William Koven

The effect of dietary arachidonic acid (20:4n - 6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae

Abstract The effects of high dietary docosahexaenoic acid (22:6 n −3, DHA) and varying arachidonic acid (20:4 n −6, AA) were tested on growth, survival and resistance to handling stress in 5–35 day old gilthead seabream larvae. Three enrichment treatments differing in their DHA/AA ratios were fed to rotifers ( Brachionus rotundiformis ) and Artemia nauplii. The high DHA (35.9% TFA) enrichment treatment (DHA-PL) contained no AA and included lipid from the heterotrophically grown DHA-rich dinoflagellate Crypthecodinium sp. A second enrichment treatment (AADHA), selected from an earlier screening study, supplemented the high DHA enrichment treatment with an AA-rich lipid (52% TFA) from the heterotrophically grown fungus Mortierella alpina. A third enrichment treatment (ALGA) was the commercial product Algamac 2000, which is devoid of AA, but includes approximately 12.9% of TFA as docosapentaenoic acid (DPA, 22:5 n −6). Rotifers fed the DHA-PL, AADHA and ALGA treatments demonstrated a range of DHA/AA ratios (20.9, 5.6 and 10.1, respectively) as did the Artemia nauplii (25.8, 3.7 and 4.6, respectively). The enriched rotifers were fed to larvae reared in 400 l V-tanks from day 5 to day 19 post-hatching. Following this period, larvae were exposed to controlled handling stress during transfer to 27 l aquaria, where they were then fed the enriched nauplii from day 20 to day 35 post-hatching. Although larval fatty acid profiles reflected the enrichment treatments, there were no marked differences ( P >0.05) in survival and growth in 5–19 day old larvae at the end of rotifer feeding. However, the larvae fed the AA enriched rotifers prior to the handling stress of transfer to the aquaria demonstrated daily and significantly ( P Artemia feeding than larvae fed the AA-deficient (DHA-PL) and ALGA-enriched rotifers. As larvae fed the ALGA, rotifers partially retroconverted DPA to AA in their tissues, the final survival (31.0%) in these larvae was markedly better ( P P Artemia . The results suggest that dietary AA fed prior to handling stress improved survival more effectively than when fed following handling stress. These findings imply, as well, the importance of early larval nutrition on later larval and juvenile survival during crowding, grading and other handling stressors.

Advanced DHA, EPA and ArA enrichment materials for marine aquaculture using single cell heterotrophs

Abstract Heterotrophically grown algae and fungal biomass and their residual materials from an industrial oil extraction process were used as components in marine larval and broodstock diets. Crypthecodinium sp. phospholipid extract and meal, used to enrich rotifers and Artemia nauplii, produced higher levels of DHA and higher DHA/EPA ratios in these zooplankters than Schizochytrium sp. algal whole cell preparation or fish oil-based emulsion. The improved enrichment resulted in enhanced growth of Atlantic halibut larvae, whereas several other marine larvae species (sea bream, European sea bass and striped bass) respond almost equally to all enrichment materials. In addition, a 60% replacement of menhaden oil with algal oil and meal in striped bass broodstock diets resulted in a similar growth increase to that obtained with standard commercial diets. Striped bass broodstock fish diets supplemented with an arachidonic acid (ArA)-rich oil obtained from heterotrophically grown fungi, Mortierella alpina , was shown to have significant benefits on the hatching rate of larvae. These findings demonstrate the potential of single cell heterotrophs as a partial substitute or replacement for fish-based ingredients in aquaculture diets.

The mode of action of Artemia in enhancing utilization of microdiet by gilthead seabream Sparus aurata larvae

Abstract In recent years, a great deal of interest has been generated in the development of a microdiet (MD) as an economic live food alternative for the larvae of commercially important species of marine fish. Despite the poor performance of microdiets (MD) when used exclusively to rear marine fish larvae, results were markedly improved when inert MDs were co-fed with live Artemia nauplii. This paper reviews the influence of Artemia on the enhancement of the ingestion, digestion and assimilation of MD during co-feeding with an emphasis on work carried out at the National Center for Mariculture (NCM) in Eilat, Israel. The accumulating information and findings at the NCM suggest two possible modes of influence by Artemia nauplii on the ingestion, digestion and assimilation of MD during co-feeding: (1) the remote influence on MD ingestion by visual and chemical stimuli and/or (2) the direct influence of nauplii biochemical composition on larval digestion and assimilation. The MD ingestion rates in seabream larvae when exposed to both Artemia visual and chemical stimuli, at various concentrations of Artemia nauplii, increased up to 120% as compared to ingestion rates in larvae that were offered MD alone. The free amino acids (FAA) alanine, glycine and arginine and the compound betaine were identified as the chemical stimuli for gilthead seabream larvae from the 14 metabolites found in the Artemia rearing medium. The effect of exogenous enzymes on digestion and assimilation in gilthead seabream larvae was demonstrated when pancreatin supplementation to a MD enhanced its assimilation by 30% and significantly improved growth while seabass (Dicentrarchus labrax) larvae were unaffected by a similar pancreatin supplementation suggesting species-specific proteolytic ability during larval development. The Artemia body composition may also contain substances exerting an influence on larval digestion and assimilation. MDs supplemented with various fractions extracted from Artemia nauplii i.e. neutral and polar lipid classes or a non-lipid fraction, separately and in combination, significantly increased MD assimilation by 10–20% in 22-d old larvae while the effect of the supplemented fractions diminished with age. In a series of follow-up studies the results indicated that Artemia phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and the free fatty acid (FFA) fraction influenced significantly (P

Lipid and n−3 requirement of Sparus aurata larvae during starvation and feeding

Abstract The n −3 fatty acids and the lipid class requirement of larval Sparus aurata were studied by comparing patterns of conservation and loss of lipid levels and their content of selected fatty acid groups in starved (for 6 days) and feeding (fed 17 days on Isochrysis enriched rotifers) larvae. There was a marked decrease in the neutral fraction while the reduction in the polar fraction was more moderate in both feeding and starved larvae. In starved fish the pattern of fatty acid loss (percent decrease in μg/mg of dry weight larvae) in the total, neutral and polar fractions was expressed as n −6 > n −9 > n −3. However, the polar lipid in fed fish exhibited a reverse trend that can be described as n −3 > n −9 > n −6. Docosahexaenoic acid (22:6 n −3) was also observed to be more strongly conserved than eicosapentaenoic acid (20:5 n −3) during starvation but more drawn upon in feeding. The results suggest a larval biochemical strategy to conserve the important n −3 fatty acids during starvation. However, their loss during feeding implies that Isochrysis -enriched rotifers do not provide sufficient levels of these fatty acids for growing Sparus aurata larvae.

Advances in the development of microdiets for gilthead seabream, Sparus aurata : a review

The performance of microdiets (MDs) for larvae of marine fish is frequently improved when they are co-fed with Artemia. This suggests that nutritional factors in the live food are positively influencing the ingestion, digestion and assimilation of the MD. This paper reviews recent advances in MD development on the gilthead seabream with special emphasis on studies that isolated, identified and tested these live food factors in MD with the aim of improving their performance. MD ingestion rates in gilthead seabream larvae increased up to 120% when the fish were exposed to the visual and chemical stimuli of various concentrations of Artemia nauplii. The free amino acids (FAA) alanine, glycine and arginine and the compound betaine were identified from the Artemia rearing medium as metabolites, which stimulated this larval response. Similarly, MD supplemented with phospholipids (PL), particularly phosphatidylcholine (PC), stimulated feeding activity and was consumed up to 45% better in young larval seabream. Moreover, dietary PC appears to have in parallel and/or in tandem a postprandial enhancing effect on lipoprotein synthesis, resulting in improved transport of dietary lipids from the mucosa of the digestive tract to the body tissues. Live food may also contribute exogenous enzymes to the digestion process or provide factors that stimulate larval pancreatic secretions or activate gut zymogens. Seabream larvae ingesting MD supplemented with porcine pancreatic extract (0.05% DW diet) showed a 30% increase in assimilation and demonstrated significantly (P<0.05) improved growth. Older seabream larvae showed 6.75 times more radioactivity in tissue lipids when fed 14C-triacylglycerol (TAG)-labeled MD supplemented with porcine lipase, while younger larvae demonstrated no improved assimilation. Factors in live Artemia may influence digestion by stimulating an endocrine response. This was shown when Artemia consumed by seabream larvae elicited a 300% increase in the level of the digestive hormone bombesin compared to levels in larvae given only a MD. On the other hand, liposomes containing the FAA methionine ingested by halibut juveniles elicited higher levels of the digestive hormone cholecystokinin (CCK) compared to juveniles ingesting liposomes containing physiological saline or fish extract. These studies suggested that mobilizing the native endocrine factors associated with the feeding and digestive processes could improve MD performance in gilthead seabream and other species by maximizing its utilization.

The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae.

Abstract The requirement for dietary ( n −3) PUFA for growth, survival and swim bladder development in gilthead seabream, Sparus aurata , larvae was tested using rotifers with various levels of ( n −3) PUFA. Four rotifer treatments differing in their ( n −3) PUFA content (0.8, 3.2, 5.1 and 8.4 mg/g dry body weight, DBW) were prepared by feeding them combinations of various oil emulsions and dried squid (rich in both EPA (20:5 n −3) and DHA (22:6 n −3)). A fifth treatment consisted of rotifers enriched on the algae Nannochloropsis sp. Regression analyses, from the emulsion treatments, were performed on various relationships between rotifer ( n −3) PUFA levels and larval phospholipid and growth. The equations describing these correlations were then used to examine whether the same parameters in larvae fed Nannochloropsis -enriched rotifers were a result of the dietary ( n −3) PUFA levels they consumed or some other nutritional factor. The results indicate that dietary ( n −3) PUFA significantly ( P n −3) PUFA (8.4 mg/g DBW) showed a four-fold increase in growth (1263% vs. 312%) over larvae offered rotifers with the lowest levels of ( n −3) PUFA (0.8 mg/g DBW). In contrast, there was no compelling evidence that EPA and/or DHA significantly ( P Nannochloropsis treatment was strongly associated with EPA and implies that, despite other sources of nutrition in the algal media, this fatty acid determines the dietary value of these rotifers for growth in Sparus aurata larvae.

The effect of dietary arachidonic acid on growth, survival, and cortisol levels in different-age gilthead seabream larvae (Sparus auratus) exposed to handling or daily salinity change

The effect of dietary arachidonic acid (ArA) on survival, growth, and cortisol level in different-age gilthead seabream larvae exposed to handling or daily fluctuating salinity was tested. Premetamorphosing (3–19 DPH) larvae were reared in 400-l V-tanks and fed one of three rotifer treatments containing ArA levels of 1.14, 2.11, or 3.87 mg g−1. At 20 DPH, the larvae were divided into two groups where each larval group was randomly divided over twelve 27-l aquaria (300 larvae per aquarium) and the stress of transfer was defined as an acute stressor. In each set of 12 aquaria, larvae were fed over 12 days three Artemia metanauplii treatments, which were tested in four aquaria per treatment, giving Artemia ArA levels of 0.59, 3.42, or 5.86 mg g−1 dry weight (DW). One set of 12 aquaria received seawater of constant salinity (25‰) and these larvae, exposed only to the stress of transfer, were considered as controls. The other set of 12 aquaria was supplied with seawater with a daily fluctuating salinity from 25‰ to 40‰ and back to 25‰, exposing the larvae to 12 days of salinity change. Another trial was carried out on 30 DPH metamorphosing larvae, which were similarly stocked in the aquaria and tested with the identical Artemia treatments as the premetamorphosing larvae study. A positive correlation was found between increasing dietary ArA level and survival at the end of the study in the control premetamorphosing (20–32 DPH) and metamorphosing (30–42 DPH) larvae (38%, 48.6%, and 77.2%, and 58%, 56.4%, and 90.4%, respectively). On the other hand, premetamorphosing and metamorphosing larvae exposed to salinity change, although exhibiting an increase in survival at the intermediate level of ArA, demonstrated a decrease in survival (55.3%, 60.0%, and 25.8%, and 70%, 83%, and 76%, respectively) when fed the highest ArA level. In the control metamorphosing larvae, basal cortisol (6.0, 8.2, and 11.4 ng g−1 DW) was independent of dietary ArA while fish exposed to salinity change demonstrated distinctly higher basal cortisol levels (7.5, 15.9, and 19.8 ng g−1 DW) that markedly increased with rising dietary ArA levels at 42 DPH. Fish exposed to salinity change and fed Artemia containing 0.59, 3.42, and 5.86 mg g−1 ArA exhibited significantly (P<0.05) decreasing SGR values (12.15, 10.68, and 9.69, respectively) while the SGR values in the control larvae (10.23, 10.92, and 9.79, respectively) were generally stable. The results showed that dietary ArA promoted survival in fish encountering only handling stress. In contrast, repetitive salinity change altered the nature of the stress response where dietary ArA appeared to upregulate cortisol synthesis coinciding with reduced growth and increased mortality.

The effect of dietary lecithin and lipase, as a function of age, on n-9 fatty acid incorporation in the tissue lipids of Sparus aurata larvae

The present study tested the effect of dietary lecithin and exogenous lipase on the incorporation of oleic acid in the tissue lipids of gilthead seabream larvae (Sparus aurata). Two of four microdiets were prepared by the addition of [14C]oleic acid as free fatty acid (FFA) to diets containing either 5% cuttlefish liver oil (CLO) or 5% soybean lecithin. Glycerol tri[1-14C]oleate was similarly incorporated in two other diets identical in lipid (4% cuttlefish liver oil, 1% soybean lecithin) and non-lipid composition but differed in that one contained a supplement of 0.05% porcine lipase. The effect of these diets was tested by following the incorporation of the label (dpm/mg larvae DBW) in the neutral and phospholipid fractions of seabream larvae at four different ages (21, 27, 32 and 45 days after hatching).A significant (p<0.05) effect of dietary lecithin on the incorporation of labelled FFA in both larval neutral and phospholipid fractions was demonstrated at all ages. This was particularly pronounced during early development (day 21) where fish fed the lecithin supplement incorporated 6.75 times more label than the diet containing [14C]oleic acid in CLO. The dietary lecithin enhancing effect diminished with age but was still significant at day 45 (2.17 times more label). In addition, the label was considerably higher in the phospholipid fraction compared to the neutral lipid, reflecting the high demand for membrane synthesis during rapid growth. Lecithin fed larvae demonstrated a higher consumption rate and efficiency of incorporation than fish consuming the cuttlefish liver oil diet, suggesting an emulsifying function for dietary phospholipid.In contrast, the supplementation with lipase showed a clear effect only in older fish where 45 day old larvae fed the lipase diet demonstrated a 3.42 times increase in radioactivity in their tissue lipids. This late lipase response may be the result of an insufficient level of dietary lecithin (M) and a short intestinal length being ineffective, in the early larval stages, in incorporating labelled free fatty acid from dietary glycerol tri[1-14C]oleate breakdown.

The importance of n-3 highly unsaturated fatty acids for growth in larval Sparus aurata and their effect on survival, lipid composition and size distribution

Abstract The effect of feeding Artemia nauplii containing different levels of n -3 HUFA on growth, survival and size distribution was tested in 22–36-day Sparus aurata larvae. Five treatments were prepared by feeding 24-h-old nauplii various percent ratios (0:100, 10:90, 25:75, 50:50, 75:25) of a commercial emulsion (high in n -3 HUFA) and a soybean oil-egg yolk emulsion (3:1 preparation, deficient in n -3 HUFA). Each treatment had five replicates and provided the following n -3 HUFA levels in Artemia : 2.6, 7.4, 12.3, 18.8, 29.8 mg/g DBW nauplii. Dietary n -3 HUFA showed a linear correlation ( P n -3 HUFA demonstrated an RGR (86.4%) and final tank biomass (30.9 g wet wt) that was more than double the values exhibited by fish fed the lowest n -3 HUFA diet (39.9%, 14.0 g wet wt, respectively). Survival, in contrast, was not linearly correlated with dietary n -3 HUFA although there was a significant survival ( P n -3 HUFA poorest diet and the majority of remaining treatments. The lesser n -3 HUFA diets (2.6, 7.4 and 12.3 mg/g DBW) nauplii produced larval populations consisting mainly of small fish (6.9 mg ± 1.9) while n -3 HUFA rich diets (18.8 and 29.8 mg n -3 HUFA/g DBW nauplii) increased the fraction of larger larvae (23.0 ± 6 mg). The level of n -3 HUFA (mg/g DBW) in larval phospholipid was highly influenced by the content of these fatty acids in the diet. However, the phospholipid n -3 HUFA levels (mg/g DBW) in larvae fed the same diet were similar regardless of fish size. Finally, by examining different size larvae in groups under the various n -3 HUFA treatments, this study found that the inverse relationship between larval lipid and moisture was a function of growth rate and not dietary n -3 HUFA.

Utilisation of yolk fuels in developing eggs and larvae of European sea bass (Dicentrarchus labrax)

Abstract Developing eggs and larvae of European sea bass ( Dicentrarchus labrax ) maintained in filtered sea water (40 gl −1 ) at 18°C, were measured for oxygen uptake, contents of free amino acids (FAA), protein, fatty acids (FA), and volumes of yolk-sac and oil globule. Newly spawned eggs had a dry weight of 90 μ g egg −1 and an egg diameter of 1.14±0.03 mm. The yolk was quickly absorbed during the embryonic and the early larval stages and was 95% depleted by 100 h post fertilisation. The depletion rates of the FAA were somewhat faster than the rate of absorption of the general yolk matter and were almost complete by 80 h post fertilisation. The oil globule was mainly absorbed after hatching following yolk absorption, and occurred concurrently with catabolism of FA from neutral lipids. Approximately 30% of the oil globule was still present at the presumed onset of exogenous feeding. Overall, FAA appeared to be a significant energy substrate during the egg stage and the early yolk-sac stage while FA from neutral lipids derived from the oil globule seemed to be the main metabolic fuel after hatching. Amino acids from protein seemed to be mobilised for energy in the last part of the yolk-sac stage. This investigation supports accumulating evidence for a common sequence of catabolic substrate oxidation in marine pelagic fish eggs that contain oil globules.