Rolf Erik Olsen

Lipid digestibility and ultrastructural changes in the enterocytes of Arctic char (Salvelinus alpinus L.) fed linseed oil and soybean lecithin

Arctic char (Salvelinus alpinus L.) were fed two isocaloric diets supplemented with 15% linseed oil or soybean lecithin, and subsequently analysed for ultrastructural changes in various intestinal segments (pyloric caeca region, midgut and hindgut) as well as apparent digestibility coefficients (ADC) of the diets. The ADC of dry matter, lipid and individual fatty acids were always highest in fish fed the soybean lecithin diet. In hindgut for example, ADC of lipid and dry matter were 94 and 96%, respectively, in fish fed the soybean lecithin diet compared to 85 and 94% in fish fed linseed oil.There were major ultrastructural differences in the enterocytes between fish fed the two experimental diets. In char fed the linseed oil diet there were substantial accumulations of lipid droplets in the enterocytes from the pyloric caeca and midgut but not in hindgut. These accumulations were not observed in fish fed the soybean lecithin diet. In some cases, deposition of fat in the enterocytes was so great that it resulted in epithelial damage with lipid droplets and cell debris appearing in the intestinal lumen. These changes are likely to be pathological and may lead to intestinal malfunction and represent a major infection route to pathogenic bacteria. On the basis of these results it is suggested that endogenous phospholipid synthesis is insufficient to maintain lipoprotein synthesis in Arctic char when fed diets containing high levels of polyunsaturated fatty acid of total lipid, and that phospholipids should be supplied with such diets.

Use of immunostimulants and nucleotides in aquaculture: a review

Currently there are numerous gaps in existing knowledge about exogenous nucleotide application to fish including various aspects of digestion, absorption, metabolism, and influences on various physiological responses, especially expression of immunogenes and modulation of immunoglobulin production. Additional information is also needed in regard to age/size-related responses and appropriate doses and timing of administration. Thus further research in these areas should be pursued. Immunostimulants Abstract It is well established that proper nutrition is essential for maintenance of normal growth and health of all animals including various aquatic species. During the last two decades increased attention has focus on immunostimulants and nucleotides to reduce susceptibility to various stressors and diseases, as well as enhance the overall health of fish. The immune response can be modulated by β-glucans and high-M-alginate. β-glucans are glucose polymers that are major structural components of the cell wall of yeast, fungi, and bacteria, but also of cereals like oat and barley. There is much structural variation in the β-glucans from these different sources, which may influence their physiological functions. Alginate is a polysaccharide composed of β-1,4-D-mannuronic acid (M) and α-L-glucuronic acid (G). In vitro as well as in vivo studies in fish show that especially β-glucans derived from fungi and yeast and alginate have immune modulating properties. Most frequently evaluated are effects on macrophage activation and on lysozyme, respiratory burst and leukocyte activity, which have been suggested to contribute to the increased resistance against infections, after immunostimulant exposure. Although more fish studies are needed, it is tempting to suggest that dietary β-glucans and alginate may be useful tools to prime the host immune system and increase resistance against invading pathogens. As no knowledge is available regarding short versus long-term effects and efficiency, more knowledge is needed on this topic.

The effect of dietary fatty acids on lactic acid bacteria associated with the epithelial mucosa and from faecalia of Arctic charr, Salvelinus alpinus (L.)

Arctic charr, Salvelinus alpinus (L.), held in fresh water, were fed four experimental diets containing different polyunsaturated fatty acids (PUFA). In addition, one group fed a diet containing only coconut oil as sole lipid source served as control. The population of aerobic heterotrophic bacteria associated with the epithelial mucosa and the faecalia was estimated using the dilution plate technique. Generally, the population level of adherent bacteria increased along the digestive tract (stomach, small intestine and large intestine). Adherent Gram‐negative and Gram‐positive bacteria seemed to be present at equal levels in all parts of the alimentary tract. Lactic acid bacteria dominated among the Gram‐positive bacteria, and they were detected in all regions of fish fed the PUFA supplemented diets. The frequency of lactic acid bacteria was highest in the digestive tract of fish fed diets with added 7·0% linolenic acid (18:3 n‐3) or 4% of a PUFA mix. A lower frequency of lactic acid bacteria was found in fish fed dietary linoleic acid (18:2 n‐6), and they were absent or present in low numbers in fish fed the coconut oil diet. It is suggested that dietary fatty acids affect the attachment sites for the gastrointestinal microbiota, possibly by modifying the fatty acid composition of the intestine wall. Numerical taxonomy procedures showed that the lactic acid bacteria Carnobacterium spp. and a Carnobacterium piscicola‐like strain were predominant, with smaller numbers of Lactobacillus plantarum, Streptococcus spp. and Leuconostoc mesenteroides present. Seven strains of Carnobacterium spp. were further identified on the basis of 16S rDNA sequence analysis, and all these strains were identified as Carnobacterium piscicola.

Atlantic Salmon (Salmo salar): The “Super-Chicken” of the Sea?

In this article, the definition of sustainability is discussed, particularly in relation to the use of marine feed resources. The current review gives an overview of the development of Atlantic salmon (Salmo salar) aquaculture and how it has evolved due to changes in legal and management framework conditions. Atlantic salmon production is characterized with high utilization of nutrients, a high yield of production, and a large demand for rendered by-products. All of these factors compare favorably to production of most terrestrial farm animals. Historically, salmon feed has contained fishmeal and fish oil as the primary protein and fat source. Rising demand for feed ingredients has not increased pressure on forage fish resources. Rather, there has been an increased use of plant protein and fat sources. Increased utilization of plant ingredients may not be as sustainable as often claimed. Provided that marine harvest is carried out within legal frames, harvesting the marine ecosystem is a sustainable operation, and at present, the only significant source of long chain n-3 fatty acids. It is concluded that Atlantic salmon farming can be compared to raising a marine “super chicken” being among the most sustainable meat products in the world food market.

Lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.)

The present study reports the effect of excessive handling stress and starvation on the lactic acid bacteria associated with the digestive tract of Atlantic salmon (Salmo salar L.). A relatively low population level (approximately 2 × 103 bacteria per gram wet tissue) of viable adherent heterotrophic bacteria was associated with the digestive tract (foregut, midgut and hindgut). Of the 752 bacterial isolates isolated from diet, water and the digestive tract, 201 isolates belonged to the carnobacteria. Of these isolates, one from the diet, one from the rearing water and 80 from the gastrointestinal tract, were further identified on the basis of 16S rDNA sequence analysis. All these isolates were identified as being Carnobacterium piscicola‐like. Daily repeated stress and starvation of the fish over 11 d had no influence on the total culturable bacterial numbers or population level of C. piscicola associated with the digestive tract. C. piscicola‐like isolates colonizing the various intestinal regions (foregut, midgut and hindgut) were also screened for their ability to produce growth inhibitory compounds active against the fish pathogen Aeromonas salmonicida. Of the 199 C. piscicola isolates tested, 139 inhibited growth of the pathogen.

Damaging effect of the fish pathogen Aeromonas salmonicida ssp. salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.

Effect of soybean oil and soybean lecithin on intestinal lipid composition and lipid droplet accumulation of rainbow trout, Oncorhynchus mykiss Walbaum

Rainbow trout (Oncorhynchus mykiss Walbaum) were fed purified diets containing fish oil for six weeks and then soybean lecithin or soybean oil for 25 days. The gastrointestinal tract segments, stomach, midgut and hindgut were then sampled for lipid and fatty acid composition and electron microscopy. Membrane lipid class composition was fairly similar in all three segments of trout fed fish oil. Hindgut contained slightly more phosphatidylserine than stomach and midgut, while midgut contained more phosphatidylcholine and less lysophospatidylcholine/sphingomyelin. Feeding soybean products appeared to marginally decrease free cholesterol. The fatty acid compositions of the main lipid classes showed significant regional differences. In control fish, stomach had higher levels of arachidonic acid (20:4n-6) and n-6 polyunsaturated fatty acids than midgut and hindgut, and lower content of docosahexaenoic acid (22:6n-3) and n-3 polyunsaturated fatty acids. Midgut phosphatidylethanolamine also had higher levels of saturated fatty acids and less n-3 polyunsaturated fatty acids than the other tissues. Feeding soybean products decreased the n-3/n-6 ratio mainly due to increases in linoleic (18:2n-6) and 20:4n-6 and decreases in 22:6n-3 and eicosapentaenoic acid (20:5n-3). Phosphatidylcholine and to a lesser extent phosphatidylethanolamine adapted more readily to dietary changes by major increases in 18:2n-6 and C20−22 n-6 polyunsaturated fatty acids. The composition of phosphatidyl-serine and -inositol appeared to be under more strict metabolic control. Linoleic acid hardly increased at all while the increase in other n-6 polyunsaturated fatty acids was less pronounced than for the other lipid classes. Regardless of lipid class, stomach resisted dietary changes more strongly than midgut and hindgut. Increases in n-6 polyunsaturated fatty acids were minor as were the loss of n-3 polyunsaturated fatty acids. The dead-end product 20:2n-6 accumulated to a higher degree in hindgut phosphatidyl-ethanolamine and -coline compared to midgut and stomach, suggesting that the activity of Δ6 desaturation is higher in the anterior part of the intestine where most of the lipid is absorbed. Feeding soybean oil caused massive accumulation of free lipid droplets in midgut enterocytes while little lipid droplets were observed in trout fed fish oil or soybean lecithin. Since both soybean products influenced intestinal composition to the same degree, altered fatty acid profiles in membranes is not responsible for the observed lipid accumulation. This supports previous observations in Arctic charr (Salvelinus alpinus L.), suggesting that fish may require exogenous phospholipids in order to sustain a sufficient rate of lipoprotein synthesis.

Epithelium‐associated bacteria in the gastrointestinal tract of Arctic charr (Salvelinus alpinus L.). An electron microscopical study

E. RINGØ, J.B. LØDEMEL, R. MYKLEBUST, T. KAINO, T.M. MAYHEW AND R.E. OLSEN. 2001.

Application of vaccines and dietary supplements in aquaculture: possibilities and challenges

The development of vaccines has proven essential for the development of a successful finfish aquaculture industry by preventing the occurrence of diseases like furunculosis and vibriosis in industrialised finfish farming. Further developments, like DNA vaccines, will aid in controlling even more diseases in the future. There are however many diseases where it is difficult to produce effective vaccines. Furthermore, many disease outbreaks may occur due to impaired animal welfare. Identifying factors associated with disease and optimizing health and welfare through biotechnological developments is likely to be an important research area in the future. The fact that dietary manipulation can affect fish gut microbiota thus improving disease resistance is well known from mammalian science, and is slowly gaining ground in finfish research. Both prebiotic and probiotic approaches have been used in fish, with particular focus on lactic acid bacteria. Positive effects include enhanced growth and feed efficiency, improved immunity and disease resistance. The synbiotic concept (using a combination of probiotics and prebiotics) is particularly promising and is gaining increased interest within the research community. Immunostimulants may also improve disease resistence via increase humoral and cellular immune responses. The most promising immunostimulants at present are β-glucans, alginate and Ergosan. Additionally, medical plant extracts and their products are receiving increased attention as immune modulators, but further studies are needed. There are also great expectations or the future usage of microalgae to control microbiota and optimize fish health.

The combined impact of plant-derived dietary ingredients and acute stress on the intestinal arachidonic acid cascade in Atlantic salmon (Salmo salar).

A study was conducted to assess the effect of substituting high levels of dietary fish oil (FO) and fishmeal (FM) for vegetable oil (VO) and plant protein (PP) on the intestinal arachidonic acid (AA) cascade in the carnivorous fish species Atlantic salmon. Four diets were fed to salmon over a period of 12 months, including a control FMFO diet, with varying replacements of plant-derived ingredients: 80 % PP and 35 % VO; 40 % PP and 70 % VO; 80 % PP and 70 %VO. Subsequently, fish were examined pre- (0 h) and post- (1 h) acute stress for blood parameters and intestinal bioactive lipidic mediators of inflammation (prostaglandins). Plasma cortisol responses were greatest in the FMFO group, while 80 % PP and 70 % VO fish exhibited increased plasma chloride concentrations. The n-3:n-6 PUFA ratio in intestinal glycerophospholipids from 70 % VO groups significantly decreased in both proximal and distal regions due to elevated levels of 18 : 2n-6 and the elongation/desaturation products 20 : 2n-6 and 20 : 3n-6. Increases in n-6 PUFA were not concomitant with increased AA, although the AA:EPA ratio did vary significantly. The 40 % PP and 70 % VO diet produced the highest intestinal AA:EPA ratio proximally, which coincided with a trend in elevated levels of PGF2alpha, PGE2 and 6-keto-PGF1alpha in response to stress. PGE2 predominated over PGF2alpha and 6-keto-PGF1alpha (stable metabolite of PGI2) with comparable concentrations in both intestinal regions. Cyclo-oxygenase-2 (COX-2) mRNA expression was an order of magnitude higher in distal intestine, compared with proximal, and was significantly up-regulated following stress. Furthermore, the 80 % PP and 70 % VO diet significantly amplified proximal COX-2 induction post-stress. Results demonstrate that high replacements with plant-derived dietary ingredients can enhance COX-2 induction and synthesis of pro-inflammatory eicosanoids in the intestine of salmon in response to acute physiological stress.