Marisol Izquierdo

Effect of broodstock nutrition on reproductive performance of fish

Abstract In many cultured fish species, particularly in those new for aquaculture, unpredictable and variable reproductive performance is an important limiting factor for the successful mass production of juveniles. An improvement in broodstock nutrition and feeding has been shown to greatly improve not only egg and sperm quality but also seed production. Gonadal development and fecundity are affected by certain essential dietary nutrients, especially in continuous spawners with short vitellogenic periods. Thus, during the last two decades, more attention has been paid to the level of different nutrients in broodstock diets. However, studies on broodstock nutrition are limited and relatively expensive to conduct. Lipid and fatty acid composition of broodstock diet have been identified as major dietary factors that determine successful reproduction and survival of offspring. Some fish species readily incorporate dietary unsaturated fatty acids into eggs, even during the course of the spawning season. Highly unsaturated fatty acids (HUFA) with 20 or more carbon atoms affect, directly or through their metabolites, fish maturation and steroidogenesis. In some species, HUFA in broodstock diets increases fecundity, fertilization and egg quality. As in higher vertebrates, vitamin E deficiency affects reproductive performance, causing immature gonads and lower hatching rate and survival of offspring. For example, elevation of dietary α-tocopherol levels has been found to reduce the percentage of abnormal eggs and increase fecundity in the gilthead seabream ( Sparus aurata ). Ascorbic acid has also been shown to play an important role in salmonid reproduction, where the dietary requirement of broodstock was higher than that of juveniles. Among different feed ingredients, cuttlefish, squid and krill meals are recognized as valuable components of broodstock diets. The protein component of cuttlefish and squid together with their optimal concentration of HUFA appear to be responsible for their positive effect on reproductive performance. Both polar and nonpolar lipid fractions of raw krill were found to effectively improve egg quality.

Impact of different dietary lipid sources on growth, lipid digestibility, tissue fatty acid composition and histology of rainbow trout, Oncorhynchus mykiss

Abstract The aim of the present study was to determine the impact of replacing at least 50% of the fish oil by alternative lipid sources in diets for rainbow trout on growth, lipid digestibility, fatty acid profiles of liver and muscle and tissue histology. Four experimental diets were formulated combining two fish oils (capelin and anchovy) with different vegetable oils (soybean, rapeseed, palm and olive) and one type of animal fat (lard), in order to obtain 60% to 80% fish oil replacement. A fifth diet prepared with pure fish oil (capelin oil) served as control. The diets were fed to apparent satiation twice a day to triplicate groups of 30 rainbow trout with an initial weight of 250 g for 64 days at 12 °C. Growth was good (TGC 3 3.7–3.9) and independent of diet. Final body weight was on average 760 g. Feed conversion ratios (FCR) ranged from 0.72 to 0.79. The only significant difference in FCR was found in the group fed an olive oil–lard combination replacing 80% of the fish oil. This diet also had a lower apparent lipid digestibility (79% vs. 91–94% for the rest of the diets). The liver and muscle fatty acid compositions were similar within groups and reflected that of the diets. But, tissue levels of 20:5 n −3 were lower and levels of 22:6 n −3 were higher than their respective dietary percentages. Histologically, a supranuclear accumulation of lipid droplets was observed in the intestinal cells of some of the groups fed diets supplemented with vegetable oils. Similarly, livers from these groups showed large amounts of lipid droplets within the hepatocytes. The present results suggest that most of the fish oil can be replaced by the alternative sources tested without compromising growth and feed utilisation. However, the histological changes observed suggest an impact of dietary lipid source on the transport and/or metabolism of fat in the fish. This requires further investigation.

Soybean and lupin seed meals as protein sources in diets for gilthead seabream (Sparus aurata) : nutritional and histological implications

The use of vegetable protein sources in diets for freshwater fish has been studied in more detail than for marine fish species. Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different vegetable protein sources, soybean and lupin seed meals. Mean feed intake and growth were not significantly influenced by type or level of plant protein in the diet. Feed utilization indexes such as feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the type of plant protein in the diet, although a general reduction of these values was observed with increased inclusion of soybean meal. Histological studies showed an increased deposition of lipid and decreased glycogen deposits in the liver with increased levels of dietary soybean meal. Protein digestibility coefficients for lupin seed meal diets were similar to the control and 10% higher than those for the soybean meal diets. A significant reduction in trypsin activity was observed in fish fed the lupin seed meal diets, and for soybean meal diets when the substitution level reached 30%. Diets including plant protein showed a higher peak of ammonia excretion rate, which appeared 2 h later than that of the fish meal diet. Highest values of dissolved ammonia were registered in fish fed a soybean meal-based diet. These results suggest that properly treated lupin meals could be an important alternative dietary protein source for gilthead seabream.

Vegetable lipid sources for gilthead seabream (Sparus aurata): effects on fish health

Abstract Commercial feeds for gilthead seabream are highly energetic, containing fish oil as the main lipid source. The steady production and raising prices of fish oil encourage the inclusion of vegetable oils in fish feeds. Fish oil could be at least partially substituted by vegetable oils in diets for marine species, being this substitution resulted in good feed utilization and maintenance of fish health, since imbalances in dietary fatty acids may alter the immunological status and stress resistance in fish. In order to evaluate the effect of vegetable oils on gilthead seabream health, fish were fed different isonitrogenous and isocaloric diets for 101 days (Experiment I) and 204 days (Experiment II). In Experiment I, diets were formulated to contain 60% of the fish oil used in the control diet (FO) as soybean oil (Diet 60SO), rapeseed oil (60RO), linseed oil (60LO) or a blend of those oils (Mix). In Experiment II, the same diets plus two which contained 80% of the fish oil as soybean oil (80SO) and linseed oil (80LO), respectively, were assayed. At the end of both experiments, basal levels of different immunological parameters were determined, including both humoral immunity (alternative complement pathway activity and serum lysozyme activity) and cellular immunity (circulating neutrophil activity and phagocytic index of head kidney macrophages). In addition, response to a confinement stress was assayed in terms of variations in plasma cortisol. The effect of dietary vegetable oils on fatty acid composition of head kidney macrophages and circulating red blood cells (RBC) was also studied. No effects of dietary vegetable oils were found in fish fed the experimental diets for a medium period. Feeding dietary vegetable oils for a long period did not affect lysozyme or neutrophil activity. However, in Experiment II, the inclusion of soybean oil reduced both serum alternative complement pathway activity (from 249 IU/ml (FO2) down to 153.8 IU/ml (60SO2)) and head kidney phagocytic activity (from 25.75% (FO2) down to 14.58% (80SO2). Inclusion of rapeseed oil reduced phagocytic activity. Fish fed vegetable oil-containing diets showed different patterns of stress response, especially those fish fed the linseed oil diets that showed a significant increase in plasma cortisol level after stress. The fatty acid composition of head kidney macrophages reflected the fatty acids content of the respective diets, but a selective incorporation of essential fatty acids into these cells was observed. The same trend was found in circulating red blood cells, indicating the important role of essential fatty acids on these cells. Sixty percent of fish oil can be replaced by a blend of different vegetable oils without affecting gilthead seabream health. However, if single vegetable oil is used to replace 60% of fish oil, fish health can be affected in terms of immunosuppression or stress resistance. Rapeseed oil affected head kidney macrophages activity, soybean oil affected serum alternative complement pathway activity and linseed oil altered stress response of fish.

Recent advances in lipid nutrition in fish larvae

Due to the importance of dietary lipid utilization for larval rearing success, increasing attention has been paid during the last years to different aspects of larval lipid nutrition such as digestion, absorption, transport and metabolism, which are frequently studied by different research groups. The present study reviews the published information on these aspects, including some recent results obtained in our laboratory, that contribute to a better understanding of larval lipid nutrition.Neutral lipase activity was found in the digesta of larval gilthead seabream as early as first feeding, followed by a significant increase which reached up 8 times the initial levels at day 15 and was clearly influenced by the fatty acid composition of dietary lipids. Accordingly, the capacity for lipid absorption by the intestinal epithelium has been also observed at the onset of exogenous feeding, although the specific location in the different digestive tract segments differ with species. Whereas the capacity to absorb lipid increases with development in live prey-fed larvae, this improvemment is delayed in larvae fed formulated diet. Increasing dietary phosphatidyl cholines levels enhanced lipid absorption regardless of whether it is of soybean or marine origin, but the latter improved hepatic lipid utilization. Enzymatic, histological and biochemical evidences suggest that marine fish larvae are able to effectively digest and absorb n-3 HUFA-rich triacylglycerols, but feeding with phosphoacylglycerols, particularly if they are rich in n-3 HUFA, would enhance phosphoacylglycerols digestion and specially lipid transport alowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Although the essentiality of n-3 HUFA for larval marine fish has been studied extensively, only recently has the importance of dietary arachidonic acid in the larvae of few species been recognised. Evidences for competitive interactions among these essential fatty acids suggest that besides a minimum dietary requirement for each essential fatty acid, their relative ratios must also be considered.

Corn gluten and meat and bone meals as protein sources in diets for gilthead seabream (Sparus aurata): Nutritional and histological implications

Abstract Two experiments were conducted to compare the effect of the partial substitution of fish meal by two different protein sources, corn gluten (CGM) and meat and bone meals (MBM) in diets for juveniles gilthead seabream. Growth, feed efficiency, protein efficiency ratio and protein productive values were not significantly affected by the source of dietary protein, although higher values were observed with diets containing increasing levels of meat and bone meal. No differences were found in fish proximate composition at the end of the experiment. Histological studies of liver tissue showed no liver alteration in fish fed diets including increasing levels of corn gluten meal. On the contrary, an increased deposition of lipids, nuclei polarization and isolated necrotic focus were found in hepatocites of fish fed diets exceeding 20% of meat and bone protein. Apparent protein digestibility (ADC) in diets containing CGM showed similar results to those obtained with the control diet. However, a significant reduction in digestibility was observed when MBM was used as partial substitute of fish meal protein. A negative correlation between dietary ash content and protein digestibility was observed. Higher amounts of nitrogen were excreted as levels of CGM and MBM increased in the diets, being significantly higher in the case of 40% substitution either with CGM or MBM protein.

Morphological aspects of intestinal cells from gilthead seabream (Sparus aurata) fed diets containing different lipid sources

Abstract The aim of the present study was to evaluate the morphological changes in the intestine of gilthead seabream fed different lipid sources. Five experimental diets containing 22% lipid and 45% crude protein were formulated: the control diet included fish oil as the only lipid source, whereas the other four diets contained linseed, soybean or rapeseed oils at two different levels of fish oil substitution, 60% or 80%. Gilthead seabream juveniles of 79 g mean body weight were fed the experimental diets for 3 months. At the end of experiment, samples of anterior intestine were taken for ultrastructural study. A morphometric study was performed to complete the morphological results. Irreversible tissue damages (necrosis or cell degeneration) could not be found in the intestinal epithelium of fish, regardless the diet fed. Microvillis were well developed, among which some absorptive vesicles were observed in fish from all diets. The major ultrastructural differences in the enterocytes of fish fed different diets were the accumulation of supranuclear lipid droplets and the formation of lipoproteins. Lipid droplet accumulation was increased by the percent of fish oil replaced. The morphometric study revealed that the cellular space occupied by lipid droplets was 2.3 times higher when fish were fed a diet containing linseed or soybean oils at a 60% replacement level than in the control fish, whereas in fish fed the vegetable oils at a 80% replacement level and rapeseed at 60% it was 5 times higher than in the control fish. In the intercellular spaces and lamina propria two different classes of lipoproteins in relation to their size were found: very low density lipoproteins (VLDL) of 30 to 85 nm in diameter and chylomicrons (QM) of over 100 nm. Fish fed a soybean oil diet, particularly at a 60% replacement level, exhibited a much higher accumulation of these granules in dilated intercellular spaces. The rest of experimental groups showed chylomicrons as the main lipoprotein. These results are discussed in relation to fatty acid compositions and cellular mechanisms involved in lipid absorption.

Effect of dietary arachidonic acid levels on growth and survival of gilthead sea bream /Sparus aurata L. larvae

In order to determine the effect of different levels of arachidonic acid (AA) in microdiets on growth and survival of gilthead seabream larvae, two experiments were carried out. In the first experiment, 17-day old larvae were fed microdiets for 14 days. In this trial, we tested four diets with a similar docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio and AA in the range of 0.1%–1.8% dry weight (d.w.) and a fifth diet with 1.0% d.w. of AA and a different DHA/EPA ratio. In the second experiment, 16-day old larvae were fed for 21 days on two microdiets with levels of 0.1% and 1.0% d.w. AA. All diets in the two experiments had the same total n−3 HUFA level. Growth was measured by total length and dry body weight of larvae. Larval lipids were extracted and fatty acid compositions of total neutral lipid (NL) and total polar lipid (PL) fractions were determined by gas chromatography. In the first experiment, the diet containing 1.8% d.w. of AA showed the best survival (P<0.05) but did not improve growth rate. In the second experiment, an increase in the AA dietary level from 0.1% to 1.0% d.w. significantly improved (P<0.05) larval growth. EPA accumulation in the PLs of the larvae was negatively affected by the inclusion of dietary AA. When working with a fixed dietary n−3 HUFA level (2.2% d.w.), the effect of dietary AA on larval growth was masked by that of the dietary DHA/EPA ratio. However, when DHA/EPA ratio in diet was unchanged (∼1.8), it was possible to improve larval growth by supplementing the diet with 1.0 d.w. of AA. The negative effect produced by the increment of dietary AA on the EPA incorporation into the larval PLs may be related to a competition interaction.

Adaptation of lipid metabolism, tissue composition and flesh quality in gilthead sea bream ( Sparus aurata ) to the replacement of dietary fish oil by linseed and soyabean oils

Linseed (LO) and soyabean (SO) oils were evaluated as fish-oil (FO) substitutes in the diets of marketable-sized gilthead sea bream (Sparus aurata). Practical diets were designed factorially with the lipid added as follows (%): FO 100, LO 60+FO 40, LO 80+FO 20, SO 60+FO 40, SO 80+FO 20. The effects of experimental diets on growth, fatty acids patterns in liver and muscle, flesh quality variables and activities of selected enzymes involved in lipid synthesis and catabolism were determined at the end of a 7-month trial. Fatty acid composition of liver and muscle generally reflected the fatty acid composition of the diets. The n-3 PUFA levels were significantly reduced by the inclusion of vegetable oils. This tendency was more pronounced for EPA than for docosahexaenoic acid. The n-3:n-6 fatty acid ratio reached the lowest values in fish fed the SO diets; this was associated with a higher liver lipid deposition. No differences were found in fillet texture and pH. However, under conditions of forced peroxidation, muscles from fish fed the SO diets had lower peroxidation levels. Vegetable oil substitution decreased lipogenesis in liver and this effect was greatest at the highest substitution level. In contrast, muscle beta-oxidation enzymes had increased activities with vegetable oil substitution. Thus, the lower hepatic lipogenesis was correlated with an increased lipid utilisation in muscle. It is concluded that growth and lipid metabolism were affected by experimental diets.

Depletion of serum alternative complement pathway activity in gilthead seabream caused by α-tocopherol and n-3 HUFA dietary deficiencies

Studies on the alternative complement pathway (ACP) activity in fish have suggested the particular relevance of this pathway as a non-specific immune mechanism being its activation directly related with the availability of certain nutrients. In the present study ACP activity in gilthead seabream fed different dietary levels of a-tocopherol and n-3 HUFA deficiencies was studied under different stress conditions (overcrowding and repetitive chasing).A reduction in ACP activity was found when diets with deficiencies in α-tocopherol and n-3 HUFA were assayed at any of the experimental conditions assayed. Levels of plasma cortisol were similar to those of fish after crowding stress subjected to dietary deficiencies at low stocking density, suggesting that dietary deficiencies produced a chronic elevation of cortisol. When fish were subjected to repetitive chasing, plasma cortisol from fish fed control diet showed a progressive increase whereas plasma cortisol levels from both dietary deficient groups showed a transitory peak 4 days after the beginning of the repetitive chasing. However, ACP activity levels of fish fed deficient diets remained lower than the control group and constant during chasing period. These results suggest that both a-tocopherol and n-3 HUFA nutritional deficiencies rather than stress are an important determinant of fish complement activity.