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Nutritional impacts on fish mucosa: dietary considerations
Nutritional impacts on fish mucosa: dietary considerations
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Jesse Trushenski Center for Fisheries, Aquaculture, and Aquatic Sciences, Southern Illinois University Carbondale, Illinois, USA Chapter Outline 8.1 Introduction 199 8.2 Lipids and gut health 200 8.3 Antinutritional factors and gut health 202 8.4 Concluding remarks 206 References 206
8.1 Introduction The alimentary canal or gut, while functioning primarily to acquire nutrients and excrete wastes, also contributes significantly to the immunocompetence of fish. Both roles, nutritional and immunological, are important in terms of the immune system of fish. First, a fish’s immune system will most certainly be compromised if it is unable to digest and absorb the energy and essential nutrients necessary for basic homeostasis, maintenance of immunologically important cells, and synthesis of various factors such as enzymes and immunoglobulins. The importance of nutritional status in determining immunocompetence cannot be overstated. Therefore, the gut’s role in digestion and absorption of nutrients is relevant, albeit indirectly, to a fish’s health. Second, the gut mucosa is a primary site of pathogen exclusion and immunological engagement. The interplay between the availability of essential nutrients and the function of the immune system is largely outside the scope of this chapter; thus, we will focus on the gut’s second, more direct role as part of the immune system (i.e., the effect of certain dietary constituents on the competency of the gut as part of the immune system). The gut has various attributes that inhibit pathogen translocation, including the production of mucus with antibacterial properties, the acidic nature of the lumen and/or apical surfaces of the enterocytes, rapid cell turnover, peristaltic activity, and the production of lysozyme (Ringø et al., 2004). Collectively, these constitute a range of mechanical and chemical barriers for infection. Beneficial microbes present within the gut serve as a microbiological barrier to infection, outcompeting pathogenic microbes for space and nutrients needed for proliferation or creating conditions in the gut that are inhospitable for pathogens (Kesarcodi-Watson et al., 2008). Additionally, the enterocytes exhibit substantial capacity for endocytosis, which is likely a major contributor to the gut’s function Mucosal Health in Aquaculture Copyright © 2015 Elsevier Inc. All rights reserved.
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as part of the immune system (i.e., antigen processing and presentation, clearance of localized infections, prevention of systemic infections, and induction of adaptive immune responses and immunological memory) (Bakke-McKellep et al., 2000). Despite these preventative mechanisms, the gut can be a site of pathogen translocation. For example, infection with Aeromonas salmonicida causes significant histological damage to the gut of Atlantic salmon (Salmo salar), suggesting that bacteria may enter the body via transcellular or paracellular pathways in the gut or by damaging enterocytes and/or dislodging them. This chapter will focus on two categories of dietary constituents, lipids and antinutritional factors, that may make such translocation events more likely and, in turn, increase susceptibility to infection and disease.
8.2 Lipids and gut health As noted above, the gut plays many roles related to nutrition, immunity, and general fish health, but these largely relate to its ability to facilitate or prevent the exchange of endogenous and exogenous materials, including nutrients, wastes, osmolytes, and pathogens. These exchanges may occur via trans- or paracellular pathways, and the relative ease with which they occur is determined by, among other things, the nature of the epithelial membranes themselves. The functional integrity of cell membranes partially depends on their lipid and fatty acid composition. This is particularly true among poikilothermic animals that routinely alter membrane composition in order to maintain fluidity and functionality under different thermal conditions – a process commonly known as “homeoviscous adaptation” (Arts and Kohler, 2009). Given that membrane function is linked to fatty acid/lipid composition and tissue composition in fish is strongly influenced by dietary composition (Turchini et al., 2009; Trushenski and Bowzer, 2012), it is reasonable to suppose that intestinal mucosal competence and fish health may be influenced by dietary lipid and fatty acid intake. In reviewing the possible avenues by which polyunsaturated fatty acids might influence the intestinal mucosa of mammals, Donnet-Hughes and colleagues (Donnet-Hughes et al., 2001) suggested that fatty acid intake might influence gut health via mechanisms related to eicosanoid synthesis, cytokine production and reactivity of immune cells, maintenance of a tight intestinal barrier, and composition of the gut microflora. More recently, it has been suggested that the lipidome (the complete lipid profile of a cell, tissue, or organism) might be targeted and harnessed by pathogens in order to avoid detection and clearance by the immune system (van der Meer-Janssen et al., 2010). Although lipid/mucosa interactions are still not fully understood, the literature suggests there are functional relationships between lipid/fatty acid intake and gut health in vertebrates, including fish. To perform optimally, cellular membranes must be strong but flexible. The balance between these two attributes determines how well a membrane performs in terms of permeability, endocytic processes, embedded enzyme function, and translocation of ions and other substrates (Arts and Kohler, 2009). Thus, cellular membranes must be comprised of elements that disrupt strict rigidity and confer strength as well as fluidity to the lipid bilayer. Cholesterol and phospholipids containing saturated fatty acids provide strength, particularly at warm temperatures. Conversely, phospholipids
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containing unsaturated fatty acids provide fluidity, particularly at cold temperatures. Long-chain polyunsaturated fatty acids (LC-PUFA) with their many double bonds and coiled or semi-coiled structures are especially relevant in this context. For example, docosahexaenoic acid (22:6n-3, DHA) is known to influence various aspects of membrane function, including membrane permeability, fusion, and elasticity as well as vesicle formation (Arts and Kohler, 2009). The fact that the lipid and fatty acid composition of fish tissues reflects dietary intake is perhaps one of the best-documented phenomena in fish nutrition and physiology (Turchini et al., 2009; Trushenski and Bowzer, 2012). Although most research investigating tissue composition in fish is focused on edible tissues, a number of studies have reported differences in composition of the intestinal tissues and their associated properties. It is known that composition of the gut epithelium varies with function along the length of the intestine in fish. Although the levels of phospholipids, cholesterol, and proteins appear to be relatively consistent throughout the intestine of trout, membranes of the mid-intestine are reportedly more fluid and richer in unsaturated fatty acids than those of the distal intestine (Pelletier et al., 1986). Various manipulations are known to affect the lipid/fatty acid composition and, presumably, the function of the gut epithelium. For example, transferring trout from freshwater to seawater causes DHA to become enriched within the brush border tissues and fluidity of the membranes to increase (Leray et al., 1984). The same effect was induced in carp held at cold temperatures by supplementing their diet with LC-PUFA-rich fish oil (Behar et al., 1989). Feeding fish oil to channel catfish (Ictalurus punctatus) altered the fatty acid composition of the brush border membranes in a similar fashion, but this was associated with reduced glucose transport in comparison to feeding with a non-fish oil, stearic acid (18:0)-rich diet (Houpe et al., 1997). Conversely, accumulation of monounsaturated fatty acids (MUFA) at the expense of LC-PUFA within the gut epithelium of European seabass (Dicentrarchus labrax) was associated with reduced activities of various brush border enzymes (Cahu et al., 2000). Interestingly, these changes were induced not by feeding diets with different fatty acid profiles, but by feeding increasing amounts of the same lipid (fish oil), suggesting that absolute levels of lipid in the diet may also influence membrane composition and function. Although compositional analyses were not conducted, the enterocytes of gilthead seabream (Sparus aurata) that were fed vegetable oils exhibited a different structure than those of fish that were fed fish oil (Caballero et al., 2003). The enterocytes of fish given rapeseed, linseed, and soybean oils contained substantially more lipid droplets and exhibited altered capacities for lipoprotein synthesis, though the enterocytes otherwise appeared morphologically normal. Few studies have investigated the relationship between lipid nutrition, membrane composition or structure, and competency of the intestinal mucosa as a pathogen barrier. However, the limited data available suggests that a functional relationship may exist. Atlantic salmon successfully completed smoltification, regardless of whether they were fed diets containing sunflower oil or fish oil (Jutfelt et al., 2007). However, basal cortisol levels suggested fish that were fed the sunflower oil diet experienced a greater degree of physiological stress during smoltification, though nutrient uptake and prevention of bacterial translocation was apparently greater in these fish.
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Arctic char Salvelinus alpinus fed diets containing marine-origin oil, linseed oil, or soybean oil exhibited differential survival rates following challenges with A. salmonicida, a pathogen thought to invade primarily by disruption of the gut epithelium (Lødemel et al., 2001). In this study, fish given the plant-origin oils exhibited reduced mortality. The authors were unable to unequivocally determine the basis for greater survival among fish served soybean or, to a lesser extent, linseed oil, but suggested changes in the microfloral community and/or increased production of mucus might have reduced adherence of A. salmonicida to the gut epithelium and subsequent infection.
8.3 Antinutritional factors and gut health The functional competency of the gut in preventing pathogen translocation is linked to the structural integrity of the enterocytes and supporting tissues. For many years, fish nutritionists have known that a variety of feedstuffs, particularly plant-derived ingredients, may be toxic, cause feed palatability problems, or impair nutrient utilization and growth performance because of various “antinutritional factors” (ANFs) (Tacon, 1997; Francis et al., 2001). Numerous plant-derivatives are known to contain ANFs, including meals and protein concentrates derived from various legumes, including pulses, oilseed, and sowing crops. However, soybean products are likely the most widely recognized for their ANF content, containing protease inhibitors, lectins, phytic acid, saponins, phytoestrogens, allergens, and other problematic constituents (Tacon, 1997; Francis et al., 2001). Soybean derivatives are also noteworthy for their association with the development of enteritis in fish, particularly salmonids (Refstie and Storebakken, 2001; Gatlin et al., 2007). Soy-induced enteritis, or inflammation of the gut, is commonly characterized by the widening, shortening, and occasional disruption or fusion of the intestinal villi; a reduced number of supranuclear vacuoles in the absorptive epithelium; the widening and infiltration of the lamina propria by various types of leukocytes; and elevated levels of lysozyme and IgM in the gut mucosa. It can also be accompanied by a more generalized immune response, involving increases in circulating immunoglobulins and other proteins, as well as increases in the number and activity of circulating leukocytes (Refstie and Storebakken, 2001). Soy-induced enteritis may interfere with nutrient absorption, but the loss of the gut’s function as a barrier to pathogen translocation and participation in antigen recognition, processing, and presentation may be of greater importance since the signs of inflammation in soyinduced enteritis are generally restricted to the distal intestine that, more than likely, is less involved in nutrient absorption than in immunological processes (Rombout et al., 2011). The effects of plant proteins on gut integrity appear to be largely restricted to soy derivatives. In a screening of various legume, oilseed, and cereal-derived feedstuffs, only soybean meal-fed Atlantic salmon exhibited altered gut histomorphology (Aslaksen et al., 2007). Similarly, rainbow trout (Oncorhynchus mykiss) that were fed lupin meal failed to exhibit signs of enteritis (Glencross et al., 2004). Since soybean-derived products can contain a variety of ANFs, it is not clear which of these is the causative
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agent in the development of soy-induced enteritis. Based on their effects in fish and other species, it has been suggested that trypsin inhibitors, phytoestrogens, or lectins may be responsible for soybean meal-related enteritis (Sanden et al., 2005), which is consistent with most experiments associating soybean meal with enteritis as the alcohol-extraction process most commonly used to prepare soybean meal does not remove these substances (Aslaksen et al., 2007). Other processing strategies effectively remove some, but not all, of the ANFs that plant products contain, resulting in products that have yielded mixed results in feeding trials. Soy protein concentrate, for example, contains lower levels of phytic acid, lectins, saponins, and oligosaccharides than soybean meal. Atlantic salmon that were fed soy protein concentrate did not exhibit enteritis, however, fish given pea protein concentrate did exhibit the classic signs of inflammation and villous atrophy of the distal intestine, along with reductions in gut enzyme activities and macronutrient digestibilities (Penn et al., 2011). Both concentrates benefit from processing, which removes or neutralizes many ANFs. However, not all ANFs are addressed equally by the processing methods specific to producing each concentrate. For example, saponins are greatly reduced in soy protein concentrate during alcohol extraction of soybean meal, but there is no equivalent step to reduce saponin levels in producing pea protein concentrate. Thus, saponins are abundant in pea protein concentrate, which induces enteritis, but not in soy protein concentrate, which does not, suggesting that saponins are a likely contributor to this response in salmonids (Penn et al., 2011). However, in another study, Atlantic salmon did not develop enteritis after being fed diets supplemented with saponins or the purported ANF oligosaccharides, stachyose and raffinose, which suggests that some other component is responsible for inducing soyrelated enteritis (Sorensen et al., 2011). In an attempt to elucidate the contribution of saponins to soybean meal-induced enteritis, Bureau and colleagues (Bureau et al., 1998) fed rainbow trout and Chinook salmon (Oncorhynchus tshawytscha) diets containing soybean meal or soy protein concentrate supplemented with an alcohol extract of soybean meal to approximate the same level of soy-derived saponins. Both diets suppressed feed intake in Chinook salmon, but not in rainbow trout. Expectedly, histological analysis of the distal intestine of Chinook salmon revealed abnormalities consistent with reduced or absent feeding, whereas the distal intestine of rainbow trout was generally unaffected by the soybean meal or soy saponin-supplemented feeds. The distal intestine of Japanese flounder (Paralichthys olivaceus) was similarly affected by saponin-supplemented feeds, but it was unclear whether the enteritis observed was the result of reduced feed intake or a specific response to dietary saponin content (Chen et al., 2011). The most significant histopathological effects of soy products appear to be primarily associated with salmonids, but there are exceptions to this generalization, and it is likely that different taxa respond differently to soy derivatives and the ANFs they contain. The distal intestines of cobia (Rachycentron canadum) (Romarheim et al., 2008), Egyptian sole (Solea aegyptiaca) (Bonaldo et al., 2006), and European seabass (Bonaldo et al., 2008) were completely unaffected by feeding soybean meal. Similarly, the intestinal integrity of hybrid striped bass (Morone chrysops x M. saxatilis) was largely unaffected by feeding soybean meal, soy protein concentrate, or soy protein
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isolate (Blaufuss and Trushenski, 2012; Laporte and Trushenski, 2012). Gilthead seabream exhibited only minor infiltration of leukocytes into the lamina propria of the distal intestine (Bonaldo et al., 2008). Generally, plant proteins are not considered as problematic in feeds for herbivorous or omnivorous fish, but interspecies variation is also observed among these nutritional guilds: soy-induced enteritis is virtually unknown in species such as channel catfish (Evans et al., 2005), but has been demonstrated in common carp (Cyprinus carpio) (Urán et al., 2008). Although there is apparently variation in the dietary constituents that induce enteritis and how fish respond to these ANFs, the signs of diet-induced enteritis are somewhat more consistent. These include changes in the morphology and functionality of the gut, particularly the distal intestine, as well as apparent broader involvement of the immune system. For example, soybean meal-induced enteritis in Atlantic salmon and is characterized by hypertrophic or hyperanaemic mucosa, shortening of the mucosal folds, and widening and infiltration of the lamina propria by various leukocytes. These effects are accompanied by changes in mucosal enzyme activities as well as an increase in IgM. Similar morphological changes were observed in both rainbow trout and Atlantic salmon that were fed soybean meal; however, inflammatory cell infiltration was less pronounced in the former (Refstie et al., 2000). Collectively, these results suggest that element(s) of soybean meal have a toxic effect on the apical membrane of fish and may delay or otherwise interfere with the production of new enterocytes. Cellular damage coupled with a reduced ability to replace damaged enterocytes implies reduced functionality of the gut in terms of nutrient absorption and pathogen exclusion. Mucosal IgM was elevated in the mid-intestine of Atlantic salmon that were fed both soy protein concentrate and soy molasses; IgM was also elevated in the distal intestine, but only among those fish fed with soy molasses (Krogdahl et al., 2000). Mucosal lysozyme activity was elevated in the mid- and distal intestine of fish given soy molasses. Under some circumstances, these responses might be associated with a state of increased “immunological readiness”; however, these authors considered elevated IgM and lysozyme activity as primarily a consequence of gut inflammation (Krogdahl et al., 2000). Conversely, Atlantic salmon fed with relatively low levels of soy and maize did not consistently exhibit significant changes in the cytophysiology of the gut. Fish that were fed soybean meal exhibited significantly increased enterocyte proliferation and migration of immature or proliferating cells up the villous fold, whereas fish fed maize exhibited the opposite, though this effect was observed in only a few individuals (Sanden et al., 2005). The same increase in cellular proliferation was observed in Atlantic salmon that were given soybean meal (Bakke-McKellep et al., 2007). However, the inflamed gut was further characterized by increased and widespread heat-shock protein (HSP)70 and caspase-3 immunohistochemical reactivities that indicate elevated attempts at cellular repair and apoptosis, respectively, in the soy-fed fish (Bakke-McKellep et al., 2007). Overall, these results reinforce the concept of enteritis as cellular response that attempts to compensate for the “cytotoxic” effect of some as-yet unidentified element(s) of soybean meal. Despite exhibiting the classical signs of soybean meal-induced enteritis in the distal intestine, nutrient transport and uptake of various nutrients were not predictably altered
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in rainbow trout and Atlantic salmon with fed soybean meal-based feeds (Nordrum et al., 2000). However, the authors stated that their results were suggestive of increased membrane permeability in the inflamed intestinal tissues (Nordrum et al., 2000). Regardless of whether greater membrane permeability is a cause or an effect of soybean meal-induced damage to the intestinal mucosa, it raises the possibility of increased vulnerability to pathogen translocation. For example, Krogdahl and colleagues (Krogdahl et al., 2000) observed significantly greater mortality among Atlantic salmon that were fed soybean meal-based feeds following A. salmonicida challenge than fish that were fed a soy protein concentrate-based feed. Moreover, lower mortality was observed in fish given the soy protein concentrate-based feed than the fish given the control fish meal-based feed. Although possible mechanisms for these observations were not addressed in this work, the authors did suggest that reduced epithelial integrity of the inflamed gut may have rendered fish fed with the soybean meal-based feed more vulnerable to infection. Alternatively, the “systemic health” of Atlantic salmon may have been generally compromised by soybean meal-induced enteritis and accompanying diarrhea, which rendered them, in their weakened state, more susceptible to disease (Krogdahl et al., 2000). Similarly, rainbow trout fed high levels (80% or more of the diet) of standard or dehulled soybean meal or soy protein concentration exhibited significantly reduced head kidney macrophage competency as measured by respiratory burst activity (Burrells et al., 1999). These changes were associated with reduced growth rates, but not with a systemic immunogenic response to soy proteins, suggesting that there may be some merit to the concept that soy-based feeds generally reduce overall fish health by affecting their nutritional status and that the immunological response to soy antigens is largely a localized event restricted to the distal intestine. Again, these effects appear to be somewhat unique to soy-derived ANF since various enzymatic and blood chemistry measures of immune function were not affected in Atlantic salmon that were fed lupin meal or hydrolyzed poultry feather meal as alternatives to fish meal, and survival following challenge with Vibrio anguillarum was also equivalent among these fish (Bransden et al., 2001). The effects of soy-derived ANFs may directly influence gut integrity and the immune system of fish; however, indirect effects observed with or without the classical signs of diet-induced enteritis may also be relevant in terms of general fish health. For example, Atlantic cod (Gadus morhua) did not exhibit major shifts in histomorphology or enzyme activities normally associated with feeding soybean meal, but other fish fed with soybean meal did exhibit increased densities of non-adherent bacteria (transient, allochthonous) compared to adherent bacteria (indigenous, autochthonous) in the gut (Refstie et al., 2006). Additionally, the taxonomic composition of the culturable gut bacteria was also altered in cod fed with soybean meal. These effects were not apparent among fish that were fed a bio-processed, enzyme-treated soybean meal. Although these authors could not provide specific evidence regarding diet-induced differences in the fish’s ability to effectively exclude pathogenic bacteria at the level of the gut mucosa, they suggested this possibility and recommended further research on this effect (Refstie et al., 2006). Conversely, in Atlantic salmon exhibiting classical soybean meal-induced enteritis, there was a significant increase in the densities of
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adherent bacteria in both the mid- and distal intestine, whereas non-adherent bacteria were not affected in the mid-intestine but significantly elevated in the distal intestine (Bakke-McKellep et al., 2007). These authors also reported shifts in the taxonomic composition of the gut microflora, but could not state whether these changes were a cause or effect of the observed enteritis (Bakke-McKellep et al., 2007).
8.4 Concluding remarks The literature on the topic of certain ANFs and gut enteritis in fish is extensive; there are far fewer investigations of the effect(s) that lipid nutrition may have on intestinal integrity and function. Undoubtedly, other ANFs and nutrients may also influence gut health in fish, but, if there is research being conducted on these topics, it is not yet readily apparent in the literature. In all cases, there is a dearth of experimentation comprehensively addressing the effects dietary constituents may have on the physiological and morphological condition of the gut mucosa and, more importantly, the effects of these nutrients or antinutrients on the functional competence of the gut in terms of nutrient acquisition as well as pathogen sampling and exclusion. Studies addressing both basic and applied aspects of nutrition as it pertains to gut health are needed and encouraged.
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Laporte, J., Trushenski, J.T., 2012. Production performance, stress tolerance, and intestinal integrity of sunshine bass fed increasing levels of soybean meal. J. Anim. Physiol. Nutr. 96, 513–526. Leray, C., Chapelle, S., Duportail, G., Florentz, Al., 1984. Changes in fluidity and 22: 6(n-3) content in phospholipids of trout intestinal brush border membrane as related to environmental salinity. Biochimica et Biophysica Acta Biomembranes 778, 233–238. Lødemel, J.B., Mayhew, T.M., Myklebust, R., Olsen, R.E., Espelid, S., Ringø, E., 2001. Effect of three dietary oils on disease susceptibility in Arctic charr (Salvelinus alpinus L.) during cohabitant challenge with Aeromonas salmonicida ssp. Salmonicida. Aquacult. Res. 32, 935–945. Nordrum, S., Bakke-McKellep, A.M., Krogdahl, Å., Buddington, R.K., 2000. Effects of soybean meal and salinity on intestinal transport of nutrients in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Phys. B 125, 317–335. Pelletier, X., Duportail, G., Leray, C., 1986. Isolation and characterization of brush border membrane from trout intestine, regional differences. Biochimica et Biophysica Acta Biomembranes 856, 267–273. Penn, M.H., Bendiksen, E.Å., Campbell, P., Krogdahl, Å., 2011. High level of dietary pea protein concentrate induces enteropathy in Atlantic salmon (Salmo salar L.). Aquaculture 310, 267–273. Refstie, S., Korsøen, Ø.J., Storebakken, T., Baeverfjord, G., Lein, I., Roem, A.J., 2000. Differing nutritional responses to dietary soybean meal in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Aquaculture 190, 49–63. Refstie, S., Landsverk, T., Bakke-McKellep, A.M., Ringø, E., Sundby, A., Shearer, K.D., Krogdahl, Å., 2006. Digestive capacity, intestinal morphology, and microflora of 1-year and 2-year old Atlantic cod (Gadus morhua) fed standard or bioprocessed soybean meal. Aquaculture 261, 269–284. Refstie, S., Storebakken, T., 2001. Vegetable protein sources for carnivorous fish: potential and challenges. Rec. Adv. Anim. Nutr. Aust. 13, 195–203. Ringø, E., Jutfelt, F., Kanapathippillai, P., Bakken, Y., Sundell, K., Glette, J., Mayhew, T.M., Myklebust, R., Olsen, R.E., 2004. Damaging effect of the fish pathogen Aeromonas salmonicida ssp. Salmonicida on intestinal enterocytes of Atlantic salmon (Salmo salar L.). Cell Tissue Res. 318, 305–311. Romarheim, O.H., Zhang, C., Penn, M., Liu, Y.-J., Tian, L.-X., Skrede, A., Krogdahl, Å., Storebakken, T., 2008. Growth and intestinal morphology in cobia (Rachycentron canadum) fed extruded diets with two types of soybean meal partly replacing fish meal. Aquacult. Nutr. 14, 174–180. Rombout, J.H.W.M., Abelli, L., Picchietti, S., Scapigliati, G., Kiron, V., 2011. Teleost intestinal immunology. Fish Shellfish Immunol. 31, 616–626. Sanden, M., Berntssen, M.H.G., Krogdahl, Å., Hemre, G.-I., Bakke-McKellep, A.M., 2005. An examination of the intestinal tract of Atlantic salmon, Salmo salar L., parr fed different varieties of soy and maize. J. Fish Dis. 28, 317–330. Sørensen, M., Penn, M., El-Mowafi, A., Storebakken, T., Chunfang, C., Øverland, M., Krogdahl, Å., 2011. Effect of stachyose, raffinose and soya-saponins supplementation on nutrient digestibility, digestive enzymes, gut morphology and growth performance in Atlantic salmon (Salmo salar L.). Aquaculture 314, 145–152. Tacon, A.G.J., 1997. Fishmeal replacers: review of antinutrients within oilseeds and pulses – a limiting factor for the aquafeed Green Revolution? In: Tacon, A.G.J., Basurco, B. (Eds.), Feeding Tomorrow’s Fish. International Centre for Advanced Mediterranean Agronomic Studies, Paris, pp. 153–182.
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Trushenski, J.T., Bowzer, J.C., 2012. Having your omega 3 fatty acids and eating them, too: strategies to ensure and improve the long-chain polyunsaturated fatty acid content of farm-raised fish. In: De Meester, F., Watson, R.R., Zibadi, S. (Eds.), Sustainable Long Chain Omega-3 Fatty Acids in Cardiovascular and Mental Health. Springer, New York, pp. 319–340. Turchini, G.M., Torstensen, B.E., Ng, W.-K., 2009. Fish oil replacement in finfish nutrition. Rev. Aquacult. 1, 10–57. Urán, P.A., Gonçalves, A.A., Taverne-Thiele, J.J., Schrama, J.W., Verreth, J.A.J., Rombout, H.W.M., 2008. Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.). Fish Shellfish Immunol. 25, 751–760. van der Meer-Janssen, Y.P.M., van Galen, J., Batenburg, J.J., Helms, B., 2010. Lipids in hostpathogen interactions: pathogens exploit the complexity of the host cell lipidome. Prog. Lipid Res. 49, 1–26.
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Jesse Trushenski Center for Fisheries, Aquaculture, and Aquatic Sciences, Southern Illinois University Carbondale, Illinois, USA Chapter Outline 8.1 Introduction 199 8.2 Lipids and gut health 200 8.3 Antinutritional factors and gut health 202 8.4 Concluding remarks 206 References 206
8.1 Introduction The alimentary canal or gut, while functioning primarily to acquire nutrients and excrete wastes, also contributes significantly to the immunocompetence of fish. Both roles, nutritional and immunological, are important in terms of the immune system of fish. First, a fish’s immune system will most certainly be compromised if it is unable to digest and absorb the energy and essential nutrients necessary for basic homeostasis, maintenance of immunologically important cells, and synthesis of various factors such as enzymes and immunoglobulins. The importance of nutritional status in determining immunocompetence cannot be overstated. Therefore, the gut’s role in digestion and absorption of nutrients is relevant, albeit indirectly, to a fish’s health. Second, the gut mucosa is a primary site of pathogen exclusion and immunological engagement. The interplay between the availability of essential nutrients and the function of the immune system is largely outside the scope of this chapter; thus, we will focus on the gut’s second, more direct role as part of the immune system (i.e., the effect of certain dietary constituents on the competency of the gut as part of the immune system). The gut has various attributes that inhibit pathogen translocation, including the production of mucus with antibacterial properties, the acidic nature of the lumen and/or apical surfaces of the enterocytes, rapid cell turnover, peristaltic activity, and the production of lysozyme (Ringø et al., 2004). Collectively, these constitute a range of mechanical and chemical barriers for infection. Beneficial microbes present within the gut serve as a microbiological barrier to infection, outcompeting pathogenic microbes for space and nutrients needed for proliferation or creating conditions in the gut that are inhospitable for pathogens (Kesarcodi-Watson et al., 2008). Additionally, the enterocytes exhibit substantial capacity for endocytosis, which is likely a major contributor to the gut’s function Mucosal Health in Aquaculture Copyright © 2015 Elsevier Inc. All rights reserved.
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as part of the immune system (i.e., antigen processing and presentation, clearance of localized infections, prevention of systemic infections, and induction of adaptive immune responses and immunological memory) (Bakke-McKellep et al., 2000). Despite these preventative mechanisms, the gut can be a site of pathogen translocation. For example, infection with Aeromonas salmonicida causes significant histological damage to the gut of Atlantic salmon (Salmo salar), suggesting that bacteria may enter the body via transcellular or paracellular pathways in the gut or by damaging enterocytes and/or dislodging them. This chapter will focus on two categories of dietary constituents, lipids and antinutritional factors, that may make such translocation events more likely and, in turn, increase susceptibility to infection and disease.
8.2 Lipids and gut health As noted above, the gut plays many roles related to nutrition, immunity, and general fish health, but these largely relate to its ability to facilitate or prevent the exchange of endogenous and exogenous materials, including nutrients, wastes, osmolytes, and pathogens. These exchanges may occur via trans- or paracellular pathways, and the relative ease with which they occur is determined by, among other things, the nature of the epithelial membranes themselves. The functional integrity of cell membranes partially depends on their lipid and fatty acid composition. This is particularly true among poikilothermic animals that routinely alter membrane composition in order to maintain fluidity and functionality under different thermal conditions – a process commonly known as “homeoviscous adaptation” (Arts and Kohler, 2009). Given that membrane function is linked to fatty acid/lipid composition and tissue composition in fish is strongly influenced by dietary composition (Turchini et al., 2009; Trushenski and Bowzer, 2012), it is reasonable to suppose that intestinal mucosal competence and fish health may be influenced by dietary lipid and fatty acid intake. In reviewing the possible avenues by which polyunsaturated fatty acids might influence the intestinal mucosa of mammals, Donnet-Hughes and colleagues (Donnet-Hughes et al., 2001) suggested that fatty acid intake might influence gut health via mechanisms related to eicosanoid synthesis, cytokine production and reactivity of immune cells, maintenance of a tight intestinal barrier, and composition of the gut microflora. More recently, it has been suggested that the lipidome (the complete lipid profile of a cell, tissue, or organism) might be targeted and harnessed by pathogens in order to avoid detection and clearance by the immune system (van der Meer-Janssen et al., 2010). Although lipid/mucosa interactions are still not fully understood, the literature suggests there are functional relationships between lipid/fatty acid intake and gut health in vertebrates, including fish. To perform optimally, cellular membranes must be strong but flexible. The balance between these two attributes determines how well a membrane performs in terms of permeability, endocytic processes, embedded enzyme function, and translocation of ions and other substrates (Arts and Kohler, 2009). Thus, cellular membranes must be comprised of elements that disrupt strict rigidity and confer strength as well as fluidity to the lipid bilayer. Cholesterol and phospholipids containing saturated fatty acids provide strength, particularly at warm temperatures. Conversely, phospholipids
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containing unsaturated fatty acids provide fluidity, particularly at cold temperatures. Long-chain polyunsaturated fatty acids (LC-PUFA) with their many double bonds and coiled or semi-coiled structures are especially relevant in this context. For example, docosahexaenoic acid (22:6n-3, DHA) is known to influence various aspects of membrane function, including membrane permeability, fusion, and elasticity as well as vesicle formation (Arts and Kohler, 2009). The fact that the lipid and fatty acid composition of fish tissues reflects dietary intake is perhaps one of the best-documented phenomena in fish nutrition and physiology (Turchini et al., 2009; Trushenski and Bowzer, 2012). Although most research investigating tissue composition in fish is focused on edible tissues, a number of studies have reported differences in composition of the intestinal tissues and their associated properties. It is known that composition of the gut epithelium varies with function along the length of the intestine in fish. Although the levels of phospholipids, cholesterol, and proteins appear to be relatively consistent throughout the intestine of trout, membranes of the mid-intestine are reportedly more fluid and richer in unsaturated fatty acids than those of the distal intestine (Pelletier et al., 1986). Various manipulations are known to affect the lipid/fatty acid composition and, presumably, the function of the gut epithelium. For example, transferring trout from freshwater to seawater causes DHA to become enriched within the brush border tissues and fluidity of the membranes to increase (Leray et al., 1984). The same effect was induced in carp held at cold temperatures by supplementing their diet with LC-PUFA-rich fish oil (Behar et al., 1989). Feeding fish oil to channel catfish (Ictalurus punctatus) altered the fatty acid composition of the brush border membranes in a similar fashion, but this was associated with reduced glucose transport in comparison to feeding with a non-fish oil, stearic acid (18:0)-rich diet (Houpe et al., 1997). Conversely, accumulation of monounsaturated fatty acids (MUFA) at the expense of LC-PUFA within the gut epithelium of European seabass (Dicentrarchus labrax) was associated with reduced activities of various brush border enzymes (Cahu et al., 2000). Interestingly, these changes were induced not by feeding diets with different fatty acid profiles, but by feeding increasing amounts of the same lipid (fish oil), suggesting that absolute levels of lipid in the diet may also influence membrane composition and function. Although compositional analyses were not conducted, the enterocytes of gilthead seabream (Sparus aurata) that were fed vegetable oils exhibited a different structure than those of fish that were fed fish oil (Caballero et al., 2003). The enterocytes of fish given rapeseed, linseed, and soybean oils contained substantially more lipid droplets and exhibited altered capacities for lipoprotein synthesis, though the enterocytes otherwise appeared morphologically normal. Few studies have investigated the relationship between lipid nutrition, membrane composition or structure, and competency of the intestinal mucosa as a pathogen barrier. However, the limited data available suggests that a functional relationship may exist. Atlantic salmon successfully completed smoltification, regardless of whether they were fed diets containing sunflower oil or fish oil (Jutfelt et al., 2007). However, basal cortisol levels suggested fish that were fed the sunflower oil diet experienced a greater degree of physiological stress during smoltification, though nutrient uptake and prevention of bacterial translocation was apparently greater in these fish.
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Arctic char Salvelinus alpinus fed diets containing marine-origin oil, linseed oil, or soybean oil exhibited differential survival rates following challenges with A. salmonicida, a pathogen thought to invade primarily by disruption of the gut epithelium (Lødemel et al., 2001). In this study, fish given the plant-origin oils exhibited reduced mortality. The authors were unable to unequivocally determine the basis for greater survival among fish served soybean or, to a lesser extent, linseed oil, but suggested changes in the microfloral community and/or increased production of mucus might have reduced adherence of A. salmonicida to the gut epithelium and subsequent infection.
8.3 Antinutritional factors and gut health The functional competency of the gut in preventing pathogen translocation is linked to the structural integrity of the enterocytes and supporting tissues. For many years, fish nutritionists have known that a variety of feedstuffs, particularly plant-derived ingredients, may be toxic, cause feed palatability problems, or impair nutrient utilization and growth performance because of various “antinutritional factors” (ANFs) (Tacon, 1997; Francis et al., 2001). Numerous plant-derivatives are known to contain ANFs, including meals and protein concentrates derived from various legumes, including pulses, oilseed, and sowing crops. However, soybean products are likely the most widely recognized for their ANF content, containing protease inhibitors, lectins, phytic acid, saponins, phytoestrogens, allergens, and other problematic constituents (Tacon, 1997; Francis et al., 2001). Soybean derivatives are also noteworthy for their association with the development of enteritis in fish, particularly salmonids (Refstie and Storebakken, 2001; Gatlin et al., 2007). Soy-induced enteritis, or inflammation of the gut, is commonly characterized by the widening, shortening, and occasional disruption or fusion of the intestinal villi; a reduced number of supranuclear vacuoles in the absorptive epithelium; the widening and infiltration of the lamina propria by various types of leukocytes; and elevated levels of lysozyme and IgM in the gut mucosa. It can also be accompanied by a more generalized immune response, involving increases in circulating immunoglobulins and other proteins, as well as increases in the number and activity of circulating leukocytes (Refstie and Storebakken, 2001). Soy-induced enteritis may interfere with nutrient absorption, but the loss of the gut’s function as a barrier to pathogen translocation and participation in antigen recognition, processing, and presentation may be of greater importance since the signs of inflammation in soyinduced enteritis are generally restricted to the distal intestine that, more than likely, is less involved in nutrient absorption than in immunological processes (Rombout et al., 2011). The effects of plant proteins on gut integrity appear to be largely restricted to soy derivatives. In a screening of various legume, oilseed, and cereal-derived feedstuffs, only soybean meal-fed Atlantic salmon exhibited altered gut histomorphology (Aslaksen et al., 2007). Similarly, rainbow trout (Oncorhynchus mykiss) that were fed lupin meal failed to exhibit signs of enteritis (Glencross et al., 2004). Since soybean-derived products can contain a variety of ANFs, it is not clear which of these is the causative
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agent in the development of soy-induced enteritis. Based on their effects in fish and other species, it has been suggested that trypsin inhibitors, phytoestrogens, or lectins may be responsible for soybean meal-related enteritis (Sanden et al., 2005), which is consistent with most experiments associating soybean meal with enteritis as the alcohol-extraction process most commonly used to prepare soybean meal does not remove these substances (Aslaksen et al., 2007). Other processing strategies effectively remove some, but not all, of the ANFs that plant products contain, resulting in products that have yielded mixed results in feeding trials. Soy protein concentrate, for example, contains lower levels of phytic acid, lectins, saponins, and oligosaccharides than soybean meal. Atlantic salmon that were fed soy protein concentrate did not exhibit enteritis, however, fish given pea protein concentrate did exhibit the classic signs of inflammation and villous atrophy of the distal intestine, along with reductions in gut enzyme activities and macronutrient digestibilities (Penn et al., 2011). Both concentrates benefit from processing, which removes or neutralizes many ANFs. However, not all ANFs are addressed equally by the processing methods specific to producing each concentrate. For example, saponins are greatly reduced in soy protein concentrate during alcohol extraction of soybean meal, but there is no equivalent step to reduce saponin levels in producing pea protein concentrate. Thus, saponins are abundant in pea protein concentrate, which induces enteritis, but not in soy protein concentrate, which does not, suggesting that saponins are a likely contributor to this response in salmonids (Penn et al., 2011). However, in another study, Atlantic salmon did not develop enteritis after being fed diets supplemented with saponins or the purported ANF oligosaccharides, stachyose and raffinose, which suggests that some other component is responsible for inducing soyrelated enteritis (Sorensen et al., 2011). In an attempt to elucidate the contribution of saponins to soybean meal-induced enteritis, Bureau and colleagues (Bureau et al., 1998) fed rainbow trout and Chinook salmon (Oncorhynchus tshawytscha) diets containing soybean meal or soy protein concentrate supplemented with an alcohol extract of soybean meal to approximate the same level of soy-derived saponins. Both diets suppressed feed intake in Chinook salmon, but not in rainbow trout. Expectedly, histological analysis of the distal intestine of Chinook salmon revealed abnormalities consistent with reduced or absent feeding, whereas the distal intestine of rainbow trout was generally unaffected by the soybean meal or soy saponin-supplemented feeds. The distal intestine of Japanese flounder (Paralichthys olivaceus) was similarly affected by saponin-supplemented feeds, but it was unclear whether the enteritis observed was the result of reduced feed intake or a specific response to dietary saponin content (Chen et al., 2011). The most significant histopathological effects of soy products appear to be primarily associated with salmonids, but there are exceptions to this generalization, and it is likely that different taxa respond differently to soy derivatives and the ANFs they contain. The distal intestines of cobia (Rachycentron canadum) (Romarheim et al., 2008), Egyptian sole (Solea aegyptiaca) (Bonaldo et al., 2006), and European seabass (Bonaldo et al., 2008) were completely unaffected by feeding soybean meal. Similarly, the intestinal integrity of hybrid striped bass (Morone chrysops x M. saxatilis) was largely unaffected by feeding soybean meal, soy protein concentrate, or soy protein
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isolate (Blaufuss and Trushenski, 2012; Laporte and Trushenski, 2012). Gilthead seabream exhibited only minor infiltration of leukocytes into the lamina propria of the distal intestine (Bonaldo et al., 2008). Generally, plant proteins are not considered as problematic in feeds for herbivorous or omnivorous fish, but interspecies variation is also observed among these nutritional guilds: soy-induced enteritis is virtually unknown in species such as channel catfish (Evans et al., 2005), but has been demonstrated in common carp (Cyprinus carpio) (Urán et al., 2008). Although there is apparently variation in the dietary constituents that induce enteritis and how fish respond to these ANFs, the signs of diet-induced enteritis are somewhat more consistent. These include changes in the morphology and functionality of the gut, particularly the distal intestine, as well as apparent broader involvement of the immune system. For example, soybean meal-induced enteritis in Atlantic salmon and is characterized by hypertrophic or hyperanaemic mucosa, shortening of the mucosal folds, and widening and infiltration of the lamina propria by various leukocytes. These effects are accompanied by changes in mucosal enzyme activities as well as an increase in IgM. Similar morphological changes were observed in both rainbow trout and Atlantic salmon that were fed soybean meal; however, inflammatory cell infiltration was less pronounced in the former (Refstie et al., 2000). Collectively, these results suggest that element(s) of soybean meal have a toxic effect on the apical membrane of fish and may delay or otherwise interfere with the production of new enterocytes. Cellular damage coupled with a reduced ability to replace damaged enterocytes implies reduced functionality of the gut in terms of nutrient absorption and pathogen exclusion. Mucosal IgM was elevated in the mid-intestine of Atlantic salmon that were fed both soy protein concentrate and soy molasses; IgM was also elevated in the distal intestine, but only among those fish fed with soy molasses (Krogdahl et al., 2000). Mucosal lysozyme activity was elevated in the mid- and distal intestine of fish given soy molasses. Under some circumstances, these responses might be associated with a state of increased “immunological readiness”; however, these authors considered elevated IgM and lysozyme activity as primarily a consequence of gut inflammation (Krogdahl et al., 2000). Conversely, Atlantic salmon fed with relatively low levels of soy and maize did not consistently exhibit significant changes in the cytophysiology of the gut. Fish that were fed soybean meal exhibited significantly increased enterocyte proliferation and migration of immature or proliferating cells up the villous fold, whereas fish fed maize exhibited the opposite, though this effect was observed in only a few individuals (Sanden et al., 2005). The same increase in cellular proliferation was observed in Atlantic salmon that were given soybean meal (Bakke-McKellep et al., 2007). However, the inflamed gut was further characterized by increased and widespread heat-shock protein (HSP)70 and caspase-3 immunohistochemical reactivities that indicate elevated attempts at cellular repair and apoptosis, respectively, in the soy-fed fish (Bakke-McKellep et al., 2007). Overall, these results reinforce the concept of enteritis as cellular response that attempts to compensate for the “cytotoxic” effect of some as-yet unidentified element(s) of soybean meal. Despite exhibiting the classical signs of soybean meal-induced enteritis in the distal intestine, nutrient transport and uptake of various nutrients were not predictably altered
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in rainbow trout and Atlantic salmon with fed soybean meal-based feeds (Nordrum et al., 2000). However, the authors stated that their results were suggestive of increased membrane permeability in the inflamed intestinal tissues (Nordrum et al., 2000). Regardless of whether greater membrane permeability is a cause or an effect of soybean meal-induced damage to the intestinal mucosa, it raises the possibility of increased vulnerability to pathogen translocation. For example, Krogdahl and colleagues (Krogdahl et al., 2000) observed significantly greater mortality among Atlantic salmon that were fed soybean meal-based feeds following A. salmonicida challenge than fish that were fed a soy protein concentrate-based feed. Moreover, lower mortality was observed in fish given the soy protein concentrate-based feed than the fish given the control fish meal-based feed. Although possible mechanisms for these observations were not addressed in this work, the authors did suggest that reduced epithelial integrity of the inflamed gut may have rendered fish fed with the soybean meal-based feed more vulnerable to infection. Alternatively, the “systemic health” of Atlantic salmon may have been generally compromised by soybean meal-induced enteritis and accompanying diarrhea, which rendered them, in their weakened state, more susceptible to disease (Krogdahl et al., 2000). Similarly, rainbow trout fed high levels (80% or more of the diet) of standard or dehulled soybean meal or soy protein concentration exhibited significantly reduced head kidney macrophage competency as measured by respiratory burst activity (Burrells et al., 1999). These changes were associated with reduced growth rates, but not with a systemic immunogenic response to soy proteins, suggesting that there may be some merit to the concept that soy-based feeds generally reduce overall fish health by affecting their nutritional status and that the immunological response to soy antigens is largely a localized event restricted to the distal intestine. Again, these effects appear to be somewhat unique to soy-derived ANF since various enzymatic and blood chemistry measures of immune function were not affected in Atlantic salmon that were fed lupin meal or hydrolyzed poultry feather meal as alternatives to fish meal, and survival following challenge with Vibrio anguillarum was also equivalent among these fish (Bransden et al., 2001). The effects of soy-derived ANFs may directly influence gut integrity and the immune system of fish; however, indirect effects observed with or without the classical signs of diet-induced enteritis may also be relevant in terms of general fish health. For example, Atlantic cod (Gadus morhua) did not exhibit major shifts in histomorphology or enzyme activities normally associated with feeding soybean meal, but other fish fed with soybean meal did exhibit increased densities of non-adherent bacteria (transient, allochthonous) compared to adherent bacteria (indigenous, autochthonous) in the gut (Refstie et al., 2006). Additionally, the taxonomic composition of the culturable gut bacteria was also altered in cod fed with soybean meal. These effects were not apparent among fish that were fed a bio-processed, enzyme-treated soybean meal. Although these authors could not provide specific evidence regarding diet-induced differences in the fish’s ability to effectively exclude pathogenic bacteria at the level of the gut mucosa, they suggested this possibility and recommended further research on this effect (Refstie et al., 2006). Conversely, in Atlantic salmon exhibiting classical soybean meal-induced enteritis, there was a significant increase in the densities of
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adherent bacteria in both the mid- and distal intestine, whereas non-adherent bacteria were not affected in the mid-intestine but significantly elevated in the distal intestine (Bakke-McKellep et al., 2007). These authors also reported shifts in the taxonomic composition of the gut microflora, but could not state whether these changes were a cause or effect of the observed enteritis (Bakke-McKellep et al., 2007).
8.4 Concluding remarks The literature on the topic of certain ANFs and gut enteritis in fish is extensive; there are far fewer investigations of the effect(s) that lipid nutrition may have on intestinal integrity and function. Undoubtedly, other ANFs and nutrients may also influence gut health in fish, but, if there is research being conducted on these topics, it is not yet readily apparent in the literature. In all cases, there is a dearth of experimentation comprehensively addressing the effects dietary constituents may have on the physiological and morphological condition of the gut mucosa and, more importantly, the effects of these nutrients or antinutrients on the functional competence of the gut in terms of nutrient acquisition as well as pathogen sampling and exclusion. Studies addressing both basic and applied aspects of nutrition as it pertains to gut health are needed and encouraged.
References Arts, M.T., Kohler, C.C., 2009. Health and condition in fish: the influence of lipids on membrane competency and immune response. In: Arts, M.T., Brett, M.T., Kainz, M.J. (Eds.), Lipids in Aquatic Ecosystems. Springer, Dordrect, pp. 237–256. Aslaksen, M.A., Kraugerud, O.F., Penn, M., Svihus, B., Denstadli, V., Jørgensen, H.Y., Hillestad, M., Krogdahl, Å., Storebakken, T., 2007. Screening of nutrient digestibility and intestinal pathologies in Atlantic salmon, Salmo salar, fed diets with legumes, oilseeds, or cereals. Aquaculture 272, 541–555. Bakke-McKellep, A.M., Press, C.M., Baeverfjord, G., Krogdahl, Å., Landsverk, T., 2000. Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal-induced enteritis. J. Fish Dis. 23, 115–127. Bakke-McKellep, A.M., Penn, M.H., Salas, P.M., Refstie, S., Sperstad, S., Landsverk, T., Ringø, E., Krogdahl, Å., 2007. Effects of dietary soybean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.). Br. J. Nutr. 97, 699–713. Behar, D., Cogan, U., Viola, S., Mokady, S., 1989. Dietary fish oil augments the function of fluidity of the intestinal brush border membrane of the carp. Lipids 24, 737–742. Blaufuss, P., Trushenski, J.T., 2012. Exploring soy-derived alternatives to fish meal: using soy protein concentrate and soy protein isolate in hybrid striped bass (Morone chrysops x M. saxatilis) feeds. N. Am. J. Aquacult. 74, 8–19. Bonaldo, A., Roem, A.J., Pecchini, A., Grilli, E., Gatta, P.P., 2006. Influence of dietary soybean meal levels on growth, feed utilization and gut histology of Egyptian sole (Solea aegyptiaca) juveniles. Aquaculture 261, 580–586.
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Bonaldo, A., Roem, A.J., Fagioli, P., Pecchini, A., Cipollini, I., Gatta, P.P., 2008. Influence of dietary levels of soybean meal on the performance and gut histology of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax). Aquacult. Res. 39, 970–978. Bransden, M.P., Carter, C.G., Nowak, B.F., 2001. Effects of dietary protein source on growth, immune function, blood chemistry and disease resistance of Atlantic salmon (Salmo salar L.) parr. Anim. Sci. 73, 105–113. Bureau, D.P., Harris, A.M., Cho, C.Y., 1998. The effects of purified alcohol extracts from soy products on feed intake and growth of chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss). Aquaculture 161, 27–43. Burrells, C., Williams, P.D., Southgate, P.J., Crampton, V.O., 1999. Immunological, physiological and pathological responses of rainbow trout (Oncorhynchus mykiss) to increasing dietary concentrations of soybean proteins. Vet. Immunol. Immunop. 72, 277–288. Caballero, M.J., Izquierdo, M.S., Kjørsvik, E., Montero, D., Socorro, J., Fernández, A.J., Rosenlund, G., 2003. Morphological aspects of intestinal cells from gilthead seabream (Sparus aurata) fed diets containing different lipid sources. Aquaculture 225, 325–340. Cahu, C.L., Zambonino-Infante, J.L., Corraze, G., Coves, D., 2000. Dietary lipid level affects fatty acid composition and hydrolase activities of intestinal brush border membrane in seabass. Fish Physiol. Biochem. 23, 165–172. Chen, W.C., Ai, Q., Mai, K., Xu, W., Liufu, Z., Zhang, W., Cai, Y., 2011. Effects of dietary soybean saponins on feed intake, growth performance, digestibility and intestinal structure in juvenile Japanese flounder (Paralichthys olivaceus). Aquaculture 318, 95–100. Donnet-Hughes, A., Schiffrin, E.J., Turini, M.E., 2001. The intestinal mucosa as a target for dietary polyunsaturated fatty acids. Lipids 36, 1043–1052. Evans, J.J., Pasnik, D.J., Peres, H., Lim, C., Klesius, P.H., 2005. No apparent differences in intestinal histology of channel catfish (Ictalurus punctatus) fed heat-treated and non-heattreated raw soybean meal. Aquacult. Nutr. 11, 123–129. Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture 199, 197–227. Gatlin, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, Å., Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D., Souza, E.J., Stone, D., Wilson, R., Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquacult. Res. 38, 551–579. Glencross, B., Evans, D., Hawkins, W., Jones, B., 2004. Evaluation of dietary inclusion of yellow lupin (Lupinus luteus) kernel meal on the growth, feed utilization and tissue histology of rainbow trout (Oncorhynchus mykiss). Aquaculture 235, 411–422. Houpe, K.L., Malo, C., Buddington, R.K., 1997. Dietary lipid and intestinal brush border membrane phospholipid fatty acid composition and glucose transport of channel catfish. Physiol. Zool. 70, 230–236. Jutfelt, F., Olsen, R.E., Björnsson, B.T., Sundell, K., 2007. Parr-smolt transformation and dietary vegetable lipids affect intestinal nutrient uptake, barrier function and plasma cortisol levels in Atlantic salmon. Aquaculture 273, 298–311. Kesarcodi-Watson, A., Kaspar, H., Lategan, M.J., Gibson, L., 2008. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274, 1–14. Krogdahl, Å., Bakke-McKellep, A.M., Røed, K.H., Baeverfjord, G., 2000. Feeding Atlantic salmon Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinal mucosa. Aquacult. Nutr. 6, 77–84.
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