Butyrate and taurine exert a mitigating effect on the inflamed distal intestine of European sea bass fed with a high percentage of soybean meal
© The Author(s) 2016
Received: 30 August 2016
Accepted: 15 November 2016
Published: 5 December 2016
Due to the paucity of oceanic resources utilized in the preparation of diets for cultured fish, commercial feed producers have been trying to replace fishmeal (FM) using alternative protein sources such as vegetable protein meals (VMs). One of the main drawbacks of using VMs in fish feed is related to the presence of a variety of anti-nutritional factors, which could trigger an inflammation process in the distal intestine. This reduces the capacity of the enterocytes to absorb nutrients leading to reduced fish growth performances.
We evaluated the mitigating effects of butyrate and taurine used as feed additives on the morphological abnormalities caused by a soybean meal (SBM)-based diet in the distal intestine of sea bass (Dicentrarchus labrax). We used three experimental diets, containing the same low percentage of FM and high percentage of SBM; two diets were supplemented with either 0.2% sodium butyrate or taurine. Histological changes in the intestine of fish were determined by light and transmission electron microscopy. Infiltration of CD45+ leucocytes in the lamina propria and in the submucosa was assessed by immunohistochemistry. We also quantified by One-Step Taqman® real-time RT-PCR the messenger RNA (mRNA) abundance of a panel of genes involved in the intestinal mucosa inflammatory response such as TNFα (tumor necrosis factor alpha) and interleukins: IL-8, IL-1β, IL-10, and IL-6.
Fish that received for 2 months the diet with 30% soy protein (16.7% SBM and 12.8% full-fat soy) developed an inflammation in the distal intestine, as confirmed by histological and immunohistochemistry data. The expression of target genes in the intestine was deeply influenced by the type of fish diet. Fish fed with taurine-supplemented diet displayed the lowest number of mRNA copies of IL-1β, IL-8, and IL-10 genes in comparison to fish fed with control or butyrate-supplemented diets. Dietary butyrate caused an upregulation of the TNFα gene transcription. Among the quantified interleukins, IL-6 was the only one to be not influenced by the diet.
Histological and gene expression data suggest that butyrate and taurine could have a role in normalizing the intestinal abnormalities caused by the SBM, but the underling mechanisms of action seem different.
KeywordsAquaculture Intestinal inflammation TEM Light microscopy Gene expression Cytokines Interleukins Soybean meal Butyrate Taurine
Due to the paucity of oceanic resources utilized in the preparation of diets for cultured fish, commercial feed producers have been trying to replace fishmeal (FM) by using alternative protein sources such as vegetable protein meals (VMs) (Tacon and Metian 2008). Vegetable products include soy, canola (rape), corn, wheat, cottonseed, lupin, sunflower, flax linseed, and peas. Of these, soybeans are promising because of their higher protein content, higher digestibility, and better amino acid profile in comparison to other grains and oilseeds (Hardy 1999; Gatlin et al., 2007; Zhang et al., 2014).
The main drawbacks of using plant-derived proteins in fish feed are related to the low level of indispensable amino acids (in particular, methionine and lysine) and to the presence of a wide variety of anti-nutritional factors, such as saponins, lectins, phytate, trypsin inhibitors, phenols, and tannins (Francis et al. 2001), which could damage the intestinal tract thus reducing nutrient absorption and fish growth (Van den Ingh et al. 1991; Knudsen et al. 2008; Urán et al. 2008a, b).
Indeed, studies on Atlantic salmon (Salmo salar), rainbow trout (Oncorhychus mykiss), common carp (Cyprinus carpio), and gilthead sea bream (Sparus aurata) have indicated that the inclusion of less-refined plant products such as soybean meal (SBM) in the diet triggers an inflammation process in the distal intestine, referred to as SBM-induced enteropathy (Baeverfjord and Krogdahl 1996; Olli and Krogdahl 1994; Van den Ingh et al. 1991; Knudsen et al. 2008; Urán, et al. 2008a, b; Venou et al. 2006). Although the provoking mechanism remains to be established, the SBM-induced enteritis is believed to be caused by a disruption of the intestinal barrier, with subsequent exposure of otherwise shielded layers of the mucosa to luminal ingredients, including food-derived and microbial antigens (Romarheim et al. 2011). The typical signs of such inflammation are a shortening of the primary and secondary intestinal mucosal folds, an increase in the number of Goblet cells, and the infiltration of inflammatory cells, particularly macrophages and eosinophilic granulocytes into the lamina propria. This situation reduces the capacity of the enterocytes lining the epithelium to absorb nutrients (Baeverfjord and Krogdahl 1996; Van den Ingh et al. 1991; Buttle et al., 2001). These effects proved to be dose-dependent in Atlantic salmon; the worst symptoms were observed at the highest inclusion level (30%), but even diets containing as low as 7.6% SBM produced morphological changes at the intestinal level (Krogdahl et al. 2003).
It has been well documented that the responsible for the off-target effects of SBM is not the soy protein but other components present in the SBM, such as the anti-nutritional factor saponin, in combination with at least one or more unidentified components (Van den Ingh et al. 1991; Baeverfjord and Krogdahl 1996; Bakke-Mckellep et al. 2000; Krogdahl et al. 2003; Knudsen et al. 2008).
Once an inflammatory immune response is initiated, proinflammatory cytokines and a panel of chemokines (secreted proteins that play a major role in the inflammatory response) instigate the coordinated expression of downstream genes (Martin et al. 2006; Kortner et al. 2012; Grammes et al. 2013; Sahlmann et al. 2013; Marjara et al. 2012; Krogdahl et al. 2015; De Santis et al. 2015). The expression of pro- and anti-inflammatory cytokine genes was quantified by Urán et al. (2008a) in the isolated intraepithelial lymphocytes of common carp (Cyprinus carpio L.), in which an intestinal inflammation was observed. The enteropathy was developed when carp continuously fed on animal protein were transferred to a diet in which 20% of the protein was replaced by SBM. After 3 weeks of feeding, the pro-inflammatory interleukin 1β (IL-1β) and tumor necrosis factor α1 (TNF-α1) genes were upregulated whereas the anti-inflammatory interleukin 10 (IL-10) was downregulated after an initial upregulation at the first week of feeding.
The effects of replacement of FM with VM, often accompanied by reduced fish performance, are not restricted to SBM inclusion solely but have been observed after inclusion of many plant protein sources in several teleost species such as gilthead sea bream, turbot, Atlantic cod, and parrot fish (Gomez-Requeni et al. 2004; Sitja-Bobadilla et al. 2005; Yun et al. 2011; Hansen et al. 2007; Lim and Lee 2009). Baeza-Ariño et al. (2014) described liver and gut alterations of gilthead sea bream, S. aurata L., fed on diets in which FM was replaced by a mixture of rice and pea protein concentrates. The results of the histological analysis showed significant changes in the case of the 90% substitution in parameters such as thickness of the gut layers, number of Goblet cells, and villi’s length and thickness, whereas the integrity of the gut structure was not significantly affected by a diet with up to 60% of replacement. In some cases, severe vacuolization was encountered, which consequently deformed enterocytes and displaced the nucleus.
The short chain fatty acid of butyrate may promote the healing of inflamed intestine through its major role in enhancing epithelial cell proliferation and differentiation and in improving the intestinal absorptive function (Canani et al. 2012; Gálfi and Neogrády 2002; Wong et al. 2006). Like other short chain or volatile fatty acids (acetic, propionic, valeric, and caproic), butyric acid is produced during the fermentation of dietary fibers by the anaerobic microbiota associated with the epithelium of the animals’ digestive tract. In addition to being the main respiratory fuel source of the intestinal bacteria, and preferred to glucose or glutamine, this four-carbon chain organic acid molecule has potential immunomodulatory and anti-inflammatory properties (Vinolo et al. 2009; Toden et al. 2007; Terova et al. 2016), and exert multiple other beneficial effects on host energy metabolism (Hamer et al. 2007; Den Besten et al. 2013; Liu et al. 2014; da Silva et al. 2016). Although the mechanisms underlying these effects are still enigmatic and subject of intense scrutiny, it is believed that they encompass the complex interplay between diet, gut microbiota, and host energy metabolism.
However, much of the research on butyrate has been focused on terrestrial vertebrates, including humans, while very few studies have been conducted in fish. In particular, little is known about the effects of butyrate used as a feed additive on fish intestinal integrity. In terrestrial farmed animals such as pig and chicken, butyrate included in the diet has had a positive influence on body weight gain, feed utilization, and composition of intestinal microflora. It exerted trophic effects on the intestinal epithelium through an increase in the villi length and crypt depth, too (Gálfi and Bokori 1990; Kotunia et al. 2004; Hu and Guo 2007). In fish, Robles et al. (2013) reported an effect of butyrate used as a feed additive in increasing the availability of several essential amino acids and nucleotide derivatives, which have been demonstrated to increase fish growth when they are added individually to the diet.
Another feed additive that has been shown to have a restorative effect on soya saponin- and soya lectin-induced enteritis in fish (Iwashita et al. 2008, 2009) is the organic acid taurine. A number of recent studies reviewed by El-Sayed (2014) and Salze and Davis (2015) have demonstrated the essentiality of dietary taurine for many commercially relevant cultured species, including marine teleosts. According to these studies, taurine is involved in many physiological functions in fish and represents an essential nutrient, which exerts powerful antioxidant and anti-inflammatory properties, and is required as a supplement in the feed when a relevant percentage of vegetable protein sources are utilized. Taurine is a neutral β-amino acid found in high concentrations in animal tissues, whereas plants contain less than 1% of the taurine levels found in animals. In mammals, taurine is mainly synthesized in liver and brain through enzymatic oxidation and direct conversion of cysteine (derived from methionine) to cysteine sulfinic acid and hypotaurine. In this pathway, the enzymes cysteine dioxygenase (CDO) and cysteinsulfinate decarboxylase (CDS) have important roles (Griffith 1987; El-Sayed 2014). Therefore, in animal taxa with limited or no activity of the CDS, such as marine fish species, a continuous supply of taurine should be provided with the diet (El-Sayed 2014; Johnson et al. 2015; López et al. 2015). Indeed, Yokoyama et al. (2001), by comparing the CSD activity and hypotaurine production in several teleost and mammalian species, found that the only fish species with a high CSD activity were rainbow trout and tilapia. However, even in these species, the enzyme activity was an order of magnitude lower than that found in mammalian species. Other data (Salze and Davis 2015) indicate that both species benefit from dietary taurine supplementation thus suggesting that levels of CSD activity are insufficient to provide the necessary amounts of taurine for maximum growth of these species.
Accordingly, the objective of the present research was to evaluate the mitigating effects of butyrate and taurine used as feed additives on the morphological abnormalities caused by a SBM-based diet in the intestine of European sea bass (Dicentrarchus labrax). In order to attain this aim, we utilized both light and transmission electron microscopy (TEM) to determine intestinal changes in fish. We also quantified the intestinal messenger RNA (mRNA) abundance of several genes involved in the mucosal inflammatory response such as TNFα, which is a cell-signaling protein (cytokine) that makes up the inflammatory acute-phase reaction, and interleukins such as IL-8, IL-1β, IL-10, and IL-6, which are well-known cytokines that regulate immune responses, inflammatory reactions, and hematopoiesis.
Formulation (%), and proximate composition of experimental diets
Wheat gluten meal
Vitamins and mineral premixb
Soybean meal (48%)
Fish meal (65%)
Proximate composition (%)
Methionine + cysteine
Fish were sampled at the end of the feeding experiment, which lasted 60 days. The experimental tanks were connected to a 20-m3 water recirculation system. The water parameters such as pH, temperature, and dissolved oxygen (DO) were strictly controlled during all the experiment. Temperature was maintained at 20 ± 0.5 °C; salinity at 22 g L−1; pH 7.2; total N–NH3 ≤ 0.1 mg L−1; N–NO2 − ≤ 0.02 mg L−1; N–NO3 − ≤ 5 mg L−1, DO 8–8.5 mg L−1, and DO saturation, over 97%.
The distal intestine of all sampled fish (six fish/diet, three from each tank) was dissected out. A small part of it was fixed in 4% buffered formalin, pH 7.2, for histological, immunohistochemical or electron microscopy examination, whereas the remaining part was conserved at –80 °C for gene expression analyses.
The experimental feed
We used three experimental diets (C, B, and T), which contained the same low percentage of fishmeal and high percentage of soybean meal, as shown in Table 1. Diets B and T were supplemented with 0.2% of sodium butyrate or taurine, respectively, whereas Diet C, which represented the control diet, was not supplemented.
Sampled intestines were fixed by immersion in 4% buffered formal, dehydrated with a graded ethanol series (20, 30, 50, 70, and 95%), embedded in paraffin, and sectioned at 4 μm. After dewaxing and rehydrating the sections, they were consecutively stained with hematoxylin-eosin. Periodic Acidic Schiff (PAS) and Alcian blue stain at pH 2.5 were used to reveal neutral and acidic mucosubstances, respectively.
The thickness of intestinal wall was measured by analysing six intestines/diet and using the average value obtained from 3 tissue sections/intestine (n = 6). The length of muscle layer and mucosa was measured (in micrometers) by using the Image J software package and then the percentages were calculated by referring to the thickness of the whole intestine wall. Statistical differences were calculated by ANOVA followed by Duncan’s post hoc test, and P < 0.05 was considered as level of significance. Statistical analysis was performed using Statistica 7.0 software (StatSoft Inc., Tulsa, OK, USA).
Immunohistochemistry for leukocyte assessment
Paraffin sections of distal intestine were dewaxed and rehydrated with a standard step of decreasing ethanol series. After washing with PBS, sections were pre-incubated for 30 min in blocking solution (2% bovine serum albumin, BSA, 0.1% Tween in PBS) and then incubated overnight at 4 °C with the polyclonal antibody rabbit anti-human CD45 (GenScript NJ, USA) diluted 1:100 in blocking solution. The washed specimens were incubated for 1 h at room temperature with the secondary antibody goat anti-rabbit (dilution 1:100 in PBS/BSA) conjugated with alkaline phosphatase (Jackson, Immuno Research Laboratories, West Grove, PA, USA). Secondary antibody alkaline phosphatase conjugated was visualized using 5-Bromo-4-Chloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium tablets (BCIP/NBT, Sigma). Mounted slides were examined under an optical microscope (Nikon Eclipse Ni, Nikon, Tokyo, Japan). Images were acquired with a DS-5 M-L1 digital camera system (Nikon). Control sections were incubated in PBS/BSA without the primary antibody. To avoid false positives due to the chromogen reaction with endogenous alkaline phosphatase, sections were treated with CH3COOH 20% applied for 15 min at 4 °C.
Specimens already fixed in formalin were transferred to a 2% Karnovsky solution at 4 °C for 2 h, post-fixed in osmium tetroxide for 1 h, and then embedded in Epoxy resins. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined with a TEM Morgagni Philips/FEI electron microscope.
One-Step Taqman® real-time RT-PCR quantification of target genes’ mRNA copies
Preparation of total RNA and first-strand (cDNA) synthesis
Total RNA was extracted from 125 mg of sea bass distal intestine (6 fish/diet). Tissue lysis and homogenization were performed in special disposable sterile tubes (GentleMACS M tubes™, MiltenyiBiotec), in order to minimize the possibility of cross-contamination between samples, and using the gentleMACSDissociator (MiltenyiBiotec). After an automated purification process by using the Maxwell® 16 Instrument and Maxwell® 16 Tissue LEV total RNA purification Kit (Promega, Milan, Italy), the RNA was isolated. This RNA purification procedure provides an easy method for efficient, automated purification of highly concentrated total RNA from up to 25 mg of tissue. Purified RNA for up to 16 samples is obtained in less than 45 min of hands-free instrument operation. No post-purification treatment with nuclease, cleanup, or concentration is required to achieve superior performance in downstream applications. The low elution volume (30–100 μL) is used to generate purified RNA in a more concentrated format for use in downstream applications such as qRT-PCR, RT-PCR, and complementary DNA (cDNA) synthesis.
The integrity and purity of total RNA were determined by a spectrophotometer NanoDrop ™ (Thermo Scientific), measuring the absorbance at 260 nm and the absorbance ratio 260/280, respectively. The integrity of RNA was verified by electrophoresis on 1% agarose gel stained with ethidium bromide.
Reverse transcription of 1 μg total RNA from each fish intestine tissue was performed with random decamers in a volume of 20 μL by using the High-Capacity cDNA Archive Kit (ThermoFisher Scientific, Milan, Italy) following the manufacturer’s instructions.
Generation of in vitro-transcribed mRNAs for target genes
Sequences and melting temperatures (Tm) of the primers used for in vitro synthesis of standard mRNAs
GenBank acc. nr.
Nucleotide sequence (5′–3′)
The forward primer was engineered to contain a T3 phage polymerase promoter gene sequence to its 5′ end and used together with the reverse primer in a conventional RT-PCR of total RNA extracted from the sea bass intestine.
PCR products were then evaluated on a 2.5% agarose gel, cloned using pGEM®-T Easy cloning vector system (Promega, Milan, Italy), and subsequently sequenced. In vitro transcriptions were performed using T3 RNA polymerase and other reagents supplied in the Promega RiboProbe In Vitro Transcription System kit according to the manufacturer’s protocol.
Generation of standard curves
The mRNAs produced by in vitro transcription were used as quantitative standards in analyzing experimental (biological) samples (Terova et al. 2009). Defined amounts of mRNAs at tenfold dilutions were subjected to real-time PCR using One-Step Taqman® EZ RT-PCR Core Reagents (ThermoFisher Scientific, Milan, Italy), including 1× Taqman® buffer, 3 mM Mn(OAc)2, 0.3 mM dNTP except dTTP, 0.6 mM dUTP, 0.3 μM forward primer, 0.3 μM reverse primer, 0.2 μM FAM-6 (6-carboxyfluorescein-labeled probe), 5 units rTH DNA polymerase, and 0.5 units AmpErase® UNG enzyme in a 25-μL reaction volume. RT-PCR conditions were 2 min at 50 °C, 30 min at 60 °C, and 5 min at 95 °C, followed by 40 cycles consisting of 20 s at 94 °C, and 1 min at 62 °C. The Ct values obtained by amplification were used to create standard curves for target genes.
Quantification of IL-1β, IL-6 IL-8, IL-10, and TNFα in biological samples
Primers and probes used for one-step quantitative real-time RT-PCR
Nucleotide sequence (5′–3′)
Data from the TaqMan® PCR runs were collected with StepOne™ Software v 2.0. Cycle threshold (Ct) values corresponded to the number of cycles at which the fluorescence emission monitored in real time exceeded the threshold limit. The Ct values were used to create standard curves to serve as a basis for calculating the absolute amounts of mRNA in total RNA.
All statistical analysis were performed by using the software IBM SPSS Statistics 21. Growth performance data were submitted to two-way analysis of variance (two-way ANOVA) considering both tank and diet effects. One-way ANOVA was applied to analyze the morphology and gene expression data. In all statistical analyses, each individual fish was considered as an experimental unit. Duncan’s test was used for post hoc analysis. Differences were considered to be statistically significant when P < 0.05.
Growth performance indexes of European sea bass fed three different diets during the feeding trial
Biomass (g) ± SD
508.20 ± 60.02
502.60 ± 73.82
528.00 ± 67.76
546.80 ± 61.14
535.20 ± 62.51
641.20 ± 60.95
0.12 ± 0.08a
0.11 ± 0.05a
0.32 ± 0.12b
2.92 ± 1.54a
2.81 ± .0.51a
1.30 ± 0.25b
1.11 ± 0.11ab
1.07 ± 0.03b
1.23 ± 0.08a
Light microscopy and immunohistochemistry
By light microscopy, we evaluated different aspects of the various anatomical portions of the distal intestine: the thickness of muscle and mucosal layers and the shape and length of the villi of the intestinal mucosa. In addition, we paid attention to the presence and distribution of Goblet cells and to the presence, size, and content of supranuclear vacuoles of enterocytes. The presence and extent of any inflammatory infiltrates were also examined.
Further investigation by using transmission electron microscopy allowed us to highlight the cell differences between the three types of samples. Different aspects of the enterocytes were investigated. From the lumen to the lamina propria of the fold, we considered the brush border, the tubulo-vesicular system occupying the apical area of enterocytes, the supranuclear vacuoles, the nuclei, and infiltrating inflammatory cells.
Gene expression analysis
Expression levels of pro and inflammatory interleukins genes (IL-1β, IL-6, IL-8, IL-10) and TNFα gene in the distal intestine of European sea bass fed different diets
Gene expression levels (transcript copies/ng total RNA)
323.19 ± 33.55a
321.30 ± 15.96a
215.09 ± 41.94b
647.14 ± 325.05
873.26 ± 176.68
603.50 ± 163.84
548.88 ± 196.73a
763.04 ± 190.70a
285.90 ± 63.58b
257.18 ± 66.55a
326.67 ± 64.73a
110.80 ± 67.70b
370.07 ± 104.81b
664.20 ± 109.52a
377.25 ± 118.41b
Although IL-1β was expressed at relatively low levels in all analyzed fish, sea bass fed with diet T (supplemented with taurine), displayed the lowest number of mRNA copies of IL-1β gene in comparison to fish fed with the other two diets. Also, the expression of IL-8 and IL-10 genes in fish fed diet T was significantly lower (P < 0.05) with respect to the fish that received control or butyrate-supplemented diets. Dietary butyrate caused an upregulation of TNFα gene transcription (Table 5); indeed, fish fed on diet B showed higher levels of TNFα mRNA copies than fish fed with the other diets (P < 0.05). Finally, among the interleukins quantified in this study, IL-6 was the only one, to be not influenced by the diet (Table 5).
The feeding experiment conducted in this study lasted 60 days which is a short period for SGR and FCR reliable evaluation. Nevertheless, SGR of fish fed with diet T was 2.7 times higher (P < 0.05) than in fish fed with diet C or B. This result is also in line with findings of Brotons Martinez et al. (2004) on sea bass fingerlings fed on a diet containing SBM and supplemented with taurine for 30 days. The dramatic differences in growth observed in our study between the taurine-supplemented group (T) and the experimental groups B and C may be due to taurine deficiency in the latter groups, thus indicating that taurine is an essential dietary nutrient, as reported in several studies reviewed by Salze and Davis (2015).
Histological samples from distal intestine of fish fed with diet B (supplemented with sodium butyrate) showed that the muscle layers were thinner than in fish fed with diets C or T. This suggests that butyrate facilitated the formation of softer fecal pellets in fish of this group. Furthermore, the complex structure of the intestinal villi included a high number of Goblet cells, and it was not pseudostratified which means that it was iperproliferative. This result is in line with previous studies (Gálfi and Bokori 1990; Kotunia et al. 2004; Hu and Guo 2007), which have demonstrated the trophic effects of butyrate on the intestinal epithelium of terrestrial animals through an increase in the villi length and crypt depth. The number of supranuclear vacuoles was less than in fish fed with diet C, and they were often heterogeneous. We observed a few intestinal Goblet cells, in fish fed with diet C with SBM, and this result is in agreement with other studies conducted in Atlantic salmon (S. salar) and rainbow trout (O. mykiss) (Baeverfjord and Krogdahl 1996; Iwashita et al. 2008). However, we found also high concentrations of absorptive vacuoles containing PAS-positive substances (probably glicoproteins, considering their acidophilic properties) in the supranuclear region of the enterocytes from fish fed with diet C. This could indicate an interruption of the digestive processes occurring within the enterocytes, since these cells are involved not only in the uptake of different nutrients but also in the digestion. For example, once inside the enterocyte, the absorbed di- and tripeptides are digested into amino acids by cytoplasmic peptidases and transported from the cell into blood; only a very small number of these small peptides enter blood intact. In contrast, other authors have reported a reduction of the same supranuclear vesicles in salmonids and in carps. In fish fed with diet C, lamina propria, muscle layer, and epithelial lining were heavily infiltrated with inflammatory cells, including macrophages and polymorphonuclear leukocytes. The important signs of inflammation in our samples were also confirmed by immunohistochemistry staining. This analysis showed a strong positivity for anti-CD45 antibody indicating a relevant diffusion of leukocytes in all the distal intestinal tissue. The fact that an antibody directed against the human protein reacted also with sea bass tissue might confirm a high structural conservation of this protein in fish as well. This hypothesis is also supported by the discovery of a CD45 homolog (Marozzi et al. 2012) in sea bass (D. labrax), whose sequence obtained from transcriptome of gills (kindly provided by Francesco Buonocore, University of Tuscia, Italy) shows 52% of identity with human CD45 isoform 1 precursor. Since a common rule of thumb is that two sequences are homologous if they share more than 30% of identity over their entire length (Pearson, 2013), we are confident that 52% is a high enough identity to support the similarity between human and sea bass CD45 sequences. Of note, a 30% identity threshold for homology underestimates the number of homologs detected by sequence similarity between humans and yeast by 33% (Pearson 2013).
The TEM observations showed partly damaged microvilli and numerous large vacuoles occupying the supranuclear cytoplasm and containing a homogeneous electrondense substance, with partial break of their membranes probably leading to the release of lytic enzymes and recall of leukocytes. Some enterocytes with pyknotic nuclei were also observed. According to Noaillac-Depeyre and Gas (1973), the supranuclear body is of complex origin. Indeed, in the adult carp, the apical cytoplasm of the enterocytes of the medium intestine shows the presence of a dense tubolovesicular network that form a voluminous supranuclear body. However, the supranuclear bodies are not related to the presence of food in the intestinal lumen since they are also present in fasting animals. Therefore, it seems that they do not result only from the running together of the food vacuoles but probably arise from the fusion of the Golgi vesicles. The supranuclear bodies would thus have a dual origin, endogenous for the hydrolytic enzymes that contain, and exogenous for the di- and tripeptides supplied by the food which are then further digested inside them (Noaillac-Depeyre and Gas 1973).
Furthermore, the aforementioned acidophilic and PAS-positive substance that we found in the vacuoles of fish fed with diet C was absent in the distal intestine of fish fed with diet B, suggesting that butyrate supplementation of the diet have had a mitigation effect in the intestine of fish.
Although the inflammatory cells were infiltrated less than in the distal intestine of fish fed with diet C and were more concentrated at the mucosal and submucosa level, we found a significant number of leukocytes as confirmed by the positive staining with the CD45 antibody. TEM analysis of enterocytes showed a focal alteration at the microvilli level. However, other areas appeared much less compromised, microvilli were regularly displayed, and a developed apical tubulo-vesicular system was evident, suggesting an intense phagocitosys activity The cells showed supranuclear vacuoles with dishomogeneous content: partly dense, like that found in the fish of group C, and partly clear; this aspect suggests that in these vacuoles a partial digestion of the content occurs.
In fish fed with diet T, the muscle layers were rather developed. The villi folds were long and regular, with enterocytes showing a limited number of vacuoles and an increased number of Goblet cells confirming a strong inflammation reaction, which were mainly concentrated in the medioapical part of the fold. Leukocytes, immunoreactive to the CD45 antibody, were still present in both, the mucosa and the submucosa, similar to those observed in fish fed with diet B. The TEM images of enterocytes were very different from those in fish fed with diets C and B, with cells showing well-displayed microvilli frequently covered by a protective mucus layer, no alterations in the structure, an apical tubulo-vesicular system, and supranuclear vacuoles filled with a heterogeneous substance, including membranous bodies suggesting that an autophagic process was in progress. To our knowledge, no other example of taurine used as a feed additive causing autophagic processes has been reported in the literature, and thus this finding should be further investigated. However, this process seems to enable enterocytes of fish fed with diet T to cope with the negative effects induced by the SBM diet and contributes to the protection of the intestinal mucosa.
The expression pattern of molecular markers of inflammatory processes such as the inflammatory cytokines supported the light and TEM microscopy observations. The highest transcription level of the pro-inflammatory IL-1β gene was found in the distal intestine of sea bass fed with diet C, indicating a local tissue reaction to SBM insult, which was less evident in fish fed on diets B or T. Cytokine IL-8 was less expressed in fish fed with diet T than in those fed with diet C or B, suggesting a protective action of taurine against oxidative stress through the reduction of the inflammatory cascade induced by oxidative stress. The transcription activity of IL-10 gene, which is known as anti-inflammatory cytokine, was strongly reduced in fish fed with diet T, remaining ,however, relatively high in fish fed with diets C and B. Among the target cytokines, only IL-6 gene expression was not affected by dietary butyrate or taurine supplementation. Proinflammatory IL-6 is of particular interest since it is expressed to a small extent in normal colonic epithelium but to a much greater degree in colonic carcinomas (Shirota et al. 1990). In human colonic cells, increased expression of both IL-6 and its receptor IL-6R have mainly an anti-apoptotic effect (Yuan et al. 2004). IL-6, indeed, upregulates the expression of anti-apoptotic protein Bcl-xl (Fujio et al. 1997; Scwarze and Hawly 1995). Yuan and colleagues (2004), clearly demonstrated that butyrate downregulates IL-6 signaling in human colonocytes by inhibiting IL-6 receptor rather than IL-6 expression. Similarly, in our study, butyrate did not influence IL-6 expression, but we cannot exclude that, even in fish, butyrate could act principally on IL-6 receptor rather than in IL-6 expression. So, this is an issue that requires further investigation.
It is well documented that taurine inhibits the overproduction of inflammatory mediators such as TNFα (Kim and Cha 2014; Locksley et al. 2001). TNFα is known to stimulate further production of itself and to induce the expression of other pro-inflammatory mediators, such as IL-6, IL-8. TNFα was expressed at higher levels in the intestine of sea bass fed with diet B, which induced an upregulation also of IL-8 but not of IL-6. Moreover, higher expression of TNFα in fish receiving B diet supported the TEM results indicating a promotive effect of butyrate in increasing cellular turnover.
Our findings on cytokine activity are mainly in agreement with the literature. Indeed, in the intestine of carps receiving soy as protein source (Urán et al. 2008a), a peak of IL-1β was observed after the first week of feeding, and the expression of this gene as well as that of TNFα remained high over the control levels for the entire experiment. In the same study, a strong upregulation of IL-10 was observed after one week of SBM feeding, but at weeks 3 and 5, expression levels were downregulated at values either lower or similar to the control (Urán et al. 2008a). In zebrafish fed on diets containing different amounts of SBM, Hedrera et al. (2013) reported a significant increase in the intestinal transcription of the mRNA coding for proinflammatory cytokines, such as IL-1β and IL-8. The transcriptional activity of IL-10, known to have an anti-inflammatory action, was increased in the group receiving feed with soy protein, too.
European sea bass fed on a diet containing a concentration of a soy protein source close to 30% (16.7% as SBM and 12.8 as full-fat soy) for 2 months developed an inflammatory status in the distal intestine. Both butyrate and taurine, at concentrations as low as 0.2%, had some mitigating effect on the symptoms of inflammation, but the underling mechanisms of action seem different. The mechanisms underlying these effects should be further investigated along with any possible synergistic effect of butyrate and taurine in the feed in normalizing the intestinal abnormalities caused by soybean meal.
CA is a student of the Ph.D. course in “Biotechnology, Bioscience and Surgical Technology” at the “Università degli Studi dell’Insubria”, Varese, Italy.
This work was funded under the seventh EU Framework Programme by the ARRAINA project No288925: Advanced Research Initiatives for Nutrition & Aquaculture. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The funding body had no role in the design of the study, collection, analysis, and interpretation of the data and in writing the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this article and its supplementary information files.
SR carried out the molecular biology and statistical analysis and participated in the feeding trial. CC and CA carried out the feeding trial and participated in the sequence alignment. GF carried out the histological study and was involved in drafting the manuscript. RG and AG carried out the immunoassays and participated in the analysis and interpretation of the data. GT conceived of the study, participated in the experimental design, and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the University of Insubria, Varese, Italy. The Committee on the Ethics of Animal Experiments of the same university approved all of the protocols performed. Fish handling was performed under tricaine methanesulfonate (MS222) anesthesia, and all efforts were made to minimize discomfort, stress, and pain to the fish.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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