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.     

Effect of diet composition on nutrient digestibility and digestive enzyme levels of gilthead sea bream (Sparus aurata L.)

An experiment was conducted to evaluate the effects of dietary composition on apparent digestibility coefficients of protein, fat and carbohydrate of gilthead sea bream. In addition α‐amylase and proteolytic activities were measured in the digestive tract of fish, held at 20±1°C. Six experimental diets were formulated containing approximately 40%, 45% and 50% protein, 11% and 21% fat and a starch level which fluctuated from 14% to 36%. Fish having an average weight of 100–130 g were used, fed at 1.5% of their body weight daily, and digestibility was measured using an indirect method. Enzyme activities were measured in the digestive tract of fish, fed diets containing a combination of 40%, 50% protein with 11%, 21% fat at 0.5, 5, 10, 24 h after a single meal. Starch digestibility was reduced with its level in the diet. It also negatively affected fat digestibility as well as protein digestibility, the last only at the high fat level. Dietary fat level had a negative effect on starch and protein digestibility. Fat affected also strongly α‐amylase levels in the digestive tract, while its effect on protease activity was of smaller magnitude. These results indicate that digestive enzyme activities and nutrient digestibility values are affected by dietary composition, carbohydrates and fat indicating the strongest effect.     

Transmission of lymphocystis disease virus to cultured gilthead seabream,Sparus aurata L., larvae

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR‐hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF‐1 cells. Whole‐mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV‐positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR‐hybridization) or in larvae (PCR‐hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR‐hybridization, ISH and IHC. After feeding on LCDV‐positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.     

Effect of phytase supplementation of a plant‐based diet on phosphorus and nitrogen bioavailability in sea bream Sparus aurata

To assess to what extent addition of phytase to a plant‐based diet results in spatio‐temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant‐based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant‐based diets used in fish aquaculture.     

Characterization and functional properties of digestive proteases in two sparids; gilthead seabream (Sparus aurata) and common dentex (Dentex dentex)

Digestive proteases present in two sparids, seabream (Sparus aurata) and common dentex (Dentex dentex), have been characterized using both biochemical and electrophoretic techniques. Although optimum pH and temperature for maximum activity of both acid and alkaline proteases were similar in the two species, important differences in total activity, as well as in thermal and pH stability were found. Specific inhibitors and SDS-PAGE zymograms were used to clarify such differences. Evidences support the existence of a more active and complex protease set in dentex. Results are discussed from the perspective of their application to the formulation of feeds for each species.     

A behavioural approach of dominance establishment in tank‐held sea bream (Sparus aurata L.) under different feeding conditions

Dominance is a strong behaviour exhibited by farmed species that very often impinges on fish growth and welfare. This study presents a behavioural approach of dominance, where colour pattern differentiation was tested as a signal of dominance presence in small sea bream population. The phenotype of dominance was first described in detail, referring to vertical dark stripes and splayed fins. Fish were kept in triplicate tanks for 21 days, during which they were exposed to three feeding conditions (well fed, limited fed and no‐fed) and continuous video recordings. Each tank was stocked with 15 individuals (~30.34 ± 1.70 g), and they were confined to the half volume of the tank via a removable net pen. Percentage of dominants per population was found up to 40%, while duration was calculated to 53.52 ± 7.44 s. Dominance behaviour was further quantified via colour pattern differentiation on sea bream body (CDA, contrast of dominance appearance). The results demonstrated that the body colour of sea bream is directly linked to species social hierarchies and such variations are visual signal of dominance rank inside the population. The fish feeding conditions had an influence on the dominance presence, but not on the dominance rank (as measured by the CDA) between fish groups. The described study provides state‐of‐the‐art knowledge on sea bream dominance. In relevance to aquaculture, dominance quantification would be a reliable tool to evaluate non‐equal food distribution in tank‐held populations, thus, avoid non‐regulate growth performance of fish before transfer to sea‐cage installations.     

Genetic variation in farmed populations of the gilthead sea bream Sparus aurata in Greece using microsatellite DNA markers

Genetic variation in seven reared stocks of gilthead sea bream Sparus aurata, originating from Greek commercial farms, was assessed using five polymorphic microsatellite markers and was compared with that of two natural populations from the Ionian and the Adriatic Seas. The total number of alleles per marker ranged from 11 to 19 alleles, and hatchery samples showed the same levels of observed heterozygosity with samples from the wild but substantially smaller allelic diversity and expected heterozygosity. The global genetic differentiation for the cultivated samples was significant as indicated by F_st analysis, which might indicate random genetic drift and inbreeding events operating in the hatcheries. On the contrary, no significant difference was found between the two wild populations. Population pairwise tests between farmed and wild stocks were also significant, with the exception of one hatchery sample, the Central Greece 1, which was not significantly different from the two wild samples perhaps due to its recent use in aquaculture from wild‐caught animals. The UPGMA tree topology grouped the wild samples together with the Central Greece 1 stock, and showed a clear division between wild and farmed sample sets for the six remaining hatchery samples. Knowledge of the genetic variation in S. aurata cultured populations compared with that in the wild ones is essential for setting up appropriate guidelines for the proper monitoring and management of the stocks either under traditional practices or for the implementation of selective breeding programmes.     

Total nitrogen,carbon and phosphorus digestibility in gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed with conventional and organic commercial feeds: implications for particulate waste production

Organic fish farming is postulated as a more environmentally friendly activity than conventional activities. However, environmental sustainability as regards waste output has not been addressed. In this respect, the most important environmental problems associated with fish farming are those related with the discharge of particulate wastes. In this study, total nitrogen, carbon and phosphorus digestibility was estimated in a wide size range of gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) fed a commercially available conventional diet and an organic feed, as a previous step to assessing particulate waste output. Neither fish size nor feed nutrient content was seen to influence nutrient digestibility in either species. The organic diet provided higher carbon but lower nitrogen and phosphorus digestibility than conventional feed in both species. Simulations under real ongrowing conditions resulted in lower values for nitrogen and higher values for carbon particulate wastes when conventional feed was supplied to both species. Particulate phosphorus wastes were 2.6‐ to 3.4‐fold higher for fish fed the organic diet. Restrictions concerning NO the raw materials used for organic diet formulation and differences in nutrient bioavailability between the diets seemed to be the underlying reason for the differences found.     

Insect larvae as feed ingredient selectively increase arachidonic acid content in farmed gilthead sea bream (Sparus aurata L.)

This work is aimed at obtaining farmed fish designed to contain targeted PUFAs. To this end, an experiment was conducted with farmed specimens of gilthead sea bream (Sparus aurata L.), which were fed meal containing different percentages of common green bottle fly (Lucilia sericata Meigen) larvae, cultured on appropriate substrates, and then the fatty acid profiles in fish muscles were determined. Results indicate that arachidonic acid content in fish muscle is significantly increased when replacing different proportions of fishmeal by larvae from L. sericata, which contains arachidonic acid up to 10.6% of total fatty acids. Thus, larvae seem to be a suitable vector for introducing target PUFA in fish muscle and it could also contribute to reduce the use of wild fish stocks for fishmeal production.     

Early assessment of gilthead sea bream (Sparus aurata) spawning dynamics by mini‐broodstocks

The implementation of any sustainable breeding program requires a maximization of effective size (Ne) to maintain inbreeding rate at minimum levels. However, the control of Ne remains a major challenge for those species whose reproduction in captivity is based on a spawning strategy where there is no control over the established matings. The present study aims to assess the spawning dynamic of gilthead sea bream (Sparus aurata) at different dates from two mini‐broodstocks (4 ♀ and 4 ♂). Here we report the use of a simple protocol based on the genotyping of fertilized eggs from a reduced set of microsatellite loci which provides a quick evaluation of daily spawning in terms of family structuring (number of active breeders and number and family size). According to our results, the daily spawning dynamics is characterized by a fluctuation in the number of active breeders, reaching maximum values (until 100%) during peak production phase. However, the proportion of possible mating (families) in a single daily spawning never exceeded more than 81%. Unbalanced parental contributions were highly frequent, with extreme cases involving the assignment of 50%–90% of the offspring to a single family. Therefore, the use of mini‐broodstocks and the grouping of spawning harvested at different dates are proposed as strategies to minimize the inbreeding risk by providing a more optimal pattern of family structuring (increase in the number of families obtained and a more balanced parental contributions) that entails a more conservative Ne/N ratio.     

Microsatellites markers to depict the reproductive and genetic patterns of farmed gilthead seabream (Sparus aurata): illustration by a case study on mass spawning

In the absence of breeding strategy, natural spawning constitutes the breeding ground for fish farmers to empirically manage their commercial broodstock. In this context, we used six microsatellite markers to characterize the genetic pattern of six commercial seabream broodfish tanks having a common history spanning four generations. The progeny of one tank single‐day mass‐spawning event, reared in two different environments, was used to estimate the genetic parameters for body weight. Limited genetic differentiation was observed among broodfish groups. A panel of nine loci allowed us to unambiguously assign 95.4% of the offspring (1692) and identify 37 parents (65% of the total broodfish). The limited effective population size (N_e = 15.3) was due to the elevated variance of parental contributions and to broodfish failing to contribute to the progeny. The fluctuation of the allele frequency highlighted the risks of genetic drift and reduction in the heterozygosity in the next generations. Heritability for body weight was moderate at commercial size (0.40 ± 0.10) and the high genetic correlation at later stages laid the groundwork for precocious selection criteria for growth. The discussion opens on the opportunity to use mass spawning for selective breeding.     

Study of the dose of thyme essential oil in feed to prolong the shelf life of gilthead seabream (Sparus aurata)

The effect of thyme essential oil (Thymus zygis, subspecies gracilis) on the quality and shelf life of gilthead seabream was studied, when added to the diet: a control diet and four experimental diets (T500, T1000, T1500, T2000) with 500, 1000, 1500 and 2000 mg kg^?1 of thyme essential oil, respectively. After 12 weeks of experimentation, the fish were stored on ice at 4 °C for 0, 7, 14 and 21 days. Physical–chemical, microbiological and sensory analyses were carried out at each sampling point to determine the degree of deterioration. A dose‐dependent effect was observed on the colour, TBARS and total volatile basic nitrogen during the storage. Microbiological counts were lower for Enterobacteriaceae and coliforms at high doses of the essential oil. The sensory analysis showed an effect on the quality index that was not dependent on the dose, which extended the shelf life of gilthead seabream from 17 to 18 days for all doses.     

Morphological and histological characterization of an ectopically mineralized structure in a gilthead sea bream Sparus aurata with opercular deformation

In this study, we describe an abnormal ectopically mineralized structure (EMS) that was found inside the skull of a juvenile Sparus aurata that also showed a bilateral opercular deformation. The overall phenotype and tissue composition were studied using micro‐CT scanning and histological analyses. The ectopic structure occupies a large volume of the brain cavity, partially extruding into the gill cavity. It shows a dense mineralization and an extracellular matrix‐rich phenotype, with variation in both the morphology and size of the cell lacunae, combined with an irregular fibre organization inside the matrix. This study is the first to report such an EMS in a juvenile teleost fish, where the tissue does not resemble any other connective tissue type described in bony fish so far. The tissue phenotype seems to rule out that the EMS corresponds to a tumorous cartilage. Yet, it is rather reminiscent of a highly mineralized structure found in cartilaginous fish, where it is suggested to be associated with damage repair.     

Plant protein and vegetable oil‐based diets modulate gilthead seabream (Sparus aurata) muscle biochemical status and proteolytic enzymes at an early postmortem stage

A 70 days feeding trial was carried out on juvenile gilthead seabream (Sparus aurata) (mean initial body weight: 362 ± 59 g) to evaluate whether the simultaneous replacement of fishmeal and fish oil by vegetable ingredients affected the muscle's proteolytic potential. Fish were fed either a fishmeal/fish oil diet, a fishmeal/vegetable oil diet, a plant protein/fish oil or a plant protein/vegetable oil diet. The use of dietary plant proteins resulted in lower sulphated glycosaminoglycans' content and lower glycogen phosphorylase activity in the muscle, while both plant proteins and vegetable oil increased muscle pH and reduced cathepsin B activity. Our results indicate that high replacement of fish meal and/or fish oil with plant ingredients has a significant impact on early postmortem metabolic processes and proteolytic potential of seabream muscle, especially when replacing both fish meal and fish oil.     

Quality of reared gilthead sea bream (Sparus aurata) during ice storage,as affected by dietary fish oil substitution; an instrumental and sensory designation approach

The fillet fatty acids (FAs) and volatile compounds (VCs) of gilthead sea bream, fed either fish oil‐based diet (FO) or plant oils (PO), were studied in dorsal and ventral fillet parts over a 12‐day ice storage. Fillet FA reflected the FA composition of the respective diets. Monounsaturated FAs were reduced with storage, while no FA differentiations occurred between fillet parts. VCs varied between diet treatments, with food chain‐transported compounds (α‐pinene and dimethylsulphide) showing higher abundance in the FO group. VCs proposed as fish spoilage indicators (trimethylamine, pentanal, propanal, 3‐methylbutanal and 1‐penten‐3‐ol) increased with storage, while concentration of carbon disulphide associated with fresh fish aroma decreased during the same period. VCs varied between fillet parts, with ventral part exhibiting higher concentrations in fat‐soluble (terpenes, aromatic hydrocarbons) and spoilage‐associated VCs. Sensory analysis revealed no significant differences between diet groups with the exception of fattiness, which was found significantly higher in the plant oil group.     

Growth and ontogenetic development of digestive functionality in black Amur bream (Megalobrama terminalis)

The function of digestive physiology during ontogenetic development is essential to ensure high survival and growth rates. In order to evaluate the digestive physiological capacity of the black Amur bream (Megalobrama terminalis), changes of morphology and digestive enzyme activity (trypsin, lipase, amylase, pepsin, leucine aminopeptidase and alkaline phosphatase) in larvae were examined from hatching to 40 days after hatching (DAH). Results indicated that fluctuation patterns differed between the total and specific activities of the digestive enzymes. The total activities of these six enzymes gradually increased throughout the fish growth. The specific activity of trypsin peaked at 5 DAH and then decreased dramatically, while it increased remarkably again from 8 to 10 DAH and remained stable level after 20 DAH. Pepsin activity was first examined in M. terminalis at 15 DAH and gradually elevated towards the end of the experiment. The specific activity of lipase displayed obvious peaks at 5 and 20 DAH. For the amylase, its specific activity reached plateau at 4 DAH, underwent sharp decrease, and remained stable after 20 DAH. In addition, we found that the specific activity of alkaline phosphatase raised significantly from hatching to 5 DAH, and tended to keep slight fluctuation after 15 DAH. From the above, we concluded that the specific activities of digestive enzymes in the larvae varied constantly from 3 to 20 DAH and turned relatively stable after 20 DAH. The present study provides effective information that is useful to improve the seedling cultivation and the technology for healthy breeding.     

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.     

Growth,digestive and absorptive abilities and antioxidative capacity in the hepatopancreas and intestine of young grass carp (Ctenopharyngodon idellus Val.) fed graded levels of dietary manganese

To study the effects of manganese on growth performance, digestive and absorptive abilities, as well as the antioxidative capacity in the hepatopancreas and intestine, young grass carp (Ctenopharyngodon idellus Val.) (264 ± 1 g) were fed diets containing graded levels of manganese at 3.65 (control), 8.62, 13.48, 18.24, 22.97 and 27.86 mg kg^?1 diet for 8 weeks. Per cent weight gain (PWG) and feed intake were the poorest in fish fed the basal diet (P < 0.05). The activities of trypsin, lipase and alkaline phosphatase in the intestine were significantly enhanced with dietary manganese level at 13.48 mg kg^?1 diet (P < 0.05). Additionally, in the hepatopancreas and intestine, the protein carbonyl and malondialdehyde contents were the lowest in fish fed the diet with dietary manganese level at 13.48 mg kg^?1 diet (P < 0.05), while the anti‐hydroxyl radical capacities, manganese superoxide dismutase (MnSOD), glutathione peroxidase and glutathione‐S‐transferase activities were significantly enhanced with dietary manganese level at 13.48 mg kg^?1 diet (P < 0.05). Moreover, the catalase activity and glutathione content in the intestine were the highest in fish fed the diet with dietary manganese level at 18.24 mg kg^?1 diet (P < 0.05). These results indicated that optimum dietary manganese promoted growth, enhanced the digestive and absorptive abilities, and improved the antioxidative capacity in young grass carp. Based on the quadratic regression analysis for PWG and intestinal MnSOD activity, the manganese requirements for young grass carp with the initial body weight of 264 g were 16.91 and 18.21 mg kg^?1 diet respectively.     

Ontogenic changes in the digestive enzyme activities and the effect of different starvation duration on the digestive enzyme activities of larval red swamp crayfish (Procambarus clarkii)

This study was performed to determine the effect of starvation and delayed feeding on activities of digestive enzymes and alkaline phosphatase (ALP) of larval red swamp crayfish (Procambarus clarkii), so as to reveal the tolerance to prolonged starvation and the recovery of digestive enzymes after delayed feeding in larval and juvenile P. clarkii. In the control group, activities of trypsin and ALP increased significantly (p < .05) with day‐age and then kept constant at 24 days after hatching (DAH) and 10 DAH, respectively, whereas the activities of amylase and pepsin increased firstly then decreased with day‐age, and the activity of lipase increased firstly then decreased and then increased again during the development period of juvenile P. clarkii (1–31 DAH). In the group with continuous starvation (CS), activities of pepsin and lipase both decreased (p < .05) after fasting, and the activities of pepsin, lipase and trypsin in the groups with delayed feeding all increased (p < .05) and recover to the levels of the control group after food supply. However, the activity of amylase increased (p < .05) in the CS group, and it decreased to normal level after food supply. The ALP activity did not significantly (p > .05) vary after starvation, whereas it decreased in the groups with delayed feeding after 1 day of food supply, and then increased back to the level similar with the control group. Results from this study could provide information for diet preparation and feeding regime in larval and juvenile red swamp crayfish culture.