Erucic acid impairs intestinal immune function of on-growing grass carp (Ctenopharyngodon idella)

Journal Pre-proof Erucic acid impairs intestinal immune function of on-growing grass carp (Ctenopharyngodon idella)

Lei Gan, Lin Feng, Wei-Dan Jiang, Pei Wu, Yang Liu, Jun Jiang, Sheng-Yao Kuang, Ling Tang, Xiao-Qiu Zhou PII:

S0044-8486(19)32632-8

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734916

Reference:

AQUA 734916

To appear in:

aquaculture

Received date:

6 October 2019

Revised date:

6 December 2019

Accepted date:

30 December 2019

Please cite this article as: L. Gan, L. Feng, W.-D. Jiang, et al., Erucic acid impairs intestinal immune function of on-growing grass carp (Ctenopharyngodon idella), aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734916

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© 2019 Published by Elsevier.

Journal Pre-proof

Erucic acid impairs intestinal immune function of on-growing grass carp (Ctenopharyngodon idella) Lei Gan a, Lin Feng a,b,c, Wei-Dan Jiang a,b,d, Pei Wu a,b,d, Yang Liu a,b,e, Jun Jiang a, Sheng-Yao Kuang f, Ling Tang f, Xiao-Qiu Zhou

a,b,c,*

a

Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China

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Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural

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University, Chengdu 611130, China c Key laboratory of Animal Disease-resistant Nutrition, Sichuan Province, China Key laboratory of Animal Disease-resistant Nutrition, Ministry of Education, China

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Key laboratory of Animal Disease-resistant Nutrition and Feed, Ministry of Agriculture and Rural Affairs,

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Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China

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China

* Corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,

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Sichuan, China. E-mail: [email protected], [email protected] (X.-Q. Zhou).

Journal Pre-proof Abstract: Erucic acid (EA) mainly occurs in rapeseed meal as an anti- nutritional factor which can deteriorate animal growth performance. To our knowledge, the deteriorated animal growth performance is closely correlated with the impaired intestinal immune function. Thus, a 60-day feeding trial was carried out to explore the relationship of EA and the intestinal immune function of on- growing grass carp (Ctenopharyngodon idella). Fish (129.17 ± 0.19 g) were distributed into 18 cages (length, width and height all equal to 1.4 m) at a

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stocking density of 15 individuals/m3 and received 6 diets 4 times a day to apparent satiety for 60 days. The

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basal diet, to which different levels of EA were added to obtain 0.00 (control), 0.29, 0.60, 0.88, 1.21 and

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1.50% EA, contained 28.73% crude protein and 6.43% crude lipid. At the end of the feeding trial, 24 fish of similar body weight from each group were used in a challenged test with Aeromonas hydrophila for 6 days

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and then collected to evaluate the effect of EA on morbidity of enteritis, intestinal immune parameters and

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intestinal inflammatory parameters. In fish intestine, our study found that EA: (1) reduced the activities of

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lysozyme (LZ) and acid phosphatase (ACP) and the contents of complement 3 (C3), C4 and

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immunoglobulin M (IgM); (2) decreased the transcript levels of liver-expressed antimicrobial peptide (LEAP)-2A, LEAP-2B, hepcidin, β-defensin-1 and mucin2; (3) aggravated inflammatory response in relation to the increased transcript levels of pro- inflammatory cytokines involved in the activation of [IκB kinase β, γ (IKKβ, γ)/inhibitor of κBα (IκBα)/nuclear factor (NF)-κBp65 and c-Rel] signalling pathway and to the decreased transcript levels of anti- inflammatory cytokines involved in the suppression of [target of rapamycin (TOR)/ribosomal protein S6 kinases 1 (S6K1) and eIF4E-binding proteins (4E-BP)] signalling pathway. EA did not alter the transcript levels of interleukin (IL)-12p35, NF-κB p52 and IKKα in the intestine of on-growing grass carp. Finally, based on a straight broken- line model and using enteritis morbidity and the activities of LZ in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) as response criterions, the maximum tolerance levels of EA for on- growing grass carp (129.17–471.18 g) were

Journal Pre-proof evaluated to be 0.53%, 0.36%, 0.44% and 0.45% diet, respectively. Keywords: Erucic acid, immune function, intestinal segments, inflammatory response, antimicrobial peptide 1. Introduction Among today’s farmed fish species, grass carp (Ctenopharyngodon idella) is regarded as the biggest one

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(FAO, 2018). Due to its large aquaculture scale, the requirement of artificial feed for this fish is also

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increasing. Grass carp can efficiently utilise the nutrients derived from plants (He et al., 2013). Thus, a large

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incorporation of plant protein source into its formulated feed may be a viable option for decreas ing its

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aquaculture cost. Unfortunately, an increasing use of plant protein source in grass carp feed did not yield the

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expected positive results (Tan et al., 2013). One of the possible reasons for the negative effect of plant protein sources on grass carp is the presence of anti- nutritional factors (ANFs) (Enami, 2011). ANFs are

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defined as inherent substances in plants, which could interfere with the balance of health and nutrient status

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of animals (NRC, 2011). The negative effects caused by ANFs on fish include depressed growth

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performance (Couto et al., 2014), disrupted intestinal structure (Chen et al., 2011) and increased enteritis morbidity after infection with pathogens (Wang et al., 2019). The ANF EA mainly occurs in rapeseed meal (Do et al., 2017). EA has been reported to interfere with the metabolism of fatty acid and subsequently cause malfunction of rat hearts (Houtsmuller et al., 1970). Our previous study reported that EA could disrupt the intestinal structural integrity of on-growing grass carp (Gan et al., 2019). However, to our knowledge, the effect of EA on the intestinal immune response of grass carp remains unclear. The immune function of fish consists of innate immune components [such as lysozyme (LZ) and liver-expressed antimicrobial peptide (LEAP)-2A] (Lieschke and Trede, 2009), and adaptive immune components [such as immunoglobulin M (IgM)] (Zhu et al., 2013). A previous study indicated that EA

Journal Pre-proof reduced rat serum α-tocopherol content (Watkins et al., 1995), and a lower level of α-tocopherol could decrease the activity of LZ in yellow catfish (Pelteobagrus fulvidraco) (Lu et al., 2016). In addition, a previous study has found that EA elevated lipid content in rats (Rahman et al., 2014), and a higher level of lipids could decrease the IgM content and down-regulate the mRNA level of LEAP-2A in grass carp (Feng et al., 2017). Additionally, fish immune function is closely related to inflammatory response which is regulated by inflammation-related cytokines [such as interleukin (IL)-8 and transforming growth factor

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(TGF)-β1] (Sun et al., 2014). In mice, Liu et al. (2012) observed that oleic acid was a major metabolite of EA. The result from Li et al. (2008) highlighted that oleic acid increased IL-8 concentration in rats.

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Meanwhile, in mice, it was shown that EA activated PPARδ (Johnson et al., 1997), which could reduce the

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level of TGF-β1 (Nagasawa et al., 2006). In fish, it was reported that the transcript levels of IL-8 and

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TGF-β1 were regulated by nuclear factor (NF)-κB and target of rapamycin (TOR) signalling pathways,

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respectively (Zhong et al., 2019). Sauer et al. (1989) reported that EA reduced carnitine content in pig hearts.

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A lower content of carnitine could stimulate NF-κB expression in rat kidneys (Kunak et al., 2016). EA also elevated plasma cholesterol content in rats (Vasdev and Kako, 1978), which could depress the transcript

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abundance of TOR in grass carp (Wang et al., 2018). As described above, we speculate that EA might impair fish intestinal immune function involved in NF-κB and TOR signalling pathways. However, the hypothesis needs to be verified.

Considering the above, it is necessary to determine the maximum tolerance level of EA for grass carp and clarify how EA poses negative effects on grass carp. For this purpose, an experiment was conducted, and morbidity of enteritis, intestinal immune parameters and intestinal inflammatory parameters in the intestine of on-growing grass carp were evaluated. 2. Methods 2.1. Diet preparation and experimental design

Journal Pre-proof Table 1 summarises the composition of basal diet which is the same to that of Gan et al. (2019). Six diets containing 0.00, 0.30, 0.60, 0.90, 1.20 and 1.50% EA were formulated. Dietary lipid levels were balanced with graded lauric acid (Yang and Dick, 1994). Final EA levels of the 6 diets were determined to be 0.00% (control), 0.29%, 0.60%, 0.88%, 1.21% and 1.50% diet with the method of gas- liquid chromatography (Sim et al., 1985). The 6 diets were stored in a freezer at 4 °C until use. Please insert Table 1 here

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This experiment was carried out according to the ethical guidelines on the protection of animals for scientific purpose from University of Sichuan Agricultural Animal Care Advisory Committee. Fish were

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obtained from a local private fishery and acclimated to pond conditions for 4 weeks. Healthy fish (initial

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weight, 129.17 ± 0.19 g) were randomly stocked into 18 cages (length, width and height were all measured

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to be 1.4 m) at a density of 30 fish per cage. Fish in triplicate cages were randomly assigned to each of the

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trial diets 4 times a day to apparent satiety for 60 days. All cages were supplied with a disc (diameter was

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measured to be 100 cm) for uneaten feed collection. During the experiment, the monitored water quality was as follows: higher than 6.0 mg L-1 (dissolved oxygen), 26.8 ± 1.6 °C (temperature) and 7.0 ± 0.4 (pH).

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At the end of 60-day feeding, 24 fish of similar body weight were subjected to immune challenge by an intraperitoneal injection of A. hydrophila (kindly provided by the Veterinary Medicine College of Sichuan Agricultural University in China) with a concentration of 1.5×108 colony- forming units (CFU) ml-1 kg-1 for 6 days according to Zhong et al. (2019). The experimental conditions were similar to those of the feeding trial. After the challenge trial, all fish were anaesthetised with benzocaine as described by Bohne et al. (2007), and the enteritis morbidity was assessed by a scoring system with a semi-quantitative method according to Song et al. (2014). After that, the digestive tracts were washed with pre-cooled physiological saline and then cut into proximal intestines (PI), mid intestines (MI) and distal intestines (DI) based on the turning point referring to Askarian et al. (2012). These samples were reserved at −80 ºC for further analysis.

Journal Pre-proof 2.2. Histological analysis Sampling of fish intestine was performed for histomorphological evaluation as described by Kokou et al. (2017). Briefly, pieces of PI, MI and DI 4 μm in thickness were stained by haematoxylin and eosin (H&E) and photographed by a Nikon TS100 light microscope. 2.3. Biochemical analysis Prior to biochemical analysis, the intestines of 6 fish per group by homogenisation in 10 volumes (w v-1 )

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of pre-cooled physiological saline were centrifuged at 6,000 g for 20 min at 4 °C to obtain supernatants as described by Huang et al. (2019). The activities of lysozyme (LZ) and acid phosphatase (ACP) were

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measured with kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to Ma et al.

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(2017). The contents of complement 3 (C3), C4 and immunoglobulin M (IgM) were determined by kits

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purchased from Zhejiang Elikan Biological Technology CO., LTD according to Li et al. (2013). The sample

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protein concentration was measured according to Bradford (1976).

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2.4. Reverse transcription polymerase chain reaction (RT-qPCR) The mRNA abundances of immune-related genes were quantified by PCR according to Jiang et al.

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(2015). Briefly, the total RNA was extracted from PI, MI and DI of 6 fish from each group by RNAiso Plus (Takara, Dalian, China) according to the manufacturer ’s protocol. The extracted RNA integrity, quantity and purity were determined respectively using 1% agarose gel with nucleotide indicator, Eppendorf BioPhotometer plus (Eppendorf AG, Humburg, Germany) and 260/280 nm optical density ratio at ≥ 1.8. Then, the total RNA was treated with Dnase I to remove genomic DNA and then reverse transcribed to cDNA with a PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to its specification. The mRNA abundances of immune-related genes from three intestinal segments were performed using RT-qPCR assays based on their specific primers from our laboratory or published sequences of grass carp (Supplemental Table S1). β-actin was recognised as a proper housekeeping gene according to our

Journal Pre-proof preliminary trial. The amplification efficiencies of target genes and β-actin were quantified using the specific gene standard curves by 10-fold serial dilutions and an efficiency of approximately 100% was detected for each primer. The mRNA transcript levels of target genes were calculated by 2−ΔΔCT with an amplified efficiency of approximately 100% of respective primer according to Livak and Schmittgen. (2001). 2.5.Western blot (WB) The procedure of WB was carried out to detect T-TOR, p-TOR and NF-κB p65 proteins in PI, MI and DI

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of fish according to Wang et al. (2019). Briefly, an approximately 50-ug protein sample was separated by gel electrophoresis (SDS-PAGE, 10%), then shifted to polyvinylidene difluoride (PVDF) membranes. The

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membranes were briefly rinsed with tris-buffered saline with Tween (TBST) containing 0.1% Tween 20,

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then blocked in 5% bovine serum albumin (BSA) in TBST for 1.5 h at room temperature and subsequently

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immersed in primary antibodies purchased from Affinity Bioscience (Golden, Colorado, USA) against

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; 1:1,000), NF-κB p65 (rabbit, anti- NF-κB p65; 1:750), β-actin (rabbit, 1:3,000) and

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anti- p-TOR Ser

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T-TOR (rabbit, anti- T-TOR; 1:1,000), phosphorylation of TOR on residue Ser2448 (p-TOR Ser 2448 ) (rabbit,

lamin B1 (rabbit, 1:1,000) overnight at 4 °C. The membranes successfully reacted with their respective

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primary antibodies and were briefly washed, followed by a 1.5-h incubation with secondary antibody (goat anti-rabbit IgG, 1:8,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactive bands were detected with an ECL kit (Beyotime Biotechnology Inc., Shanghai, China). The bands were quantified by ImageJ 1.43 (National Institutes of Health, Bethesda, MD, USA). A similar result was achieved by repeating the experiment 3 times. Statistical analysis Statistical analysis regarding the effects of EA on intestinal immune-related indicators of fish was performed with general linear models (GLM) procedure (SAS Institute, Inc., 2006), and P < 0.05 was considered significant. Orthogonal polynomial contrast was applied to measure the linear and quadratic

Journal Pre-proof effect of EA according to Hooft et al. (2011), and the assessment of maximum tolerance levels of EA were performed by SAS with a broken- line regression model according to Robbins et al. (2006). Pearson correlation coefficient analysis was performed using PROC CORR in SAS. 3. Result 3.1. The effect of EA on enteritis morbidity and intestinal histology of fish Enteritis morbidity is summarised in Fig. 1 and 2. Our study showed that EA (≥ 0.60% diet) markedly

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facilitated enteritis morbidity in grass carp. Moreover, an increase of enteritis morbidity was detected with dietary EA increment (P < 0.01). Afterward, our study also showed that typical pathological symptoms, such

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of fish fed dietary EA levels up to 0.60% diet (Fig. 3).

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Please insert Fig. 1 here

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Please insert Fig. 2 here Please insert Fig. 3 here

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as higher epithelial goblet cells (GC) and blood capillary hyperaemia (BH), were monitored in the intestine

3.2. The effect of EA on the activities and contents of intestinal immune-related indicators of fish

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As shown in Table 2, the activities of LZ and ACP, and the contents of C3 in three intestinal segments of fish decreased in a linear (P < 0.001) and quadratic (P < 0.05) fashion with dietary EA increment. Incremental EA levels also linearly (P < 0.001) decreased the contents of C4 and IgM in PI, and both linearly (P < 0.001) and quadratically (P < 0.05) decreased the contents of C4 and IgM in MI and DI of fish. Please insert Table 2 here 3.3. The effect of EA on the transcript levels of intestinal antimicrobial peptides of fish The transcript levels of LEAP-2A, LEAP-2B, hepcidin, β-defensin-1 and Mucin2 in three intestinal segments of fish were markedly decreased with incremental EA level (P < 0.05) (Fig. 4). 3.4. The effect of EA on the transcript levels of genes related to intestinal inflammatory response of fish

Journal Pre-proof Incremental EA levels markedly elevated the transcript levels of IL-1β, tumour necrosis factor (TNF)-α, IFN-γ2, IL-6, IL-8, IL-12p40, IL-15, IL-17D, NF-κBp65, C-Rel, IKKβ and IKKγ and decreased the transcript levels of IL-10, IL-11, IL-4/13A, IL-4/13B, TGF-β1, TGF-β2, TOR and S6K1 in three intestinal segments of fish (P < 0.05). No statistical differences of IL-12p35, NF-κBp52 and IKKα transcript levels were monitored among each EA groups (P > 0.05) (Fig. 4). Please insert Fig. 4 here

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3.5. The effect of EA on the protein expressions of nuclear NF-κB p65, p-TOR Ser2448 and T-TOR of fish intestine

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A linear increase in the protein expressions of nuclear NF-κB p65 (P < 0.01) and a linear reduction in the

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ratios of p-TOR(Ser2448 ) protein levels to T-TOR protein levels (P < 0.01) in fish intestines were verified

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with EA level increment (Fig. 5 and 6).

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Please insert Fig. 5 here

3.6. Correlation analyses

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Please insert Fig. 6 here

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The correlation coefficients of genes related to the immune function of fish intestines are shown in Supplemental Table. S2. Our data showed a positive correlation between the protein level of nuclear NF-κB p65 and the transcript levels of IL-1β, TNF-α, IFN-γ2, IL-6, IL-8, IL-12p40, IL-15 and IL-17D. Moreover, nuclear NF-κB p65 protein levels and C-Rel transcript levels were adversely correlated with the transcript level of IκBα, which was adversely correlated with the transcript levels of IKKβ and IKKγ. Meanwhile, our data also showed a positive correlation between the ratio of p-TOR(Ser2448 ) protein level to T-TOR protein level and the transcript levels of IL-10, IL-11, IL-4/13A, IL-4/13B, TGF-β1, TGF-β2 and S6K1. Moreover, the transcript levels of 4E-BP1 and 4E-BP2 were adversely correlated with the ratio of p-TOR(Ser2448 ) protein level to T-TOR protein level.

Journal Pre-proof 4. Discussion ANFs could not be ignored in the plant protein sources of aquafeed as they pose many negative effects on fish species, which include inhibited growth (Chen et al., 2011) and induced enteritis (Knudsen et al., 2008). EA is a major ANF in rapeseed meal (Do et al., 2017). Our previous study found that EA could inhibit the growth performance of grass carp (Gan et al., 2019). The inhibited growth of fish is closely related to the occurrence of enteritis (Gu et al., 2018).

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4.1 EA and its effects on intestinal immune function and enteritis morbidity

Hyperaemia is a reliable bioindicator of enteritis in fish intestines (Huang et al., 2019). Our study

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showed that EA (≥ 0.60% diet) markedly facilitated enteritis morbidity in grass carp. Afterward, we further

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monitored the influence of EA on fish intestinal pathological symptoms. Higher intestinal epithelial goblet

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cell numbers (GC) (O’Hara and Sharkey, 2007) and blood capillary hyperaemia (BH) (Byelinska et al., 2018)

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could be regarded as pathological symptoms of fish intestines. Our study showed that EA caused similar

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pathological symptoms to those mentioned above in three intestinal segments of fish. The reasonable explanations for these apparent symptoms induced by EA in fish intestine might be as follows: (1) EA

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facilitated enteritis morbidity of fish, which might be linked to vitamin A (VA) deficiency. Carroll (1959) noted that EA could stimulate the occurrence of VA deficiency symptoms in rats. Zhang et al. (2017) concluded that VA deficiency facilitated enteritis morbidity of grass carp. (2) EA increased the intestinal epithelial goblet cell numbers of fish, which might be linked to oleic acid. A previous study performed on rat intestines reported that the chain-shortened product of EA was oleic acid (Liu et al., 2012). Reddy and Naidu (2016) suggested that higher levels of oleic oil could increase the number of goblet cells in rat colons. (3) EA caused blood capillary hyperaemia, which might be linked to the accumulation of lipids. Gorrill and Walker (1974) observed that EA induced the accumulation of lipids in lambs. Results from our laboratory found that excessive lipid levels caused blood capillary hyperaemia in fish intestines (Feng et al., 2017). To

Journal Pre-proof our knowledge, the occurrence of these pathological symptoms in fish were tightly linked to the impaired intestinal immune function (Li et al., 2018). Fish immune function mainly consists of innate immune function (such as LZ and antibacterial peptides) and adaptive immune function (such as IgM) (Uribe et al., 2011). The present study observed that EA decreased the activities of LZ and ACP and the contents of C3, C4 and IgM, and down-regulated the transcript levels of antibacterial peptides LEAP-2A, LEAP-2B, hepcidin, β-defensin-1 and Mucin-2 in three

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intestinal segments of fish, suggesting that EA could impair the intestinal immune function of fish. Similar to the result of our study, other ANFs, such as phytic acid and gossypol, could also impair the intestinal

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immune function of grass carp (Zhong et al., 2019; Wang et al., 2019). The negative effect caused by EA

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might be related to reactive oxygen species (ROS). A previous study found that EA could increase the ROS

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level in fish intestines (Gan et al., 2019). Jiang et al. (2017) reported that ROS impaired the immune

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function of Mytilus edulis. Additionally, in fish, the impaired intestinal immune function was also closely

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related to the aggravation of inflammatory response, which was regulated by pro- inflammatory cytokines and anti- inflammatory cytokines, and the NF-κB and TOR signalling pathways were involved in the process

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(Li et al., 2018). In three intestinal segments of grass carp, our study observed that EA elevated the transcript levels of pro- inflammatory cytokines IL-1β, TNF-α, IFN-γ2, IL-6, IL-8, IL-12p40 (not IL-12p35), IL-15 and IL-17D related to the activation of the NF-κB (not NF-κB p52) signalling pathway, and decreased the transcript levels of anti- inflammatory cytokines IL-10, IL-11, IL-4/13A, IL-4/13B, TGF-β1 and TGF-β2 related to the inhibition of TOR signaling pathway. This suggested that EA could aggravate inflammatory response in fish intestines partially associated with the activation of NF-κB (not NF-κB p52) and inhibition of TOR signalling pathways. Similar results were also detected in other experiments. For example, aggravated intestinal inflammatory response was caused by soya-saponins in juvenile turbot (Scophthalmus maximus) (Gu et al., 2018) and Atlantic salmon (Salmo salar L) (Krogdahl et al., 2015); soybean

Journal Pre-proof β-conglycinin in Jian carp (Cyprinus carpio var. Jian) (Zhang et al., 2013); soybean glycinin in Jian carp (Jiang et al., 2015) and soybean antigenic protein in obscure puffer (Takifugu fasciatus) (Yao et al., 2019). In the present study, several results regarding EA-aggravated inflammatory response of grass carp intestine attracted our attention, and the possible explanations were as follows. First, EA only elevated the transcript levels of IL-12p40 (not IL-12p35) in the intestines of grass carp, which might be related to prostaglandin E2 (PGE2). In mice, it has been found that IL-1β stimulates the secretion of PGE2

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(Molina-Holgado et al., 2000), which could elevate the transcript levels of IL-12p40 (not IL-12p35) (Sheibanie et al., 2004). Our trial results showed that EA elevated the transcript level of IL-1β in grass carp

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intestines. Second, EA elevated the transcript levels of IKKβ and IKKγ (not IKKα), which might be related

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to PKCζ. In rats, it was observed that TNF-α increased PKCζ activity (Estève et al., 2002), which could

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elevate the transcript levels of IKKβ and IKKγ (not IKKα) (Peng et al., 2007). Our results showed that EA

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elevated the transcript level of TNF-α in the intestines of grass carp. Third, EA elevated the transcript levels

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of NF-κBp65 and C-Rel (not NF-κBp52) in three intestinal segments of grass carp, which might be related to IKKα. In mice, Bonizzi et al. (2004) reported that IKKα activated NF-κBp52. Our study found that EA

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did not alter the transcript level of IKKα in the intestines of grass carp. 4.2 The maximum tolerance level of EA in fish The broken- line analysis (SAS) of enteritis morbidity and LZ in PI, MI and DI revealed that the maximum tolerance levels of dietary EA were 0.53%, 0.36%, 0.44% and 0.45% diet, respectively (Fig. 7), which were lower than that based on the broken- line analysis (SAS) of PWG from the growth trial (Gan et al., 2019). These data noted that infections with intestinal pathogenic bacteria could decrease the maximum tolerance level of EA for on-growing grass carp. A similar phenomenon was also detected in other experiment from our laboratory regarding gossypol (one kind of ANFs) (Wang et al., 2019), which showed that the maximum tolerance level of gossypol based on IgM for on- growing grass carp in challenge trial was

Journal Pre-proof lower than that based on PWG in growth trial. Please insert Fig. 7 here 5. Conclusion In conclusion (Fig. 8), our study found that EA could impair the intestinal immune function of on-growing grass carp, which might partly be involved in two pathways: (1) weakening the immune response partly by decreasing the activities of LZ and ACP and the contents of C3 and C4 and inhibiting the

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transcript levels of antimicrobial peptides; (2) aggravating inflammatory response partly by up-regulating the transcript levels of pro- inflammatory cytokines and down-regulating the transcript levels of

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anti- inflammatory cytokines, which might be related to the activation of NF-κB (not NF-κBp52) and the

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inhibition of TOR signalling pathways. Finally, the straight broken- line analysis (SAS) of enteritis morbidity,

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and the activities of LZ in PI, MI and DI revealed that the maximum tolerance levels of dietary EA for

Acknowledgments

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Please insert Fig. 8 here

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on-growing grass carp were 0.53%, 0.36%, 0.44% and 0.45% diet, respectively.

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This research was financially supported by National Key R&D Program of China (2018YFD0900400), the Earmarked Fund for China Agriculture Research System (CARS-45), Outstanding Talents and Innovative Team of Agricultural Scientific Research (Ministry of Agriculture), Foundation of Sichuan Youth Science and Technology Innovation Research Team (2017TD0002), Key Research and Development Plan in Sichuan Province (2018NZ0007), Supported by Sichuan Science and Technology Program (2019YFN0036). The authors would like to thank the personnel of these teams for their kind assistance.

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Journal Pre-proof Huang, C., Feng, L., Jiang, W.-D., Wu, P., Liu, Y., Zeng, Y.-Y., Jiang, J., Kuang, S.-Y., Tang, L., Zhou, X.-Q., 2019. Deo xynivalenol decreased intestinal immune function related to NF -κB and TOR signalling in juvenile grass carp (Ctenopharyngodon idella). Fish Shellfish Immun. 84, 470-484. Jiang, W.-D., Hu, K., Zhang, J.-X., Liu, Y., Jiang, J., Wu, P., Zhao, J., Kuang, S.-Y., Tang, L., Tang, W.-N., 2015. Soyabean glycinin depresses intestinal growth and function in juvenile Jian carp (Cyprinus carpio var Jian): protective effects of glutamine. Br. J. Nutr. 114, 1569-1583. Jiang, Y., Tang, X., Sun, T., Wang, Y., 2017. BDE-47 exposure changed the immune function of haemocytes in Mytilus edulis: An explanation based on ROS-mediated pathway. Aquat. Toxicol. 182, 58-66. Johnson, T.E., Ho lloway, M.K., Vogel, R., Rutledge, S.J., Perkins, J.J., Rodan, G.A., Sch midt, A., 1997. Structural requireme nts and cell-type specificity for ligand activation of peroxisome proliferator-activated receptors. J. Steroid. Biochem. 63, 1-8. Knudsen, D., Jutfelt, F., Sundh, H., Sundell, K., Koppe, W., Frø kiaer, H., 2008. Dietary soya saponins increase gut permeability and play a key role in the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.). Br. J. Nutr. 100, 120-129.

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Krogdahl, Å., Gajardo, K., Kortner, T.M., Penn, M., Gu, M., Berge, G.M., Bakke, A.M., 2015. Soya saponins induce enteritis in Atlantic salmon (Salmo salar L.). J. Agric. Food Chem. 63, 3887-3902.

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Kunak, C.S., Ugan, R.A., Cadirci, E., Karakus, E., Po lat, B., Un, H., Halici, Z., Saritemu r, M., At maca, H.T., Karaman, A., 2016. Nephroprotective potential of carn itine against glycerol and contrast -induced kidney injury in rats through modulation of oxidative stress, proinflammatory cytokines, and apoptosis. Br. J. Radiol. 89, 20140724.

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young grass carp (Ctenopharyngodon idella). Fish Shellfish Immun. 76, 333-346. Li, T., Zhao, B., Wang, C., Wang, H., Liu, Z., Li, W., Jin, H., Tang, C., Du, J., 2008. Regulatory Effects of Hydrogen Sulfide on 1081-1087.

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IL-6, IL-8 and IL-10 Levels in the Plas ma and Pu lmonary Tissue of Rats with Acute Lung In jury. Exp Biol Med. 233, Lieschke, G.J., Trede, N.S., 2009. Fish immunology. Current Biology. 19, R678-R682. Liu, J., Liang, S., Liu, X., Brown, J.A., Newman, K.E., Sunkara, M., Morris, A.J., Bhatnagar, S., Li, X., Pu jol, A., 2012. The absence of ABCD2 sensitizes mice to disruptions in lipid metabolism by dietary erucic acid. J Lipid Res. 53, 1071-1079. Livak, K.J., Sch mittgen, T.D., 2001. Analysis of relat ive gene exp ression data using real-t ime quantitative PCR and the 2− ΔΔCT method. methods. 25, 402-408. Lu, Y., Liang, X.-P., Jin, M., Sun, P., Ma, H.-N., Yuan, Y., Zhou, Q.-C., 2016. Effects of dietary vitamin E on the growth performance, antio xidant status and innate immune response in juvenile yellow catfish (Pelteobagrus fulvidraco). Aquaculture. 464, 609-617. Ma, S., Sun, Y., Wang, F., M i, R., Wen, Z., Li, X., Meng, N., Li, Y., Du, X., Li, S., 2017. Effects of tus sah immunoreactive substances on growth, immunity, disease resistance against Vibrio splendidus and gut microbiota profile of Apostichopus japonicus. Fish Shellfish Immun. 63, 471-479. Molina-Holgado, E., Ort iz, S., Mo lina-Holgado, F., Guaza, C., 2000. Induction of COX-2 and PGE2 biosynthesis by IL-1β is mediated by PKC and mitogen-activated protein kinases in murine astrocytes. Br. J. Pharmacol. 131, 152-159. Nagasawa, T., Inada, Y., Nakano, S., Tamu ra, T., Takahashi, T., Maruyama, K., Yamazaki, Y., Kuroda , J., Shibata, N., 2006. Effects of bezafibrate, PPA R pan-agonist, and GW501516, PPA Rδ agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet. Eur J Pharmacol. 536, 182-191.

Journal Pre-proof National Research Council (NRC), 2011. Nutrient Requirements of Fish and Shrimp. National Academy Press, Washington, DC, pp. 57–101. O’Hara, J.R., Sharkey, K.A., 2007. Pro liferat ive capacity of enterochromaffin cells in guinea -pigs with experimental ileitis. Cell Tissue Res. 329, 433-441. Peng, Y., Sigua, C.A., Gallagher, S.F., Murr, M.M., 2007. Protein kinase C-ζ is crit ical in pancreatitis-induced apoptosis of Kupffer cells. J Gastrointest Surg. 11, 1253-1261. Rah man, H., Nasreen, L., Hab ib, K., Rah man, N., 2014. Effects of Dietary Coconut Oil on Erucic Acid Rich Rapeseed Oil-induced Changes of Blood Serum Lipids in Rats. Curr Nutr Food Sci 10, 302-307. Reddy, K.V.K., Naidu, K.A., 2016. Oleic acid, hydro xytyrosol and n -3 fatty acids collectively modulate colitis through reduction of oxidative stress and IL-8 synthesis; in vitro and in vivo studies. Int Immunopharmacol. 35, 29-42. Robbins, K.R., Saxton, A.M., Southern, L.L., 2006. Estimation of nutrient requirements using broken -line reg ression analysis. J Anim Sci. 84, E155-E165. Sauer, F.D., Kramer, J.K.G., Forester, G.V., Butler, K.W., 1989. Palmitic and erucic acid metabolis m in isolated perfused hearts

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modulation of intestinal microbiota in grouper Epinephelus coioides. Aquaculture. 430, 50-56. Tan, Q., Liu, Q., Chen, X., Wang, M., Wu, Z., 2013. Growth performance, biochemical ind ices and hepatopancreatic function of grass carp, Ctenopharyngodon idellus, would be impaired by dietary rapeseed meal. Aquaculture. 414-415, 119-126.

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gossypol reduced intestinal immunity and aggravated inflammation in on -growing grass carp (Ctenopharyngodon idella). Fish Shellfish Immun. 86, 814-831.

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Journal Pre-proof Zhong, J.-R., Feng, L., Jiang, W.-D., Wu, P., Liu, Y., Jiang, J., Kuang, S.-Y., Tang, L., Zhou, X.-Q., 2019. Phytic acid d isrupted intestinal immune status and suppressed growth performance in on -growing grass carp (Ctenopharyngodon idella). Fish Shellfish Immun. 92, 536-551. Zhu, L.-Y., Nie, L., Zhu, G., Xiang, L.-X., Shao, J.-Z., 2013. Advances in research of fish immune-relevant genes: a co mparat ive

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overview of innate and adaptive immunity in teleosts. Dev. Comp. Immunol. 39, 39-62.

Journal Pre-proof Table 1 Composition and nutrients content of the basal diet. Ingredients Fish meal

%

Nutrients content

5.10

Crude protein

Casein Gelatin

23.86 6.50

Crude lipid4 Crude fibre 4

DL-Met (99%)

0.21

Ash

L-Trp (99.2%) Fish oil

0.05 2.52

5

n-3 n-65

Soybean oil α-starch

1.81 22.00

Available phosphorus Gross energy (cal/g)

Corn starch

22.39

Cellulose Vitamin premix1 Mineral premix2

5.00 1.00 2.00

Choline chloride (50%) Ethoxyquin (30%)

1.00 0.05

Ca(H2 PO4 )2 3 Erucic acid premix

1.51 5.00

6.43 4.26

4

5.80 1.04 0.96 0.40 4285.19

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6

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1

% 28.73

4

Per kilogram of vitamin premix (g kg-1 ): retinyl acetate (500,000 IU g-1 ), 0.39; cholecalciferol (500,000 IU

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g- 1 ), 0.20; DL-a-tocopherol acetate (50%), 23.23; menadione (22.9%), 0.83; cyanocobalamin (1%), 0.94; D-biotin (2%), 0.75; folic acid (95%), 0.17; thiamine nitrate (98%), 0.10; ascorhyl acetate (95%),9.77;

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niacin (99%), 3.44; meso-inositol (98%), 28.23; calcium-D-pantothenate (98%), 3.85; riboflavin (80%), 0.73;

Per kilogram of mineral premix (g kg-1 ): MnSO 4 .H2 O (31.8% Mn), 2.6600; MgSO 4 .H2 O (15.0% Mg),

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2

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pyridoxine hydrochloride (98%), 0.45. All ingredients were diluted with corn starch to 1 kg.

200.0000; FeSO 4 .H2O (30.0% Fe), 12.2500; ZnSO 4 .H2 O (34.5% Zn), 8.2500; CuSO 4 .5H2 O (25.0% Cu), 0.9560; calcium iodate (3.25% I), 1.54; Na2 SeO 3 (44.7% Se), 0.0168. All ingredients were diluted with corn starch to 1 kg. 3

Erucic acid premix was added to obtain graded levels of erucic acid, and the amount of lauric acid was

added to guarantee the equal lipid level in each experimental treatment according to Yang et al. (1994). 4

Crude protein, crude lipid, crude fibre and ash contents were measured values.

5

n-3 and n-6 were referred to Zeng et al. (2016), and calculated according to NRC (2011).

6

Available phosphorus was referred to Wen et al. (2015), and calculated according to NRC (2011).

Journal Pre-proof Table 2 Immune-related parameters in the proximal intestine, middle intestine and distal intestine of grass carp fed diets containing different EA levels1 . Dietary EA levels (% diet) 0.00(control) 0.29

P-values 0.60

0.88

1.21

1.50

SEM

Linear

Quadratic

LZ

140.37

139.81

125.83

112.73

102.73

76.28

10.43

< 0.001

< 0.05

ACP

348.80

343.01

304.97

255.67

206.77

159.10

19.57

< 0.001

< 0.05

C3

27.33

27.08

23.54

22.77

16.89

13.04

1.77

< 0.001

< 0.001

C4

14.74

14.32

12.52

11.68

8.65

8.13

0.93

< 0.001

0.15

IgM

57.51

56.11

51.00

48.40

41.09

35.99

3.70

< 0.001

0.07

LZ

171.26

170.17

159.88

135.57

126.21

92.59

7.97

< 0.001

< 0.001

ACP

399.88

395.25

374.88

362.18

327.60

271.93

< 0.001

< 0.001

C3

30.36

30.00

27.51

25.79

24.73

18.15

1.10

< 0.001

< 0.001

C4

16.08

15.95

14.68

11.94

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13.39

9.34

6.87

0.74

< 0.001

< 0.001

IgM

71.71

71.47

66.72

65.34

53.19

37.55

1.89

< 0.001

< 0.001

LZ

193.38

181.68

167.61

156.76

102.64

86.83

10.13

< 0.001

< 0.001

ACP

430.26

428.92

401.62

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PI

386.31

317.10

274.36

17.92

< 0.001

< 0.001

C3

34.17

32.96

28.44

27.08

19.75

14.12

1.56

< 0.001

< 0.001

C4

27.64

27.24

22.33

21.38

13.84

8.82

1.49

< 0.001

< 0.001

IgM

83.57

82.04

71.19

66.88

52.50

43.43

5.53

< 0.001

< 0.05

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LZ, lysozyme (U mg-1 protein); ACP, acid phosphatase (U mg-1 protein); C3, complement 3 (mg g-1

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DI

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MI

protein); C4, complement 4 (mg g-1 protein); IgM, immunoglobulin M (mg g-1 protein). P- values indicate a

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significant linear or quadratic dose response relationship (P < 0.05).

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Fig. 1.

Fig. 1. Compared to control, EA induced enteritis symptom after challenged with A. hydrophila in on-growing grass carp

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(Ctenopharyngodon idella). (A) control, (B) 0.29% diet, (C) 0.60% diet, (D) 0.88% diet, (E) 1.21% diet and (F) 1.50% diet.

Journal Pre-proof Fig. 2.

*P < 0.01

50%

40% 35% 30% 25%

20% 15% 10%

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The enteritis morbidity in intestine

45%

0%

0.00

0.29

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5% 0.60

0.88

1.21

1.50

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Dietary EA levels (% diet)

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Fig. 2. Effects of dietary EA (% diet) on enteritis morbidity of on-growing grass carp (Ctenopharyngodon idella) after

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infection of Aeromonas hydrophila. P-value indicates a significant linear dose response relationship (P < 0.01).

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Fig. 3. PI:

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MI:

DI:

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Fig. 3. Histological analysis of intestines of on-growing grass carp (Ctenopharyngodon idella) fed diets supplemented with

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different levels of EA. (A) control, (B) 0.29% diet, (C) 0.60% diet, (D) 0.88% diet, (E) 1.21% diet and (F) 1.50% diet.

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Abbreviations for PI, MI and DI: GC, goblet cell; BH: blood capillary hyperaemia.

Journal Pre-proof Fig. 4. 0.88

1.21 1.50

0.00

0.29 0.60

MI 0.88

1.21 1.50

0.00

0.29 0.60

DI 0.88

1.21 1.50

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0.29 0.60

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0.00

0

1.5

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-1.5

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Immune-related signaling molecules

Cytikines

Antimicrobial peptides

PI EA (% diet) LEAP-2A LEAP-2B hepcidin β-defensin-1 Mucin2 IL-10 IL-11 IL-4/13A IL-4/13B TGF-β1 TGF-β2 IL-1β TNF-α IFN-γ2 IL-6 IL-8 IL-12p35 IL-12p40 IL-15 IL-17D NF-κBp65 NF-κBp52 c-Rel IkBα IKKα IKKβ IKKγ TOR S6K1 4E-BP1 4E-BP2

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Fig. 4. Heat-map of the mRNA levels of antimicrobial peptides, cytokines, NF-κB signalling pathway-related molecules and TOR signalling pathway-related molecules in the PI, MI and DI of on-growing grass carp (Ctenopharyngodon idella) fed

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graded levels of EA. The values of upregulation (red) and downregulation (green) (i.e., from 1.5 to −1.5) represent log2 fold changes. N=6 for each EA level. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5.

Fig. 5. Effects of dietary EA levels (% diet) on protein levels of nuclear NF-κB p65 in the PI (A), MI (B) and DI (C) of on-growing grass carp (Ctenopharyngodon idella). Data represent means of 6 fish in each group, error bars indicate S.E. P-value indicates a significant linear dose response relationship (P < 0.01).

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Fig. 6.

Fig. 6. Effects of dietary EA levels (% diet) on protein levels of total target of rapamycin (T-TOR) protein phosphorylation at Ser

2448

(p-TOR Ser

2448

) in the PI (A), MI (B) and DI (C) of on-growing grass carp (Ctenopharyngodon idella). Data

represent means of 6 fish in each group, error bars indicate S.E. P-value indicates a significant linear dose response relationship (P < 0.01).

Journal Pre-proof Fig. 7. B LZ in PI (U mg-1 prot)

The enteritis morbidity in the intestine (%)

A

Parameters L=10.97 U=-33.9 R=0.53; z1=(x>R)*(R-x); Model y=L+U*(z1)

Parameters L=140.1 U=52.2 R=0.36; z1=(x>R)*(R-x); Model y=L+U*(z1)

Dietary erucic acid levels (% diet)

Dietary erucic acid levels (% diet)

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Dietary erucic acid levels (% diet)

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Parameters L=170.7 U=69.3 R=0.44; z1=(x>R)*(R-x); Model y=L+U*(z1)

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D LZ in DI (U mg-1 prot)

LZ in MI (U mg-1 prot)

C

Parameters L=187.5 U=98.6 R=0.45; z1=(x>R)*(R-x); Model y=L+U*(z1)

Dietary erucic acid levels (% diet)

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Fig. 7. Broken-line regression analysis (SAS) of enteritis morbidity (A), and LZ in the PI (B), MI (C) and DI (D) of on-growing grass carp (Ctenopharyngodon idella) from each treatment. Parameters are defined as L=plateau, U=slope and

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R=breaking point.

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Fig. 8.

Fig. 8. The potential signaling pathways of EA damaging the immune function of fish intestine.

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, might through

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, not through;

Journal Pre-proof Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as

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potential competing interests:

Journal Pre-proof Highlights 1. Inclusions of erucic acid (EA) at levels < 0.36% EA in diets are safe for on-growing grass carp. 2. EA (≥ 0.36% diet) induced the occurrence of enteritis and impaired the intestinal immune function in on-growing grass carp. 3. EA (≥ 0.36% diet) impaired the intestinal immune function of on- growing grass carp partly related

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to nuclear factor (NF)-κB (not NF-κB p52) and target of rapamycin (TOR) signalling pathways.