Eduardo Almansa

Influence of broodstock gilthead seabream (Sparus aurata L.) dietary fatty acids on egg quality and egg fatty acid composition throughout the spawning season

The influence of broodstock dietary lipids on egg quality and egg fatty acid composition throughout the spawning season of gilthead seabream was investigated. For this purpose, the fish were fed for 7 months either a control diet (diet C) or a diet deficient in n−3 highly unsaturated fatty acids (n−3 HUFA) but rich in both oleic (18:1n−9) and linolenic (18:3n−3) acids (diet D). Eggs spawned by both groups of fish were sampled at the beginning, middle and end of the spawning season and the fatty acid composition of their neutral (NL) and polar lipids (PL) determined. In the early season, percentages of fertilized and hatched eggs, relative proportions of NL and PL as well as their fatty acid compositions, were not affected by the lipid composition of the broodstock diet. However, the eggs spawned during the middle and late seasons showed marked differences among the two groups of fish, clearly reflecting the influence of dietary fatty acids. This influence was more evident in the neutral lipid fraction than in the polar lipids. No correlation was found between the number of buoyant eggs and eicosapentaenoic (20:5n−3, EPA), docosahexaenoic (22:6n−3, DHA) fatty acids or total n−3 HUFA contents in egg phospholipids. However, a negative correlation was detected when percentages of fertilized eggs were compared with the levels of 18:1n−9, 18:3n−3 and with the ratio 18:1n−9/n−3 HUFA present in the phospholipids. Our results indicate the importance of maintaining not only the level of n−3 HUFA in egg membrane phospholipids, but also the balance between n−3 HUFA and other fatty acids such as 18:1n−9 and 18:3n−3, in order to obtain a high spawning quality.

Lipid and fatty acid composition of ovaries from wild fish and ovaries and eggs from captive fish of white sea bream (Diplodus sargus)

Abstract The fatty acid composition of ovaries from wild white sea bream and ovaries and eggs of captive white sea bream were investigated to estimate the fatty acid requirements of this species. The total lipid (TL) content in wild fish ovaries was similar to that found in ovaries and eggs from captive fish. The general pattern of the fatty acid distribution in lipid of ovaries from wild fish and ovaries and eggs from captive fish was similar. In total lipid, no significative differences in total monoenes, n −6, n −3 and n −3 highly unsaturated fatty acids (HUFA) were found. However, the relative percentage of specific fatty acids differed between wild and captive fish. Thus, arachidonic acid (20:4 n −6, AA) percentage was higher in wild ovaries than in ovaries and eggs from captive fish, whereas eicosapentaenoic acid (20:5 n −3, EPA) showed the opposite trend. In consequence, wild fish presented a lower EPA/AA ratio in their ovaries when compared with ovaries and eggs of captive fish. These differences were extended to all lipid classes studied (triacylglycerol (TG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The present study suggests that essential fatty acids, specially EPA and AA of the diet supplied to the captive white sea bream broodstocks, do not seem to be in an appropriate proportion for this species.

Growth and survival of cuttlefish (Sepia officinalis) of different ages fed crustaceans and fish. Effects of frozen and live prey

Abstract Three feeding experiments, using live mysid shrimp, grass shrimp or fish fry as prey for 1-, 30- and 60-day-old cuttlefish were conducted to determine the efficiency of each dietary source in relation to cuttlefish size and age. Additionally, a fourth experiment using fish fry and grass shrimp, but previously frozen, was also conducted. The results showed that when 1-day-old cuttlefish were fed mysids, grass shrimp or fish for 4 weeks, mysids were the best prey, but only during the first week. From this moment until the end of the experiment, the best growth rate was when cuttlefish were fed grass shrimp. Cuttlefish fed fish fry showed the poorest growth rate throughout the experiment. Similarly, cuttlefish aged 30 or 60 days fed grass shrimp or fish fry had the best growth rates when fed grass shrimp. When cuttlefish were fed live fish, survival increased with size of cuttlefish (73.3%, 91.7% and 100% for 1, 30 and 60 days cuttlefish, respectively). In the fourth experiment, using frozen diets, overall acceptance of each diet (feeding rates) was the same for fish and shrimp. However, lower growth was obtained when cuttlefish were fed fish compared to grass shrimp. This lower growth was due to a lower food conversion (28% vs. 41%). Since cephalopod paralarvae and juvenile most likely need prey rich in polyunsaturated fatty acids (PUFA), phospholipids and cholesterol, and a moderate content in neutral lipids, we have analyzed the biochemical compositions of the different prey to evaluate the influence of this factor on growth and survival.

Effects of culture density and live prey on growth and survival of juvenile cuttlefish, Sepia officinalis

The effects of culture density on growth and survival of juvenile cuttlefish were tested. Groups of 1, 3 and 5 hatchlings were placed in small containers with bottom surface of 80 cm2, obtaining individual densities of 125, 375 and 625 cuttlefish m−2, respectively. Additionally, groups of 5 hatchlings were placed in containers with 2 different bottom areas (80 and 240 cm2), providing culture densities of 625 and 42 cuttlefish m−2, respectively. A total of 120 hatchlings were used and experiments lasted for 40 days. No differences were found in growth between any of the densities tested throughout the experiment until 35 days old. After this, cuttlefish placed in isolation grew significantly larger. A second experiment was conducted in a flow through system, using two rectangular tanks with bottom surface of 0.5 m2. Two groups of 25 cuttlefish hatchlings were used in this experiment, which lasted for 40 days. Both groups were fed live juvenile shrimp (Crangon crangon) during the first 5 days. Afterwards, one group was fed live fish fry of different species, while the other continued to be fed shrimp. After day 10 and until the end of the experiment, hatchlings fed shrimp grew significantly larger than those fed fish fry. Survival of hatchlings fed shrimp or fish fry after 40 days was of 100% and 68%, respectively. Total protein content of both prey types was similar. Therefore, the higher polar lipid content, especially due to the higher phosphatidylcholine and phosphatidylethanolamine levels observed in the shrimp, compared to fish fry could possibly be one of the major factor to explain the significantly higher growth rates for S. officinalis juveniles fed shrimp. Also, the percentage of polar lipids in the shrimp (47.4%) was closer to the one of juvenile cuttlefish (38.1%) than the composition of polar lipids in fish fry (10.4%). This could also be an important factor to explain the poor growth and survival obtained when feeding fish fry to the cuttlefish.

Effect of dietary supplementation with shrimp on skin pigmentation and lipid composition of red porgy (Pagrus pagrus) alevins

A feeding experiment was conducted on red porgy alevins to investigate the influence of dietary supplementation with shrimp on pigmentation and lipid composition of carcass (muscle and skin) and eyes. To this purpose, red porgy alevins with an initial mean weight of 2.8±1.3 g were divided into six groups. Three groups were fed gilthead seabream commercial pellet (P, 100% pellet), whereas the other three were fed with the commercial pellet and shrimp Pleisonika sp. (PS, 88% pellet:12% shrimp) to provide the diet with a source of carotenoids. After 4 months of feeding, the results showed that PS-fish groups displayed a pink-coloured skin similar to that of the wild fish, whereas P-fish groups showed a dark-grey-coloured skin. Furthermore, a higher total content of carotenoids was found in carcass and eyes from PS-fish groups with respect to P-fish groups. No significant differences in growth, survival or other gross external signs were found between treatments. Total lipid (TL), triacylglycerol (TG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of red porgy carcass exhibited a specific fatty acid profile. However, the general patterns of the fatty acid distribution in lipids of carcass and eyes from both groups of fish (P and PS) were similar, and only slight differences were found mainly in carcass for some of the fatty acids. We conclude that the natural carotenoids supplied by the shrimp were effectively assimilated by the red porgy and allowed the cultured fish to acquire a skin coloration similar to that of wild fish.

Characterization of intestinal Na+–K+-ATPase in the gilthead seabream (Sparus aurata L.). Evidence for a tissue-specific heterogeneity

Abstract Gilthead seabream intestine contains both a Mg 2+ -dependent Na + –K + -ATPase which is completely inhibited by 1×10 −3 M ouabain, and also a residue-ATPase activity that is entirely ouabain-insensitive. The maximal activity of intestinal Na + –K + -ATPase (35.15 μ mol inorganic phosphate (P i ) mg protein −1 h −1 ) was observed in the microsomal fraction at 35°C, pH 7.5, 2–5 mM MgCl 2 , 5 mM ATP, 10 mM K + and 200 mM Na + . The intestinal Na + –K + -ATPase of gilthead seabream exhibits similar characteristics to other teleost Na + –K + -ATPases regarding pH dependence, Mg 2+ /ATP optimal ratios and ouabain sensitivity, but exhibits unusual sensitivity to ionic strength and cation promoted cooperative activation and higher affinity for magnesium and ATP when compared to other marine and euryhaline teleosts. The Arrhenius plot for intestinal Na + –K + -ATPase showed a break point at 15.41°C, with similar activation energies above and below the discontinuity point. The analysis of polar lipid fatty acid composition was correlated to the break point in the Arrhenius plot, suggesting a regulatory role of the lipid microenvironment on the enzyme activity. Finally, the kinetic characteristics, temperature-activity relationship, fatty acid composition and substrate dependence of intestinal Na + –K + -ATPase are compared with literature data and discussed on the basis of the differences between species and also between osmoregulatory tissues. It is concluded that the Na + –K + -ATPase of Sparus aurata is differently and specifically expressed between the osmoregulatory organs.

Age Validation in Octopus vulgaris Beaks Across the Full Ontogenetic Range: Beaks as Recorders of Life Events in Octopuses

ABSTRACT This study demonstrates the daily deposition of increments in Octopus vulgaris beaks for both lateral wall surfaces (LWS) and rostrum sagittal sections (RSS). Forty-nine marked wild animals kept in aquaria (weight range, 158–3,521 g) and 24 captive-reared known-age individuals (paralarvae, 0–98 days old; adults, 200–734 days old) were studied, encompassing for the first time the full age range of the species, including known-age individuals older than 1 mo. The daily deposition of beak increments was validated in the LWS by injection of Calcofluor, and in the RSS by environmental marking (thermal, confinement, capture, and stress of the chemical marking process). A total of 111 successful validations (when beak increments corresponded precisely to days elapsed) were achieved, and the maximum validated periods were 57 days (LWS) and 112 days (RSS). In the pelagic stage and transition to the settlement stage, a new pattern of microincrements that record age was demonstrated in the lateral hood surfaces of upper jaws, where stress checks were observed. In the benthic stage, tip erosion in beak RSS results in some underestimation of age; however, the demonstration that RSS can record environmental stress renders it a potentially useful tool for documenting life events.

Dietary Effect on the Proteome of the Common Octopus (Octopus vulgaris) Paralarvae

Nowadays, the common octopus (Octopus vulgaris) culture is hampered by massive mortalities occurring during early life-cycle stages (paralarvae). Despite the causes of the high paralarvae mortality are not yet well-defined and understood, the nutritional stress caused by inadequate diets is pointed out as one of the main factors. In this study, the effects of diet on paralarvae is analyzed through a proteomic approach, to search for novel biomarkers of nutritional stress. A total of 43 proteins showing differential expression in the different conditions studied have been identified. The analysis highlights proteins related with the carbohydrate metabolism: glyceraldehyde-3-phosphate-dedydrogenase (GAPDH), triosephosphate isomerase; other ways of energetic metabolism: NADP+-specific isocitrate dehydrogenase, arginine kinase; detoxification: glutathione-S-transferase (GST); stress: heat shock proteins (HSP70); structural constituent of eye lens: S-crystallin 3; and cytoskeleton: actin, actin-beta/gamma1, beta actin. These results allow defining characteristic proteomes of paralarvae depending on the diet; as well as the use of several of these proteins as novel biomarkers to evaluate their welfare linked to nutritional stress. Notably, the changes of proteins like S-crystallin 3, arginine kinase and NAD+ specific isocitrate dehydrogenase, may be related to fed vs. starving paralarvae, particularly in the first 4 days of development.

Biometrical relationships in developing eggs and neonates of Octopus vulgaris in relation to parental diet

Captive Octopus vulgaris adults were fed three mono-diets based on pilchard, crab and squid and allowed to grow until reproduction under controlled temperature. Spawns from each dietary treatment were isolated, and the embryonic development, egg length, width and wet weight, in addition to neonate dry weight, dorsal mantle length and ventral mantle length were monitored. Pilchard-diet spawns developed faster in terms of thermal time. Initial egg wet weight was higher for squid and crab diets. Irrespective of the parental diet, eggs passed through a swelling process so that egg width and wet weight increased in a nonlinear way, whereas egg length was left nearly unaffected. Egg length and initial wet weight showed a high correlation with neonate dry weight. Egg length, even at advanced incubation, can be used as a good proxy for neonate dry weight, this fact having potential implications for the ecological and aquaculture research on O. vulgaris.

Selective polyunsaturated fatty acids enrichment in phospholipids from neuronal-derived cell lines

Most studies aimed at exploring the molecular and cellular properties of plasma membranes in neural tissues make use of cell lines. However, cell membrane lipid composition of cell lines is notably different from that of brain tissues where they presumably derive from. Using septal-derived SN56 cells and hippocampal-derived HT22 cells, we demonstrated that cell lines exhibit lower contents of saturated (18:0) and long polyunsaturated fatty acids (PUFA; 20:4n-6 and especially 22:6n-3), as well as higher monounsaturated fatty acid contents (mainly 18:1n-9), compared to mouse brain. Also, cell lines exhibited higher contents of sterol esters and lower contents of cholesterol and phospholipids, especially phosphatidylethanolamine and phosphatidylserine. We have also evaluated the effects of different (n-3/n-6) PUFA enrichments on fatty acid and phospholipid contents in these cell lines. Our results show that enrichment of culture medium with 22:6n-3 and 20:4n-6 in a 70/30 proportion during 48 h, using fat-free bovine serum albumin as vehicle, successfully readjusted fatty acid profiles in cell line-polar lipids to values found in natural nerve cells. Interestingly, no differences in cell survival were observed upon enrichment. The generalization of these methodologies would allow a more feasible adaptation of cellular models to the study of in vivo nerve physiology.