Alicia Estévez

Recent developments in the essential fatty acid nutrition of fish

Abstract Because of competitive interactions in the metabolism of polyunsaturated fatty acids, tissue and bodily requirements for each of the three dietary essential fatty acids in marine fish, 22:6 n −3, 20:5 n −3 and 20:4 n −6, cannot be meaningfully considered in isolation. Rather, it is necessary to consider requirements in relative as well as absolute amounts, i.e., in terms of the ratio of 22:6 n −3:20:5 n −3:20:4 n −6. This is illustrated by recent research in our laboratories which has suggested that the optimal dietary ratio of 22:6 n −3:20:5 n −3 in sea bass larvae is circa 2:1 with the optimal dietary ratio of 20:5 n −3:20:4 n −6 being circa 1:1. The optimal dietary ratio of 22:6 n −3:20:5 n −3 in turbot and halibut larvae is similarly circa 2:1 but the optimal dietary ratio of 20:5 n −3:20:4 n −6 in these species is 10:1 or greater. In addition, studies with salmon parr point to dietary 18:3 n −3 and 18:2 n −6 being important in determining the optimal tissue ratio of 20:5 n −3:20:4 n −6 for successful parr–smolt transition. We deduce that differences in essential fatty acid requirements for different species of fish reflect different dietary and metabolic adaptations to different habitats and consider how such knowledge can be exploited to develop improved diets for fish, especially in their early stages of development.

Lipid nutrition of marine fish during early development: current status and future directions

Abstract Research on the dietary requirements of marine fish larvae has evolved from considerations of optimal dietary levels of n −3 HUFA to considerations of optimal dietary ratios of the two principal HUFAs, 22:6 n −3 and 20:5 n −3, and more recently to considerations of optimal dietary levels and ratios of all three dietary essential fatty acids, 22:6 n −3, 20:5 n −3 and 20:4 n −6. Our present understanding of the requirements and optimal dietary balance of 22:6 n −3, 20:5 n −3 and 20:4 n −6 is reviewed. Limitations of enriching live feed are considered, particularly from the point of view of achieving an optimal balance between levels of phospholipids and triacylglycerols in enriched live feeds that generate an optimal blend of essential fatty acids and energy-yielding fatty acids. It is concluded that the ideal marine fish larval diet is one containing circa 10% of the dry weight as n −3 HUFA-rich, marine phospholipids with less than 5% triacylglycerols, as exemplified by the lipid compositions of marine fish egg yolk, marine fish larvae themselves and their natural zooplankton prey. Such diets provide 22:6 n −3, 20:5 n −3 and 20:4 n −6 in the desired levels and ratios and simultaneously satisfy known requirements for phospholipids, inositol and choline. Approaches to developing marine fish larval diets more closely resembling this “gold standard” diet are considered.

Optimising lipid nutrition in first-feeding flatfish larvae

Although global production of flatfish has increased in recent years, both in terms of numbers of fish and diversification into new species, problems still remain with low survival rates and difficulties with metamorphosis. This short review highlights some advances made in optimising lipid nutrition in an attempt to overcome some of these problems. Copepod nauplii are the best live prey for first-feeding flatfish larvae. Rotifers can provide a useful method of essential fatty acid delivery but Artemia are poorer in this regard, especially at first feeding. Copepods are nutritionally beneficial due to their naturally high levels of the essential highly unsaturated fatty acids (HUFA), 20:5n-3 (eicosapentaenoic acid; EPA) and 22:6n-3 (docosahexaenoic acid; DHA), which are predominantly in the form of phospholipids. Rotifers can be enriched with fish oil emulsions to provide compositions similar to copepods, while enriched Artemia are difficult to enrich with high levels of DHA and the HUFA tend to be located in triglycerides rather than phospholipids. There is considerable evidence that the superior efficacy of copepods and rotifers is largely due to the digestibility and availability of HUFA supplied as pre-formed phospholipids. In addition to the essentiality of EPA and DHA, the requirement for 20:4n-6 (arachidonic acid; ARA) should also be considered. Improvements in dorsal pigmentation in turbot and halibut can be achieved by providing ratios of DHA/EPA of >2:1 but, perhaps more importantly, an EPA/ARA ratio of >5:1. This suggests that eicosanoids are involved in the control of pigmentation and this is further supported by the use of enrichments containing 18:3n-6, the elongation product of which (20:3n-6), is a potent inhibitor of ARA-derived eicosanoid formation, as is EPA.

The larval development of Maja squinado and M. brachydactyla (Decapoda, Brachyura, Majidae) described from plankton collected and laboratory-reared material

The morphology of the different larval stages (zoea I, zoea II and megalopa) of the majid crab Maja squinado Herbst, 1788 reared under laboratory conditions is described. The description of M. brachydactyla Balss, 1922 larvae is also revised, both from individuals reared in laboratory conditions and those collected from plankton samples. The larval stages of M. squinado are morphologically similar to those of the Atlantic species M. brachydactyla; the larval morphology does not justify the separation of M. squinado into two species. Larvae of M. brachydactyla obtained from the wild were similar to those reared under laboratory conditions. The morphology of the zoeal and megalopal stages of M. squinado and M. brachydactyla described in this paper have been compared with previous descriptions of the genus Maja, including M. brachydactyla (described as M. squinado).

Development temperature has persistent effects on muscle growth responses in gilthead sea bream

Initially we characterised growth responses to altered nutritional input at the transcriptional and tissue levels in the fast skeletal muscle of juvenile gilthead sea bream. Fish reared at 21–22°C (range) were fed a commercial diet at 3% body mass d−1 (non-satiation feeding, NSF) for 4 weeks, fasted for 4d (F) and then fed to satiation (SF) for 21d. 13 out of 34 genes investigated showed consistent patterns of regulation between nutritional states. Fasting was associated with a 20-fold increase in MAFbx, and a 5-fold increase in Six1 and WASp expression, which returned to NSF levels within 16h of SF. Refeeding to satiation was associated with a rapid (<24 h) 12 to 17-fold increase in UNC45, Hsp70 and Hsp90α transcripts coding for molecular chaperones associated with unfolded protein response pathways. The growth factors FGF6 and IGF1 increased 6.0 and 4.5-fold within 16 h and 24 h of refeeding respectively. The average growth in diameter of fast muscle fibres was checked with fasting and significant fibre hypertrophy was only observed after 13d and 21d SF. To investigate developmental plasticity in growth responses we used the same experimental protocol with fish reared at either 17.5–18.5°C (range) (LT) or 21–22°C (range) (HT) to metamorphosis and then transferred to 21–22°C. There were persistent effects of development temperature on muscle growth patterns with 20% more fibres of lower average diameter in LT than HT group of similar body size. Altering the nutritional input to the muscle to stimulate growth revealed cryptic changes in the expression of UNC45 and Hsp90α with higher transcript abundance in the LT than HT groups, whereas there were no differences in the expression of MAFbx and Six1. It was concluded that myogenesis and gene expression patterns during growth are not fixed, but can be modified by temperature during the early stages of the life cycle.

Prostaglandin (F and E, 2- and 3-series) production and cyclooxygenase (COX-2) gene expression of wild and cultured broodstock of senegalese sole (Solea senegalensis)

Prostaglandin levels in different tissues and cyclooxygenase (COX-2) gene expression were compared between wild and cultured Senegalese sole (Solea senegalensis) broodstock in which a significantly different fatty acid profile, particularly lower tissue levels of arachidonic acid (ARA, 20:4n-6) and higher levels of eicosapentaenoic acid (EPA, 20:5n-3) in the cultured fish compared to wild had already been described. This is the first report of the COX-2 mRNA expression in Senegalese sole. Cyclooxygenase (COX-2) mRNA expression and prostaglandin (2- and 3-series) levels were determined in tissues from 32 broodstock fish, 16 (8 males and 8 females) from each origin wild and cultured (G1). Transcripts of COX-2 were highly expressed in gills, sperm-duct (s-duct), testis, oviduct and spleen compared to liver, kidney and muscle. Differences in COX-2 transcripts expression were found in response to the origin of the fish and expression was significantly higher in s-duct and gills from wild fish compared to cultured. Wild fish showed significantly higher levels of total 2-series PGs and lower levels of 3-series compared to cultured fish. The significance of the lower COX-2 expression and lower PG 2-series production in some of the tissues of cultured fish was discussed in relation to the previously described differences in fatty acid profile (lower tissue levels of ARA and higher levels of EPA and EPA/ARA ratio in cultured fish) and the reproductive failure to spawn viable eggs from G1 cultured Senegalese sole compared to successful spawning from captive wild broodstock.

Isolipidic diets differing in their essential fatty acid profiles affect the deposition of unsaturated neutral lipids in the intestine, liver and vascular system of Senegalese sole larvae and early juveniles

Abstract How lipid content and composition in the diet is utilized by the various organs and tissues of fish is reflected in their structure, such as the intestine through which dietary lipids are digested and absorbed, the vascular system which is involved in their transport, and the liver where lipids are stored and metabolized. However, no study has been conducted to compare the effect of different diets containing different levels of highly unsaturated fatty acids (HUFA) and essential fatty acids (EFA) on lipid deposition in fish larvae and early juveniles. Thus, we evaluated the effects of six isolipidic diets (enriched Artemia salina ), differing in their fatty acid profile, on the lipid accumulation patterns in selected target tissues (intestine, liver and vascular system) in Senegalese sole ( Solea senegalensis ) larvae and early juveniles. Results showed that the profile of fat accumulation in these three tissues was significantly affected by the dietary treatments, the developmental stage of the fish (premetamorphosis, metamorphosis or postmetamorphosis), as well as by the interaction between these two factors that were responsible for changes in the histological organization of the tissues. Histological results revealed that a slight variation in the EFA levels (e.g. EPA, DHA or ARA) or in their ratios (EPA/DHA, ARA/EPA, ARA/DHA, (n-3)/(n-6) HUFA, OA/PUFA) modified the metabolism of lipids and disrupted the pattern of lipid accumulation in the target tissues, leading to intestinal and hepatic steatosis.

Effects of graded levels of arachidonic acid on the reproductive physiology of Senegalese sole (Solea senegalensis): Fatty acid composition, prostaglandins and steroid levels in the blood of broodstock bred in captivity

Previous studies on Senegalese sole (Solea senegalensis) indicated that cultured broodstock (first generation, G1) have lower tissue levels of arachidonic acid (20:4n-6, ARA) than wild counterparts. ARA is metabolized to form prostaglandins (PGs) that are involved in steroid production and follicle maturation in fish. In the present study the effects of different dietary levels of ARA on blood lipid and fatty acid composition, prostaglandin (PGF2α, PGF3α, PGE2 and PGE3) levels and plasmatic steroid levels (11-ketotestosterone, 11-KT, testosterone, T and estradiol, E2) in G1 Senegalese sole were studied. For this purpose, 12 groups of ten fish (1:1 male and female), were fed six diets (each diets was fed to two groups) with different dietary ARA levels over nine months (diets A=0.7, B=1.6, C=2.3, D=3.2, E=5.0, F=6.0% ARA). ARA and CHOL levels in blood showed a significant increase in an ARA dose related manner (P<0.05) whereas EPA and EPA/ARA ratio were reduced. In males, steroid (11-KT and T) levels increased significantly with increasing dietary ARA in a dose dependent manner, whereas in females E2 did not show any change related to dietary ARA content. Plasma concentration of 3-series PGs (i.e., PGE3 and PGF3α) were reduced in parallel to increased ARA levels in blood (P<0.05) and levels of PGs 3-series were always higher than 2-series PGs (PGE2 and PGF2α). In conclusion there is an effect of dietary ARA on steroid production of Senegalese sole males, which might have important consequences in the reproduction of cultured fish.

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis)

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco (ES, INVE) or Aquagrow Gold (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparβ), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.

Dietary fatty acid composition affects food intake and gut-brain satiety signaling in Senegalese sole (Solea senegalensis, Kaup 1858) larvae and post-larvae.

Little is known how dietary lipids affect food intake during larval development of fish, especially with regard to fatty acid (FA) composition. In fact, very little work has been done on appetite regulation and food intake in fish larvae in general, due to biological and technical difficulties associated with this type of studies. A new method using fluorescent microspheres as markers was developed in this study to evaluate food intake and prey selectivity of Senegalese sole larvae and post-larvae. Food intake was quantified in fish fed Artemia metanauplii enriched with oils differing in FA profile: cod liver oil (CLO), linseed oil (LSO), soybean oil (SBO) or olive oil (OO). The fish did not preferentially ingest a specific diet when presented with a choice. However, pre-metamorphic larvae from the CLO treatment ingested more metanauplii per g body weight, while differences in post-larvae were not significant. These findings were developed further by analyzing mRNA levels of a range of putative anorexigenic (pyya, pyyb, glp1, cckl, cart1a, cart1b, cart2a, cart4, pomca, pomcb, crf) and orexigenic (gal, npy, agrp2) genes, to identify those which are significantly affected by feeding and/or dietary FA composition. The variety of expression patterns observed highlighted the complexity of appetite regulatory mechanisms. In general, fish fed the CLO diet tended to show gene expression patterns most dissimilar to the remaining treatments. Expression in pre-metamorphic larvae was generally less in accordance with the putative function of the genes than in post-larvae, which could suggest a yet underdeveloped regulatory system.