**3. Morphophysiological effects at fish gut of soy protein inclusion in aquafeed**

Although soy protein has widely been used in aquafeed as a cheap alternative protein source for FM; however, the presence of some ANFs in SBM restricts its level of inclusion in aquafeed. High inclusion of soybean ingredients causes several negative effects on palatability and intestinal

**Figure 3.** (a) Intestinal histology (posterior, intestine, 20 X) of rainbow trout (*O. mykiss*) fed FM-based control diet showing normal condition of intestine (Kumar et al., unpublished data). (b) Intestinal histology (posterior, intestine, 20 X) of rainbow trout (*O. mykiss*) fed low SBM (10.3%)-based diet showing inflamed muscularis, leukocyte infiltration of the lamina propria leading to swelling and mucosal fold fusion (bridging). Increased prevalence of globlet cells possibly to secrete more mucous to protect the epithelium. Asterisk denotes inflammation (Kumar et al., unpublished data). (c) Intestinal histology (posterior, intestine, 20 X) of rainbow trout (*O. mykiss*) fed high SBM (20.7%) based diet showing villi and lamina propria highly inflamed (leading to much wider mucosal folds), muscularis inflamed, villi shortened, disorganization of epithelium, reduction in supranuclear absorptive vacuoles, mucosal fold fusion (bridging) and some structural disintegration. By far, this treatment led to the most changes (Kumar et al., unpublished data).

digestive enzymes compared to fish fed FM. Also, lower gene expression levels of the digestive enzymes in the hepatopancreas were observed in the SBM fed red sea bream compared with the FM fed fish, which is in line with the report in yellowtail; orally administrated FM increased the trypsin and lipase gene expressions in the pyloric caeca, but not in fish administrated SBM [51]. Perera and Yúfera [52] reported that early SBM feeding of *S. aurata* larvae significantly affects the activity of most pancreatic enzymes in a time-of-exposure dependent form. More than 10 days of SBM feeding (i.e., beyond 14 dph) delayed the normal development of *S. aurata* larvae digestive capacities as the activities of all trypsin, chymotrypsin, and amylase were significantly reduced. This is opposed to the typical response of juvenile and adult fish to SBM. Protease inhibitors present in SBM can partially abrogate the activity of trypsin and chymotrypsin in the proximal intestine [53], and juveniles of *S. aurata* [54] and other fish such as Atlantic salmon [55, 56] exhibit a rapid compensatory increase in activities of these enzymes. However, SBM-induced increase in trypsin activity in juvenile fish is more marked in the distal intestine and has been attributed to a reduced ability to reabsorb the pancreatic enzymes [11] and the upregulation of trypsin-like activity by immune cells [56]. Therefore, other plausible explanation for our observations is that these intestinal processes are not fully functional in early larvae. Conversely, lipase activity was relatively insensitive to SBM in *S. aurata* larvae, as reported before in postsmolt Atlantic salmon [55]. The observed decrease in pancreatic proteases may be responsible for less lipase inactivation in the digestive

The Potential Impacts of Soy Protein on Fish Gut Health http://dx.doi.org/10.5772/intechopen.92695 97

tract explaining the stable lipase activity.

**4. Soy protein inclusion impacts on gut-associated immunity**

The gastrointestinal tract carries many functions in teleost; among them, defense is possibly one of the most important functions. Gut acts as a physical barrier to pathogen entry that also contains a gut-associated lymphoid tissue (GALT). Teleost gut-associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The gut microbes play a critical role in the development and maturation of GALT, which in turn mediate a variety of host immune functions [49]. Recent work on the structure of GALT and other intestinal cell populations, the absorption of macromolecules from the intestinal lumen, and the production of specific mucosal antibodies strongly suggests, however, that the gut of these lower vertebrates is immunocompetent. Fish intestine, especially the posterior segment, is immunologically active and armored with various immune cell types, including B cells, macrophages, granulocytes, and T cells [57, 58]. Studies on the gut-associated immunity are important for the aquaculture industry for several reasons. First, the gut is one of the main portals of entry of pathogens. Second, farmed fish are generally fed commercial pellets, which give farmers the ability to manipulate fish health by incorporating drugs, vaccines, and different feed ingredients or additives into the feed. Third, the gut immune system of teleost's allows microbial colonization by symbionts, and this microbial community can be regarded as a mechanism to modulate fish pathogens [59]. Many studies attempt to reveal the effects of inclusion of plant origin ingredients and different feed additives on gut-associated immunity in fish. In this current chapter, an attempt has made to discuss the effects of soy protein ingredients inclusion in aquafeed on gut-associated immunity of various fish species.

**Figure 4.** (a-1) Cross-section of anterior intestine (X 40, HE) of Amberjack (*Seriola dumerili*) fed FM-based control diet showing increased fold height (hF). (a-2) Cross-section of anterior intestine (X 400, HE) of Amberjack (*Seriola dumerili*) fed FM-based control diet showing increased enterocyte height (hE) and microvillus height (hMV). (b-1) Cross-section of anterior intestine (X 40, HE) of Amberjack (*Seriola dumerili*) fed 50% FM replaced with SBM-based diet showing reduced fold height (hF). (b-2) Cross-section of anterior intestine (X 400, HE) of Amberjack (*Seriola dumerili*) fed 50% FM replaced with SBM-based diet. Showing reduced enterocyte height (hE) and microvillus height (hMV).

morphology of fish. From previous researches, it is well documented that high inclusion of soybean meal (>40%) causes several intestinal morphological changes such as the reduction in mucosal folding, reduced fold height, enterocyte height, microvillus height, loss of mucosal integrity, abnormal vacuolization, and inflammatory cell infiltration (**Figures 3a–c** and **4a–1, a–2, b–1, b–2**) in aquatic animals [5–8, 36, 45–49]. The degree of morphological changes in the intestine depends on the inclusion level of SBM which is also correlated with the cultured fish species. Reduced fold height, enterocyte height, and microvillus height reduced the area of nutrient absorption in the intestine which finally affects the fish performances.

Feed nutrients must be digested for their utilization, and pancreatic digestive enzymes have essential roles for the digestion; trypsin and chymotrypsin are the main pancreatic proteases, lipase is the major pancreatic lipolytic enzyme, and amylase is known as the major pancreatic digestive enzyme for carbohydrates (Murashita et al. [50]). Inclusion of SBM that also affects the digestive enzyme secretion of different fish species is well documented. Murashita et al. [50] reported that red sea bream fed SBM showed lower content and activity of four pancreatic digestive enzymes compared to fish fed FM. Also, lower gene expression levels of the digestive enzymes in the hepatopancreas were observed in the SBM fed red sea bream compared with the FM fed fish, which is in line with the report in yellowtail; orally administrated FM increased the trypsin and lipase gene expressions in the pyloric caeca, but not in fish administrated SBM [51]. Perera and Yúfera [52] reported that early SBM feeding of *S. aurata* larvae significantly affects the activity of most pancreatic enzymes in a time-of-exposure dependent form. More than 10 days of SBM feeding (i.e., beyond 14 dph) delayed the normal development of *S. aurata* larvae digestive capacities as the activities of all trypsin, chymotrypsin, and amylase were significantly reduced. This is opposed to the typical response of juvenile and adult fish to SBM. Protease inhibitors present in SBM can partially abrogate the activity of trypsin and chymotrypsin in the proximal intestine [53], and juveniles of *S. aurata* [54] and other fish such as Atlantic salmon [55, 56] exhibit a rapid compensatory increase in activities of these enzymes. However, SBM-induced increase in trypsin activity in juvenile fish is more marked in the distal intestine and has been attributed to a reduced ability to reabsorb the pancreatic enzymes [11] and the upregulation of trypsin-like activity by immune cells [56]. Therefore, other plausible explanation for our observations is that these intestinal processes are not fully functional in early larvae. Conversely, lipase activity was relatively insensitive to SBM in *S. aurata* larvae, as reported before in postsmolt Atlantic salmon [55]. The observed decrease in pancreatic proteases may be responsible for less lipase inactivation in the digestive tract explaining the stable lipase activity.

## **4. Soy protein inclusion impacts on gut-associated immunity**

The gastrointestinal tract carries many functions in teleost; among them, defense is possibly one of the most important functions. Gut acts as a physical barrier to pathogen entry that also contains a gut-associated lymphoid tissue (GALT). Teleost gut-associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The gut microbes play a critical role in the development and maturation of GALT, which in turn mediate a variety of host immune functions [49]. Recent work on the structure of GALT and other intestinal cell populations, the absorption of macromolecules from the intestinal lumen, and the production of specific mucosal antibodies strongly suggests, however, that the gut of these lower vertebrates is immunocompetent. Fish intestine, especially the posterior segment, is immunologically active and armored with various immune cell types, including B cells, macrophages, granulocytes, and T cells [57, 58]. Studies on the gut-associated immunity are important for the aquaculture industry for several reasons. First, the gut is one of the main portals of entry of pathogens. Second, farmed fish are generally fed commercial pellets, which give farmers the ability to manipulate fish health by incorporating drugs, vaccines, and different feed ingredients or additives into the feed. Third, the gut immune system of teleost's allows microbial colonization by symbionts, and this microbial community can be regarded as a mechanism to modulate fish pathogens [59]. Many studies attempt to reveal the effects of inclusion of plant origin ingredients and different feed additives on gut-associated immunity in fish. In this current chapter, an attempt has made to discuss the effects of soy protein ingredients inclusion in aquafeed on gut-associated immunity of various fish species.

Research conducted on the modulation of gut immune response due to the inclusion of soy products in aquafeed predominantly concentrate on innate immune parameters. Several molecules involved in innate immunity are found in the intestine of different fish species, such as lysozymes in Asian sea bass (*Lates calcarifer*) [60] and Atlantic salmon (*Salmo salar* L.) [61]; complement components in grass carp (*Ctenopharyngodon idella*) [62], rainbow trout (*Oncorhynchus mykiss*) [63, 64], and Asian sea bass [65]; cytokines in Atlantic cod (*Gadus morhua*) [66] and rainbow trout [67]; lectins in several species (reviewed in [68]); or antimicrobial peptides (AMPs) in rainbow trout [69] and grouper (*Epinephelus coioides*) [70].

the feeding trial period, with the BSBM diet producing higher levels in the long-term sample (60 day). A previous study on Atlantic salmon (*Salmo salar*) showed increased lysozyme activity in the intestinal mucosa due to the dietary inclusion of soybean molasses, indicating a potential inflammatory response and was potential activation of leukocytes [35]. Kim and Austin [78] also found high lysozyme activity in rainbow trout intestinal mucus samples after the administration of probiotics compounds which may be closely related to bioprocessed plant-based ingredients. Therefore, increased lysozyme levels may also be indicative of intes-

The Potential Impacts of Soy Protein on Fish Gut Health http://dx.doi.org/10.5772/intechopen.92695 99

The mucosal immune system in fish includes certain immunocompetent cells and factors in the intestinal mucous membrane. Of these factors, the interleukins (ILs), interferon regulatory factors (IRFs), and tumor necrosis factors (TNFs) are the main immune-relevant factors linked to inflammation in the distal intestine in fish [79]. Recently Miao et al. [80] reported the substitution effects of dietary SBM on the mucosal immune system in northern snakehead through measuring the gene expression of certain inflammatory cytokines (IL-1β, IL-8, IL-10, and IL-17F) in the distal intestine. After 63-day feeding, trial results indicated that dietary soybean meal affected the gene expression of certain factors. The up-regulated relative expression of IL-1β in the fish fed diet group containing 75% defatted fishmeal replacement with SBM was consistent with the observations in Atlantic salmon [77, 81]. However, the level of IL-1β observed in the same diet group was only 1.6-fold higher than that in FM-based control diet, while that observed in Atlantic salmon was 20-fold higher [79]. The effect of dietary soybean meal on the expression of IL-1β reflects the fish species and stages due to the different tolerance capability for soybean meal [79].

Healthy gut microbiota is essential to promote host health and well-being. Before the 1970s, there were some controversies regarding the existence and role of an indigenous microbiota in fish. However, it is now well established that fish and other aquatic animals have a microbiota in the GI tract (for review, see; [21, 23, 82–92]). The intestinal microbiota of fish, as is the case of mammals, is classified as autochthonous (indigenous) or allochthonous bacteria [90, 93]. The autochthonous bacteria are those able to colonize the host's gut epithelial surface or are associated with the microvilli, while the allochthonous bacteria are incidental visitors in the GI tract and are expelled after some time without colonizing [90, 93]. Several factors affect the gut microbiota in fish including host factors, environmental factors, microbial factors, etc. However, until recently, among different influencing factors affecting the fish microbiota, water and diet (environmental factors) have been studied extensively [49]. In this section, we address the effect of dietary soybean products on intestinal bacterial community of finfish and crustaceans (**Table 1**).

Research conducted until recently on SBM inclusion effects on gut microbiota of fish indicated that SBM modulated the intestinal microbiota toward developing an undesirable microbial community that can induce mucosal inflammation [110, 111]. Heikkinen et al. [94] reported that rainbow trout fish fed FM- and SBM-based diets for 4 weeks showed decreased number

tinal innate immunity and gut health enhancements.

**5. Effects of soy protein inclusion on gut microbiota**

**5.1. In salmonids**

The immune status of a fish's intestinal mucosa is closely associated with inflammation, which is mediated by cytokines. Cytokines, such as IL-1, IL-10, and IL-16 have a fundamental role in the regulation of inflammatory responses in fish throughout the infection process [31, 32]. Many studies have shown that IL-1 and IL-16 are increased in inflammatory bowel disease and there was a positive association between disease activity [71, 72]. While the deprivation of IL-10 evokes the development of inflammatory bowel disease, the decrease of IL-10 can aggravates local inflammation [73, 74]. Wang et al. [75] reported the increased expression levels of IL-1 and IL-16 mRNA with the SBM level in the diet, whereas the IL-10 mRNA expression level decreased with the SBM level in the diet of orange-spotted grouper (*Epinephelus coioides*). At the same time, infiltrate leucocytes were observed in the intestinal epithelium in grouper fed diets contained SBM. Furthermore, the degree of intestine inflammation was positively correlated with IL-1 and IL-16 mRNA expression levels but negatively correlated with expression of IL-10 mRNA. Their results suggest that SBM can cause intestinal inflammation by increasing proinflammatory cytokine levels and decreasing anti-inflammatory cytokine levels.

Krogdahl et al. [35] examined the effect of solvent and alcohol extracted SBM in the diets of Atlantic salmon, and their results indicated that fish fed solvent-extracted SBM showed higher mortality rate when challenged by *A. salmonicida*. In addition, fish fed alcoholextracted SBM revealed increased levels of both lysozyme and IgM in the mid and distal intestinal mucosa. In Atlantic salmon, Lilleeng et al. [76] showed significantly downregulated TGF-β gene expression after on day feeding of extracted SBM (460 g kg−<sup>1</sup> ), whereas reduced expression of interferon-inducible lysosomal thiol reductase (GILT) was observed followed by 3 days feeding. The authors assumed that the downregulation of TGF-β and GILT might be due to the failure to maintain mucosal integrity in the distal intestine. Sahlmann et al. [77] investigated transcriptomic profiling in Atlantic salmon feeding of SBM (200 g kg−<sup>1</sup> ) containing diet for 1 week. On days 3 and 5, a prominent change in gene expression patterns was observed. Immune-related genes were upregulated during the first 5 days: GTPase IMAP family members; NF-kB-related genes; and regulators of T-cell and B-cell function. These immune genes expression profiles suggest that intestinal inflammation is induced within a week upon administration of an SBM-containing diet, which may in turn negatively influence the growth performance of salmonids.

Bruce et al. [29] evaluated processed soybean meal ingredients (defatted soybean meal, bioprocessed soybean meal [BSBM], and commercial soy protein concentrate [CSPC]) inclusion in the diets of rainbow trout on intestinal immunity. They reported no significant differences in intestinal immunoglobulin concentrations (p = 0.41) or gut leukocyte phagocytosis at day 15 samplings (p = 0.41). Intestinal lysozyme activity showed some modulation throughout the feeding trial period, with the BSBM diet producing higher levels in the long-term sample (60 day). A previous study on Atlantic salmon (*Salmo salar*) showed increased lysozyme activity in the intestinal mucosa due to the dietary inclusion of soybean molasses, indicating a potential inflammatory response and was potential activation of leukocytes [35]. Kim and Austin [78] also found high lysozyme activity in rainbow trout intestinal mucus samples after the administration of probiotics compounds which may be closely related to bioprocessed plant-based ingredients. Therefore, increased lysozyme levels may also be indicative of intestinal innate immunity and gut health enhancements.

The mucosal immune system in fish includes certain immunocompetent cells and factors in the intestinal mucous membrane. Of these factors, the interleukins (ILs), interferon regulatory factors (IRFs), and tumor necrosis factors (TNFs) are the main immune-relevant factors linked to inflammation in the distal intestine in fish [79]. Recently Miao et al. [80] reported the substitution effects of dietary SBM on the mucosal immune system in northern snakehead through measuring the gene expression of certain inflammatory cytokines (IL-1β, IL-8, IL-10, and IL-17F) in the distal intestine. After 63-day feeding, trial results indicated that dietary soybean meal affected the gene expression of certain factors. The up-regulated relative expression of IL-1β in the fish fed diet group containing 75% defatted fishmeal replacement with SBM was consistent with the observations in Atlantic salmon [77, 81]. However, the level of IL-1β observed in the same diet group was only 1.6-fold higher than that in FM-based control diet, while that observed in Atlantic salmon was 20-fold higher [79]. The effect of dietary soybean meal on the expression of IL-1β reflects the fish species and stages due to the different tolerance capability for soybean meal [79].
