorophyta, Volvocales) microalga

A feeding experiment was conducted over 9 weeks with seven groups of 30 (fish per group) unpigmented gilthead seabream, Sparus aurata (L. 1875) (initial mean weight = 145.2 ± 12.3 g). Three experimental diets were prepared by adding to a basal diet free of carotenoid (final pigment content of around 40 mg per kg feed): (i) a biomass of the carotenogenic Chlorella vulgaris (Chlorophyta, Volvocales); (ii) a synthetic astaxanthin; and (iii) a mixture (1:1) of microalgal biomass and synthetic astaxanthin. At 3‐week intervals, five fish were sampled from each tank for total carotenoids analysis in skin and muscle. The carotenoid pigments (total amount = 0.4%) identified in the carotenogenic alga were lutein (0.3%), β‐carotene (1.2%), canthaxanthin (36.2%), astaxanthin, free and esterified forms (55.0%), and other pigments (7.3%). Carotenoid pigments were significantly deposited in the four skin zones studied during the feeding trial: the forefront between the eyes, the opercule, along the dorsal fin and in the abdominal area. In the muscle, regardless of the astaxanthin source, the amount of carotenoids measured was very low (less than 1 mg kg^?1) and differences not significant. Moreover, no muscle pigmentation was evident, and there was no variation in the amount of carotenoid analysed in skin tissue, through the trial, for each treatment. It was concluded that supplementing the feed with C. vulgaris would be an acceptable practice in aquaculture to improve the market appeal of the gilthead seabream.     

Effect of diets containing a purified soybean trypsin inhibitor on growth performance,digestive proteases and intestinal histology in juvenile sea bream (Sparus aurata L.)

Juvenile sea bream were fed on diets containing 0.0, 2.0 or 4.0 g kg^?1 of a soybean trypsin inhibitor (SBTI) for 30 days. The growth performance, total protease activity and intestinal histology were studied after 0, 15 and 30 days of dietary treatment. No significant differences were found in the weight gain, specific growth rate (SGR) and feed conversion rate in fish fed on inhibitor‐supplemented diets when compared with those fed on an inhibitor‐free diet. Only the SGR at day 15 decreased significantly with protease inhibitor inclusion, although this effect was not observed at day 30. In relation to proteolytic activity at day 15, the total protease activity in the distal intestine decreased in fish fed on inhibitor‐supplemented diets. Zymograms of these extracts showed that the SBTI reduced the intensity of some proteolytic fractions in the distal intestine. A noticeable reduction in the protease activity of the intestinal content in fish fed on the highest level of soybean inhibitor (4.0 g kg^?1) was also observed. However, at day 30, the inhibition effect on these active bands was not detected, and the total protease activity was similar to that in fish fed on an inhibitor‐free diet. Histological examination revealed no perceptible differences in the intestinal structure between any of the diet groups. In addition, all fish were maintained under experimentation for 10 more days and fed on an inhibitor‐free diet to determine whether the possible effects caused by the protease inhibitor could be reverted. The administration of SBTI‐supplemented diets did not affect sea bream growth performance or intestine histology after 30 days, and only a decrease in the total alkaline protease activity was found at day 15.     

Evaluation of corn gluten meal as a protein source in diets for gilthead sea bream (Sparus aurata L.) juveniles

A 12‐week growth trial was conducted to evaluate corn gluten meal as an alternative protein source to fish meal in diets for gilthead sea bream juveniles. The experimental diets were formulated to be isonitrogenous and isoenergetic and to have 20%, 40%, 60% and 80% of fish meal protein, the only protein source in the control diet, replaced by corn gluten meal. At the end of the growth trial only the group fed the diet with 80% corn gluten protein exhibited significantly reduced growth and feed efficiency compared with the fish meal‐based diet. This was most likely due to a dietary amino acid deficiency in that diet. A trend was noticed for feed efficiency to improve with the replacement of fish meal protein in the diets up to 60%. There were no significant differences among groups in protein and energy retention (as percentage of intake). At the end of the trial whole body water content of the experimental groups was significantly lower and the lipid content of groups including 60% and 80% corn gluten protein was significantly higher than that in the control. No other differences were observed in whole body composition among groups. Apparent digestibility coefficients (ADC) of the diets were evaluated in a separate trial. The ADC of dry matter of the experimental diets was significantly higher than in the control diet; there were no significant differences among diets in the ADC of energy and protein, except for the ADC of protein of diet with 80% corn gluten protein, which was significantly lower than the control. The results of this study indicate that corn gluten meal can replace up to 60% fish meal protein in diets for gilthead sea bream juveniles with no negative effects on fish performance.