Nano-delivery of trace minerals for marine fish larvae: influence on skeletal ossification,and the expression of genes involved in intestinal transport of minerals,osteoblast differentiation,and oxidative stress response

Currently, the larviculture of many marine fish species with small-sized larvae depends for a short time after hatching, on the supply of high-quality live zooplankton to ensure high survival and growth rates. During the last few decades, the research community has made great efforts to develop artificial diets, which can completely substitute live prey. However, studies aimed at determining optimal levels of minerals in marine larvae compound feeds and the potential of novel delivery vectors for mineral acquisition has only very recently begun. Recently, the agro-food industry has developed several nano-delivery systems, which could be used for animal feed, too. Delivery through nano-encapsulation of minerals and feed additives would protect the bioactive molecules during feed manufacturing and fish feeding and allow an efficient acquisition of active substances into biological system. The idea is that dietary minerals in the form of nanoparticles may enter cells more easily than their larger counterparts enter and thus speed up their assimilation in fish. Accordingly, we evaluated the efficacy of early weaning diets fortified with organic, inorganic, or nanoparticle forms of trace minerals (Se, Zn, and Mn) in gilthead seabream (Sparus aurata) larvae. We tested four experimental diets: a trace mineral-deficient control diet, and three diets supplemented with different forms of trace minerals. At the end of the feeding trial, larvae growth performance and ossification, and the level of expression of six target genes (SLC11A2β, dmt1, BMP2, OC, SOD, GPX), were evaluated. Our data demonstrated that weaning diets supplemented with Mn, Se, and Zn in amino acid-chelated (organic) or nanoparticle form were more effective than diets supplemented with inorganic form of minerals to promote bone mineralization, and prevent skeletal anomalies in seabream larvae. Furthermore, nanometals markedly improved larval stress resistance in comparison to inorganic minerals and upregulated mRNA copy number of OC gene. The expression of this gene was strongly correlated with mineralization degree, thus confirming its potency as a good marker of bone mineralization in gilthead seabream larvae.     

Effect of krill phospholipids versus soybean lecithin in microdiets for gilthead seabream (Sparus aurata) larvae on molecular markers of antioxidative metabolism and bone development

The objective of the present study was to compare the effectiveness of dietary marine phospholipids (MPL) obtained from krill and soybean lecithin (SBL) on the rearing performance and development of seabream (Sparus aurata) larvae. Larvae were fed from 16 to 44 day posthatching (dph) five formulated microdiets with three different levels (50, 70 and 90 g kg^–1) of phospholipids (PL) obtained either from an MPL or from a SBL source. Larvae‐fed MPL show a higher survival, stress resistance and growth than those‐fed SBL, regardless the dietary PL level. Overall, the increase in MPL up to 70 g kg^–1 total PL in diet was enough to improve larval gilthead seabream performance, whereas even the highest SBL inclusion level (90 g kg^–1 PL) was not able to provide a similar success in larval growth or survival. Inclusion of SBL markedly increased the peroxidation risk as denoted by the higher TBARs in larvae, as well as a higher expression of CAT, GPX and SOD genes. Moreover, SBL tends to produce larvae with a lower number of mineralized vertebrae and a lower expression of osteocalcin, osteopontin and BMP4 genes. Finally, increasing dietary MPL or SBL lead to a better assimilation of polyunsaturated fatty acids in the larvae, n‐3HUFA (especially 20:5n‐3) or n‐6 fatty acids (especially 18:2n‐6), respectively. In conclusion, MPL had a higher effectiveness in promoting survival, growth and skeletal mineralization of gilthead seabream larvae in comparison with SBL.     

Effect of dietary microminerals in early weaning diets on growth,survival, mineral contents and gene expression in gilthead sea bream (Sparus aurata,L) larvae

In previous studies, combined inclusion of Zn, Mn and Se in early weaning diets improved larval growth, but suggested a potential toxicity by one of these elements. The present study aimed to determine the effect of the single inclusion of Zn, Mn, Se or Cu, their combination (Control+) or their absence (Control?) on larval diets. At the end of the trial, survival was significantly (p < .05) lowest in fish fed C+ diet (17.16 ± 7% mean ± SD), followed by that of larvae fed Mn diet (21.91 ± 7%). The highest survival was obtained by Cu diet (35.27 ± 15%), followed by C? diet (34.58 ± 9%). Cu and Se supplementation significantly improved total length and body weight, in comparison with the C? fish. On the contrary, fish fed Mn and C+ showed the lowest growth. Supplementation with Zn or Cu significantly increased CuZnsod, whereas gpx was significantly upregulated in fish fed Se and C+ diets. ARA/EPA level was significantly highest and DHA/EPA lowest in larvae fed the Cu diet in fish fed C+ diet. The results pointed out the importance of supplementation with Cu, as well as Se and Zn, on early weaning diets for gilthead sea bream, and the potential toxic effect of Mn.     

Rotifers enriched with iodine,copper and manganese had no effect on larval cod (Gadus morhua) growth,mineral status or redox system gene mRNA levels

It currently remains unclear if rotifers contain sufficient mineral levels to meet larval fish requirements. In this study, rotifers were enriched with a commercial enrichment (control), or with additional iodine, iodine and copper, or iodine, copper and manganese, and the effects of feeding these rotifers to Atlantic cod (Gadus morhua) larvae from 3 to 18 days post hatch were investigated. Rotifer enrichment with minerals was successful, but Mn enrichment also increased rotifer zinc levels. No differences were observed between treatments in larval growth or survival, or in the mRNA levels in the majority of the redox system genes analysed. Only Zn levels increased in cod larvae in response to mineral enrichment of rotifers. Apart from Zn, little evidence was found to suggest that cod larvae require increased concentrations above the control rotifer levels of the essential elements studied here.     

Biomarkers of bone development and oxidative stress in gilthead sea bream larvae fed microdiets with several levels of polar lipids and α‐tocopherol

Although dietary marine phospholipids are able to improve culture performance of marine fish larvae in a further extend than soybean lecithin, both types of phospholipids (PL) markedly increase oxidative risk. The inclusion of a fat‐soluble antioxidant such as the vitamin E α‐tocopherol could allow a better control of oxidative stress. The objective of this study was to determine the combined effect of graded levels of α‐tocopherol with different levels and sources of krill phospholipids (KPL) and soybean lecithin (SBL) on growth, survival, resistance to stress, oxidative status, bone metabolism‐related genes expression and biochemical composition of sea bream larvae. Sea bream larvae were completely weaned at 16 dph and fed for 30 days seven microdiets with three different levels of PL (0, 40 and 80 g kg^?1 diet) and two of α‐tocopherol 1500 and 3000 mg kg^?1 diet. Sea bream larvae fed diets without PL supplementation showed the lowest survival, growth and stress resistance, whereas increase in PL, particularly KPL, markedly promoted larval survival and growth. However, feeding SBL markedly increased TBARs and GPX gene expression increasing the peroxidation risk in the larvae. Besides, KPL inclusion improved incorporation of n‐3 HUFA and, particularly, EPA into larval tissues, these fatty acids being positively correlated with the expression of BMP‐4, RUNX 2, ALP, OC and OP genes and to bone mineralization for a given larval size class. The increase in dietary α‐tocopherol tends to improve growth in relation to the n‐3 HUFA levels in the diet, denoting the protective role of this vitamin against oxidation. Indeed, dietary α‐tocopherol decreased the oxidative stress in the larvae as denoted by the reduction in larval TBARs contents and gene expression of SOD and CAT, but not GPX. Thus, increase in dietary α‐tocopherol effectively prevented the formation of free radicals from HUFA, particularly EPA, but did not affect the incidence of bone anomalies or the expression of genes related to osteogenetic processes.     

Changes in mineral concentrations in amberjack Seriola dumerili larvae during seed production: high concentrations of certain minerals in rotifers do not directly affect the mineral concentrations in larvae

This study examined the changes in mineral concentrations in amberjack Seriola dumerili larvae during seed production taking into consideration the mineral concentrations of rotifers in the larval rearing tanks (tank rotifers), since the concentrations of certain minerals in tank rotifers have been reported to be very high. In the present study, the concentrations of Ca, Mg, Fe, Zn and Mn in tank rotifers were generally higher than in freshly enriched rotifers. The extent of the increase in tank rotifers varied depending on the mineral, larval production facility and larval production trial. Concentrations of Ca in the larvae increased as they grew, those of P and Mn increased up to ca. 30 days post-hatching (DPH), those of Mg and Fe increased up to ca. 20 DPH and then decreased, and those of Zn continuously decreased through the seed production periods (to ca. 40 DPH). However, there were no clear relationships between the mineral concentrations of the tank rotifers and the larvae. These results indicate that high concentrations of minerals in tank rotifers are not directly reflected in amberjack larvae. This suggests that amberjack larvae maintain their mineral status by controlling the uptake and/or excretion of food minerals.     

Mineral Requirements of Penaeus vannamei: A Preliminary Examination of the Dietary Essentiality for Thirteen Minerals

Diets containing a complete mineral premix or mineral premixes from which Ca, P, Na, CI, K, Mg, Mn, Fe, Zn, Cu, I, Se, or Cr were individually deleted were fed to juvenile Penaeus vunnamei (mean initial weight 79.5 mg) over a 4-week experimental period. Final mean percent weight gain ranged from 1,472 (Cu-free mineral premix) to 1,991% (Na-free mineral premix) with an average of 1,812% for all treatments. Survival during the first two week period and the second two week period was greater than 92% and 88%, respectively. There were no significant differences observed among treatments for growth and survival data.
Upon termination of the growth trial, the hepatopancreas and carapace were dissected from the shrimp and evaluated for ash, Ca, P, Na, K, Mg, Mn, Fe, Zn and Cu content. Of the minerals analyzed, the individual deletion of Mg, Mn, Fe, Zn and Cu from the mineral supplement resulted in reduced tissue mineralization in P. vannamei whereas, the deletion of Ca and P from the mineral mix did not produce significant responses. Deletion of Se from the mineral premix resulted in a significant increase in the ash content of the carapace. Based on the information provided by this and other studies, further investigation of the dietary requirements of P. vannamei for Ca, P, Fe, Mg, Mn, Zn, Cu and Se is recommended.     

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.     

Different levels of organic trace minerals in diets for Nile tilapia juveniles alter gut characteristics and body composition,but not growth

Thirty groups of twenty juveniles each of Nile tilapia (Oreochromis niloticus, initial average weight 23.93 ± 0.80 g) were fed diets containing increasing amounts (0%, 25%, 50%, 75%, and 100% of levels used by the tilapia feed industry) of organic trace mineral (Fe, Zn, Mn, Cu, Se) supplementation for 56 days. Growth was not affected by the dose of mineral supplementation. However, body protein increased in fish fed intermediate doses of organic minerals, reaching a maximum at 40.44% supplementation. Doses >50% showed mild damage to the intestinal tissue. Body Fe, Zn and Cu deposition were highest at 28.32%, 24.43% and 78.45% supplementation, respectively. Tilapia could modulate intestinal morphology when fed different doses of organic minerals. As the supplementation doses increased, hemosiderin staining increased in the distal intestine and total body Fe content, indicating that the distal region of the intestine plays an important role in iron excretion by tilapia. Supplementation of organic trace minerals at intermediate doses does not harm Nile tilapia growth, but excessive trace mineral supplementation can impair body protein retention and promote histological alterations in the gut.     

Optimum krill phospholipids content in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of krill phospholipids (KPL) for sea bream (Sparus aurata) larvae, and its influence on larval development and digestive enzymes activity. Larvae were fed five formulated microdiets with five different levels of KPL. Complete replacement of live preys with the experimental microdiets for seabream larvae produced high survival and growth rates, particularly in fish fed the highest levels of KPL. In the present study, increase in dietary KPL up to 120 g kg^?1 (100 g kg^?1 total PL) significantly improved larval survival and growth, whereas further increase did not improve those parameters. An increase in alkaline phosphatase, trypsin and lipase activity with the elevation of KPL up to 120 g kg^?1 was also found denoting a better functioning of digestive system. Besides, there was a linear substrate stimulatory effect of dietary KPL on phospholipase A2 activity. Finally, increasing dietary KPL lead to better assimilation of n‐3 HUFA especially eicosapentaenoic acid, reflected in the higher content of these fatty acids in both neutral and polar lipids of the larvae. In summary, KPL were found to be an excellent source of lipids for seabream larvae. Optimum inclusion levels of this ingredient in microdiets to completely substitute live preys at this larval age were found to be 120 g kg^?1 KPL.     

Replacing rotifers with a microparticle diet from first feeding in yellowfin seabream,Acanthopagrus latus (Houttuyn), larvae

The possibility to replace or reduce rotifer use with a microparticle diet (MPD) from first feeding in yellowfin seabream larvae was investigated. The experiment consisted of five treatments, a rotifer (100%) control treatment (100R), three experimental treatments, which received a fixed ration of MPD supplemented with 75%, 50% and 25% of the amount rotifer fed in the control treatment (treatments 75R‐MPD, 50R‐MPD and 25R‐MPD respectively), and finally a treatment with the fixed MPD ration only (treatment 0R‐MPD). The results indicated that feeding regimes 100R, 75R‐MPD and 50R‐MPD did not differ significantly in the total length, final survival and stress test resistance. The highest dry weight was achieved in treatments 100R and 75R‐MPD at the end of the experiment compared with treatments 50R‐MPD, 25R‐MPD and 0R‐MPD. The 0R‐MPD treatment showed the lowest growth and survival. Yellowfin seabream larvae were able to ingest inert food directly from the moment of first feeding.     

Effects of different dietary selenium sources (sodium selenite,selenomethionine and nanoselenium) on growth performance,muscle composition,blood enzymes and antioxidant status of common carp (Cyprinus carpio)

This experiment was conducted to compare the effects of different sources of selenium (Se) on growth, muscle Se concentration, muscle proximate composition, blood enzymes and antioxidant status of common carp, Cyprinus carpio. Organic Se (selenomethionine, SeMet), inorganic Se (sodium selenite, Na₂SeO₃), or nanoselenium were each added to the basal diet at 0.7 mg Se kg^?1 diet. Four groups of fish with an average weight of 9.69 ± 0.12 g were fed one of the experimental diets for 8 weeks. Nano‐Se diet resulted in better growth performance (P < 0.05). Fish fed with Se‐supplemented diets showed a higher (P < 0.05) Se content in the muscle and liver tissues. Fish fed nano‐Se had the highest liver glutathione peroxidase, superoxide dismutase and catalase activities (P < 0.05). Liver malondialdehyde level was significantly (P < 0.05) lower in fish fed on SeMet and nano‐Se diets as compared to control. The carp fed on nano‐Se diet had the lowest values of aspartate transaminase and alanine transaminase. Lactate dehydrogenase activity was significantly lower in fish fed on SeMet and nano‐Se diets. This study shows that nano‐Se acts more efficiently on growth performance and antioxidant defence system of common carp than organic and inorganic sources of Se.     

Effects of dietary zinc sources and levels on growth performance,tissue zinc retention and antioxidant response of juvenile common carp (Cyprinus carpio var. Jian) fed diets containing phytic acid

Plant protein‐based diets are widely used for common carp (Cyprinus carpio var. Jian). The plant source antagonist, such as phytate, negatively affects the bioavailability of trace minerals, but this aspect was not considered in previous studies on this species. A 10‐week growth trial was conducted to investigate the effects of inorganic Zinc (ZnSO₄·7H₂O, Zn‐S) and organic Zn (2‐hydroxy‐4‐(methylthio) butanoic Zinc, Zn‐M) on growth performance, Zn retention and antioxidant responses of common carp (initial weight 10.0 ± 0.03 g). Eleven semi‐purified diets containing 7 mg/kg phytic acid were prepared with 0, 5, 20, 35, 50 and 65 mg/kg Zn from Zn‐S and Zn‐M (12.8 mg/kg Zn in basal level), respectively. The results showed that either Zn‐S or Zn‐M inclusion improved growth performance, vertebral Zn deposition and antioxidant responses of fish. The Zn requirement based on WGR, vertebrae Zn and plasma SOD were 47.0, >77.8 and 48.5 mg/kg, and 63.3, 42.2 and 36.8 mg/kg, respectively, with Zn‐S and Zn‐M as Zn sources. The relative bioavailability of Zn‐M was 2.44‐ (vertebrae Zn deposition) and 1.74‐fold (plasma SOD activity) the availability of Zn‐S in the diet of common carp. The lower growth performance and poor bioavailability in vertebrae of Zn‐S group were related to the chelating of zinc with phytic acid.     

Hyperaccumulation of selenium in hybrid striped bass: a functional food for aquaculture?

One method of increasing the value of aquacultured product is to produce fillets that are fortified with minerals that are beneficial to human health – that is enhance the functionality of an already healthy product. A good candidate mineral in this regard is selenium (Se) which is of vital importance to normal metabolism in humans. In order to evaluate the dose response and tissue accumulation of supplemental dietary Se, a study was undertaken with hybrid striped bass (HSB). Animals were fed diets supplemented with either organic (0–3.2 mg kg^?1 as SelPlex^®) or inorganic (0.2 and 0.4 mg kg^?1 as sodium selenite) Se for 6 weeks. Because basal fishmeal‐based diets contained 1.22 mg Se kg^?1, doses of Se delivered equated to 1.22–4.42 mg kg^?1. At trial end, greatest weight gain was observed in fish receiving 0.2 mg Se kg^?1, irrespective of form (organic/inorganic). Se accumulation in HSB liver and fillet revealed a classical dose‐response once a threshold level of 0.2 mg Se kg^?1 was surpassed. Greatest tissue accumulation of Se was observed in fish fed the 3.2 mg Se kg^?1 level (P > 0.0001). A 100 g portion of Se‐enhanced HSB fillet would contain between 33 and 109 μg Se, amounting to a dietary intake of between 25 and 80 μg Se; a level that would satisfy present daily intake recommendations. Comparison of tissue Se levels indicated that the muscle provides a more conspicuous gauge of dietary Se dose‐response than does liver. Dietary treatments of between 0.4 and 1.6 mg organic Se kg^?1 reduced (P < 0.024) plasma glutathione peroxidase (GSH‐Px) activity. No differences were observed in ceruloplasmin, lysozyme or GSH‐Px activities between organic and inorganic Se when delivered at the 0.2 mg Se kg^?1 level. Ceruloplasmin, lysozyme and GSH‐Px levels were elevated (P ≥ 0.025) in fish fed the diet containing 0.4 mg inorganic Se kg^?1.     

Enhancement of gilthead seabream (Sparus aurata) larval growth by dietary vitamin E in relation to two different levels of essential fatty acids

The objective of this study was to determine the effect of dietary vitamin E on gilthead seabream (Sparus aurata) growth and survival, at two different highly unsaturated fatty acids (HUFAs) levels. Eighteen days old gilthead seabream larvae were fed four formulated experimental diets combining two different dietary levels of HUFAs (M: medium 2.5 + 1.5, DHA + EPA, H: high 5 + 2.5 DHA + EPA g per 100 g) with two different levels of vitamin E (M: medium 540 mg kg^?1, H: high 2900 mg kg^?1): MM, MH, HM, HH (HUFA/vitamin E). After 2‐week feeding trial, the average survival rate was 52.6% and there were no significant differences found among treatments. Increase in vitamin E up to high level markedly improved larval growth, particularly when dietary HUFA levels were lower, suggesting a higher protection value when these fatty acids are more limiting. At medium dietary HUFA levels, increase in vitamin E from medium to high level enhanced larval growth performance in terms of total length. Moreover, increase in vit E enhanced HUFAs content in the larval polar lipids denoting the anti‐oxidative effect of vitamin E.     

The effect of dietary exogenous digestive enzymes on ingestion,assimilation, growth and survival of gilthead seabream (Sparus aurata,Sparidae, Linnaeus) larvae

The success of microdiets commonly used in the cultivation of marine fish larvae is limited to serving as partial replacements for live food. This limited success is thought to be associated with a reduced digestive ability due to an incompletely developed digestive system. The enhanced growth obtained from live food has been partially attributed to the digestive enzyme activity of the food organism. The present study was designed to test the effect of an exogenous digestive enzyme incorporated. into a microdiet on the growth of Sparus aurata. Larval gilthead seabream, 20–32 days old, were fed ¹⁴C labelled microdiets containing a commercial pancreatic enzyme at different concentrations (0, 0.1 and 0.05g / 100 g dry diet). Rates of ingestion and assimilation were measured and their relationship to dry weight was determined. Our results show that the success of the microdiet as a food for larval gilthead seabream was limited by the larva's low ingestion rate which only approached its maintenance requirement. In addition, the presence of digestive enzyme in the microdiet enhanced its assimilability by 30%. Larval growth over ten days was 0, 100 and 200% on microdiet free of added enzymes, one with added enzymes and a live food regime, respectively. It is our opinion that successful development of microdiets for Sparus aurata must be based on diets improved both in digestibility and attraction to the larvae. Further studies are now underway to determine the nutritional requirements of gilthead seabream larvae using the experimental method developed in the present study. This research was carried out in partial fulfillment of the requirements for the M.Sc. degree.     

Mesocosm: A Reliable Technology for Larval Rearing of Diplodus puntazzo and Diplodus sargus sargus

Species diversification is today considered as a major issue for the sustainable development of the Mediterranean aquaculture. For successful propagation of any species however, larval rearing is considered a bottleneck and therefore the development of appropriate tools is essential. Mesocosm is a semi-intensive technology that facilitates larval rearing of several species integrating principles of both intensive and extensive aquaculture, which solves biological problems and many of their technical, human and economical consequences. The extensive (and now even the semi-extensive) strategy is used in the most critical segments of the rearing process during the early developmental stages, when larvae are still extremely weak, sensitive to intensive environment, easily stressed and difficult to feed. The intensive strategy is used as soon as larvae are considered mature enough to be reared easily using classical methods. The technology was used for the rearing of two species, with potential for aquaculture, the sharpsnout seabream (Diplodus puntazzo) and the white seabream (Diplodus sargus sargus). Three groups of each species were monitored for a period of 50–70 days post hatching. Survival for both species was about 54% at the end of the trials. Sharpsnout seabream larvae reached 19.6 ± 0.9 mm total length and 107.2 ± 31.9 mg body weight 50 days post hatching. White seabream larvae 60 days post hatching reached 32.7 ± 2.7 mm total length and 450 ± 70 mg body weight. In order to verify the economical viability of the technology, the individual production cost for each species was estimated and reached ¢0.027 for white seabream and ¢0.043 for sharpsnout seabream. Results indicate the reliability of the technology for the larval rearing of the two species.     

Skeletal Anomalies of Pacu,Piaractus mesopotamicus,Larvae from a Wild‐caught Broodstock

Skeletal anomalies in fish are frequent, representing a major problem for the aquaculture industry. The anomalies usually arise in the larval phase and worsen, persist, or recover over the ensuing stages of development. In addition to impairing larval quality, they compromise the growth, health, and food conversion of fish, lowering their economic value. This study evaluated the occurrence of skeletal anomalies in pacu larvae during the first 30 d of development. The newly hatched larvae of six breeding pairs taken from the Paranapanema River (offspring A, B, C, D, E, and F) and subjected to induced spawning were reared separately for 30 d, feeding on live prey (Artemia nauplii). Nearly 20 larvae from each breeding pair were killed and stored 1, 2, 4, 6, 8, 10, 13, 16, 19, 22, 25, and 30 days post‐hatching. The larvae were stained to differentiate cartilage and bones and examined under a stereo microscope. A total of 1310 larvae were checked for the presence (1) or absence (0) of anomalies in the head, vertebral column, and fins. Associations between growth parameters (total length [TL] vs. standard length [SL]; SL vs. vertebral column length [VL]) were obtained by regression. Correspondence analysis was used to correlate data on the different parameters evaluated. On completion of observation, larvae from pair E had the highest SL, whereas offspring F were the shortest. All types of skeletal anomalies were detected in the larvae irrespective of the offspring, affecting the normal growth of pacu larvae obtained from wild‐caught breeders.     

Visualization of musculature of the larval greater amberjack Seriola dumerili (Risso) using whole‐mount immunostaining with special reference to cranial muscles

Muscles play important roles in feeding, respiration and swimming during not only adult stage but also larval stage in fish. However, there is no information of the muscle development in larval greater amberjack, Seriola dumerili (Risso). Here, we investigated muscle development in the greater amberjack, focusing primarily on the cranial muscles from 0 days post hatch (dph) to 12 dph using a modified whole‐mount immunohistochemical staining method. We found that the muscles required for feeding develop by 3 dph, when the larvae begin to feed. Subsequently, muscle composition in the dorsal branchial arches changes to the adult form between 5 and 8 dph. At 8 dph, all the muscles required for feeding and respiration appear, whereas the dorsal, pelvic and caudal fin muscles required for swimming develop later. This report provides fundamental information on larval greater amberjack muscle development, which will enable the detection of abnormal larval muscles and improve larval rearing techniques by modifying environmental conditions.     

Optimum soybean lecithin contents in microdiets for gilthead seabream (Sparus aurata) larvae

The aim of the present study was to determine the optimum dietary levels of soybean lecithin (SBL) for seabream (Sparus aurata) larvae, and its influence on production performance and digestive enzymes activity. Larvae were fed five formulated microdiets with five levels of SBL. Complete replacement of live preys with the experimental microdiets for seabream larvae at 16 dph produced over 55% survival rates, particularly in fish fed with the highest levels of SBL. Moreover, increase in dietary SBL up to 80 g kg⁻¹ significantly improved larval growth, leading to high final total length and body weight. An increase in alkaline phosphatase activity with the elevation up to 80 g kg⁻¹ SBL was also found denoting a better maturation of the digestive system. Besides, there was a stimulatory effect of dietary SBL on PLA2 activity. Finally, increasing dietary SBL lead to better utilization of dietary highly unsaturated fatty acid, as it was reflected in their higher content in both neutral and polar lipid of the larvae. In summary, elevation of dietary SBL up to 80 g kg⁻¹ in microdiets for seabream significantly improved digestive enzymes activities, enterocyte maturation, utilization and deposition of dietary essential fatty acids and larval growth, as a consequence of a better digestion, absorption, transport and deposition of dietary nutrients.