Daniel Montero

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.

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.

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.

Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress

Abstract High stocking density is an aquaculture-related situation which causes crowding stress in fish and potentially affecting the immune system. Protection against disease can be induced through immunostimulation, enhancing defence mechanisms. Vitamins C and E are among the most important nutrients influencing the immune system, although the effect of these vitamins in the amelioration of stress in fish has not been studied in detail. An experiment was carried out to study the effect dietary supplementation of vitamin C or vitamin E on the immune system of fish under a crowding stress situation in a commercial farm. A commercial diet for this species was supplemented with vitamin C or vitamin E and these diets were assayed at high stocking density (over 40 kg m−3) and the results were compared with those from fish fed the commercial diet and held at farm conditions (20 kg m−3). Some biological and immunological parameters were studied at the end of the experimental period. Fish held at high stocking density showed lower final weight than fish held at lower stocking density. These also showed higher plasma cortisol levels (over 6 ng cortisol ml−1 plasma for all the diets assayed) than those fish held at lower density (1.13 ng cortisol ml−1 plasma). High stocking density produced higher serum lysozyme activity values in fish fed the commercial diet compared with those fish held at lower density (67.49 and 36.84 units ml−1, respectively). This effect was not observed in fish held at high stocking density but fed vitamin C and vitamin E supplemented diets (lysozyme activity: 21.97 and 19.71 units ml−1, respectively). High stocking density also appeared to decrease serum alternative complement activity. In those fish fed the vitamin E supplemented diet, levels were similar to fish held at low stocking density, suggesting the role of this vitamin in this defense mechanism.

Background colour influence on the stress response in cultured red porgy Pagrus pagrus

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

Serum haemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies

Gilthead sea bream, Sparus aurata, were subjected to different stressful situations and the haemolytic activity mediated by the alternative complement and the agglutinating serum activity were tested as indicators of immunocompetence. The results show that both parameters were significantly compromised after chronic or repeated acute stress as well as in groups fed with vitamin E or Ω3 highly unsaturated fatty acids (HUFA) deficient diets and in infected fish. These results suggest that both haemolytic and agglutinating activity are suitable indicators for immunocompetence as they are non-specific responses induced by non-specific stressors. In addition, a comparison is performed with other immune indicators subjected to the same stressors.

Replacement of dietary fish oil by vegetable oils affects humoral immunity and expression of pro-inflammatory cytokines genes in gilthead sea bream Sparus aurata.

Commercial gilthead sea bream feeds are highly energetic, fish oil traditionally being the main lipid source. But the decreased fish oil production together with the increased prices of this oil encourages its substitution by vegetable oils, imposing new nutritional habits to aquaculture species. Partial replacement of fish oil by vegetable oils in diets for marine species allows good feed utilization and growth but may affect fish health, since imbalances in dietary fatty acids may alter fish immunological status. The effect of dietary oils on different aspects of fish immune system has been reported for some species, but very little is known about the effect of dietary oils on immune-related genes expression in fish. Thus, the objective of this study was to elucidate the role of dietary oils on the expression of two pro-inflammatory cytokines, Tumor Necrosis Factor-α (TNF-α) and Interleukine 1β (IL-1β) on intestine and head kidney after exposure to the bacterial pathogen Photobacterium damselae sp. piscicida. For that purpose, 5 iso-nitrogenous and iso-lipidic diets (45% crude protein, 22% crude lipid content) were formulated. Anchovy oil was the only lipid source used in the control diet (FO), but in the other diets, fish oil was totally (100%) or partially (70%) substituted by linseed (rich in n-3 fatty acids) or soybean (rich in n-6 fatty acids) (100L, 100S, 70L, 70S). Fish were fed experimental diets during 80 days and after this period were exposed to an experimental intestinal infection with the pathogen. Serum and tissue samples were obtained at pre-infection and after 1, 3 and 7 days of infection. RNA was extracted and cDNA was synthesized by reverse transcription from intestine and head kidney and the level expression of TNF-α and IL-1β were assayed by using quantitative real time PCR. The expression level of genes analysed was represented as relative value, using the comparative Ct method (2(-ΔΔCt)). Serum anti-bacterial activity was measured as serum bactericidal capacity and lysozyme activity. Reduction of FO tends to reduce basal (pre-infection) genetic expression of both cytokines. However, complete FO replacement caused an over expression of both pro-inflammatory cytokines, particularly after 3 days of induced infection in fish fed soybean oil based diets. On the other hand, fish fed diets with low content of n-6 fatty acids showed better serum bactericidal capacity after infection, suggesting that the substitution of fish oil by vegetable oils containing high levels of n-6 fatty acids may induce imbalances on fish immune response, leading to a lower potential response against infections.