M.J. Caballero

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.

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.

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.

Combined effect of lipid level and fish meal quality on liver histology of gilthead seabream /Sparus aurata

Abstract Effect of eight diets comparing three different lipid levels (15, 22 and 27%) and two fish meal qualities were studied on growth and liver histology. Fish meal quality was judged by the content of biogenic amines and temperature processing techniques. The experiment included a comparison of pelleted feed with extruded feed for the 22% lipid diet. A total of 1140 gilthead seabream of 70 g average initial body weight were randomly stocked in 500-l fiberglass tanks in duplicate groups of 60 fish. After 2 months of experiment, the fish were transferred to 1-m3 tanks. Fish were fed twice a day to apparent satiation for 6 months until they reached about 400 g (commercial size). Fish fed diets containing high quality fish meal showed, in general, a higher growth than those fish fed with low quality fish meal. For diets containing high quality fish meal, the fish fed 22 and 27% dietary lipid had significantly higher growth than those fish fed 15% dietary lipid. On the contrary, in diets containing low quality fish meal, only fish fed 27% dietary lipid showed significantly the higher growth rate. Fish fed the pelletized diets showed a lower growth than those fish fed extruded diets. Livers from fish fed diets containing high quality fish meal and 27% lipid showed foci of swelling hepatocytes that were not found for low quality fish meal at the same dietary lipid content. Ultrastructurally, these foci were characterized to present irregular nuclei displaced to periphery of hepatocytes and large lipid droplets in the cytoplasm. Livers from fish fed high and low fish meal qualities with 22% lipid showed similar morphological characters of hepatocytes to those that fed 15% lipid, but the difference was observed in the nuclei displacement.

Growth, feed utilization and body lipid content of gilthead seabream (Sparus aurata) fed increasing lipid levels and fish meals of different quality

The present study shows the effects of feeding gilthead seabream, 70 to 400 g, with nine diets containing three different lipid levels (15, 22 and 28% on a dry weight basis), combined with two types of fish meal of different quality. In addition, feed processing effects (extruded vs. pelletized) were also compared for the diet containing 22% lipid. The diets prepared with the high quality fish meal promoted better growth than those prepared with the low quality one. Protein utilization was enhanced by a sparing effect of dietary lipid at 22%. This lipid level needed to be increased up to 28% with low quality meal to effectively spare dietary protein. A significant increase in fish growth with higher dietary lipid levels, in both high and low quality fish meal diets, was only related to a significant increase in total body lipid content in high quality fish meal diets, and an excess of lipid may have been the cause of hepatocyte abnormalities in fish fed the highest lipid levels. The effect of pellet processing was apparent in low quality fish meal diets, where a significantly better growth performance was obtained in fish fed the extruded diet. There were no significant differences among all treatments in feed intake, and values were similar to those suggested by commercial fish feed producers, for similar fish size and water temperatures. Values for feed conversion ratio (FCR) were lower than 1.6 for all diets.

Development of red porgy Pagrus pagrus visual system in relation with changes in the digestive tract and larval feeding habits

Abstract Red porgy larvae, like other sparids such as red seabream are visual feeders. The normal development of the visual system is essential for successful prey capture and predator avoidance, leading to increased larval growth and survival. The aim of this work is to characterise the development of visual organs in relation to changes in the digestive system and feeding habits. Twenty-five larvae from hatching to day 29 were daily collected from the rearing tank, fixed in formalin, embedded in paraffin, 5 μm sectioned and stained with haematoxylin and eosin (H&E) and Periodic Acid Shift Reactive-Haematoxiline (PAS-Hx). Light microscopy was used to study changes in ocular morphology with respect to digestive system development. At hatching, eye and digestive systems of Pagrus pagrus larvae have no function. However, at day 3 post-hatch, when the mouth opens, the larvae must be ready for prey capture and digestion. Despite this, few day 3 larvae had food in the digestive tract. At day 4 photoreceptors were well developed in the eye, pigmentation pattern was complete and thus the visual system was completely ready for prey capture. This development coincided with detection of digestive activity in the midgut and most of larvae starting to take food. The results of this study suggest that adequate development of the visual system is important to establish the start of exogenous feeding. Besides, the appearance of rod cells increases larval photosensitivity and suggests that changes in lighting regimes could be necessary throughout the larval phase.

Reduced gut bacterial translocation in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides (MOS)

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg⁻¹ dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.

Response to confinement in sea bass (Dicentrarchus labrax) is characterised by an increased biosynthetic capacity of interrenal tissue with no effect on ACTH sensitivity

Two experiments were carried out to investigate the influence of confinement stress on plasma cortisol levels and on the sensitivity of the interrenal cells to adrenocorticotropic hormone (ACTH) stimulation in sea bass Dicentrarchus labrax. Confining sea bass at 70 kg m(-3) for 24 h resulted in elevated plasma cortisol levels at all times (0.1, 1, 4 and 24 h) and corresponded to a reduced cortisol content in head-kidney homogenates after 0.1 and 1 h of confinement. An increased activity of the interrenal cells was also indicated by the enlarged nuclear diameters measured after 1 and 4 h of confinement. In vitro superfusion experiments showed that 4 h of confinement resulted in an increased basal unstimulated release of cortisol from head-kidney tissues compared with that in unstressed control fish. Although the stimulation factor (cortisol release as percent increase above basal) of the stressed fish was significantly lower than in controls, no difference in the maximal stimulated release (in absolute amounts) was evident between stressed and control fish. Care must be taken when interpreting superfusion data, as to whether the stressor actually leads to a reduction in interrenal sensitivity, or is due to an alteration in the basal release of cortisol.

Vegetable lipid sources affect in vitro biosynthesis of triacylglycerols and phospholipids in the intestine of sea bream (Sparus aurata).

Despite the good growth performance of several fish species when dietary fish oil is partly replaced by vegetable oils, recent studies have reported several types of intestinal morphological alterations in cultured fish fed high contents of vegetable lipid sources. However, the physiological process implied in these morphological changes have not been clarified yet, since alterations in the physiological mechanisms involved in the different processes of lipid absorption could be responsible for such gut morphological features. The objective of the present study was to investigate the activities of reacylation pathways in fish, the glycerol-3-phosphate and the monoacylglycerol pathways, in order to clarify the intestinal triacylglycerol (TAG) and phospholipid biosynthesis to better understand the morphological alterations observed in the intestine of fish fed vegetable oils. Intestinal microsomes of sea bream fed different lipid sources (fish, soyabean and rapeseed oils) at three different inclusion levels were isolated and incubated with L-[(14)C(U)]glycerol-3-phosphate and [1-(14)C]palmitoyl CoA. The results showed that in this fish species the glycerol-3-phosphate pathway is mainly involved in phospholipid synthesis, whereas TAG synthesis is mainly mediated by the monoacylglycerol pathway. Feeding with rapeseed oil reduced the reacylation activity in both pathways, explaining the high accumulation of lipid droplets in the supranuclear portion of the intestinal epithelium, whereas soyabean oil enhanced phosphatidylcholine synthesis, being associated with the increase in VLDL found in previous studies.

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation. Fish were fed 4 g kg(-1) MOS (Bio-Mos(®) Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents. Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet. The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.