Selenium deficiency impaired structural integrity of the head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella)

Accepted Manuscript Selenium deficiency impaired structural integrity of the head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) Lin Zheng, Wei-Dan Jiang, Lin Feng, Pei Wu, Ling Tang, Sheng-Yao Kuang, Yun-Yun Zeng, Xiao-Qiu Zhou, Yang Liu PII:

S1050-4648(18)30511-4

DOI:

10.1016/j.fsi.2018.08.038

Reference:

YFSIM 5496

To appear in:

Fish and Shellfish Immunology

Received Date: 3 July 2018 Revised Date:

6 August 2018

Accepted Date: 17 August 2018

Please cite this article as: Zheng L, Jiang W-D, Feng L, Wu P, Tang L, Kuang S-Y, Zeng Y-Y, Zhou X-Q, Liu Y, Selenium deficiency impaired structural integrity of the head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella), Fish and Shellfish Immunology (2018), doi: 10.1016/ j.fsi.2018.08.038. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Selenium deficiency impaired structural integrity of the head kidney, spleen and skin in

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young grass carp (Ctenopharyngodon idella) ACCEPTED MANUSCRIPT

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Lin Zheng a,1, Wei-Dan Jiang a,b,c, Lin Feng a,b,c, Pei Wu a,b,c, Ling Tang d, Sheng-Yao Kuang d, Yun-Yun Zeng

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a,b,c

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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

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c

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Agricultural University, Chengdu 611130, China

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, Xiao-Qiu Zhou a,b,c*, Yang Liu a,b,c*

Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan

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d

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

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Sichuan, China. Tel.: +86 835 2885157; fax: +86 835 2885968.

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E-mail addresses: [email protected], [email protected] (X.-Q. Zhou); [email protected] (Y. Liu).

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

These two authors contributed to this work equally.

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Abstract This study focused on the effects of dietary MANUSCRIPT selenium deficiency on structural integrity of the head ACCEPTED

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kidney, spleen and skin in young grass carp (Ctenopharyngodon idella). A total of 540 healthy grass carp

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(mean weight 226.48 ± 0.68 g) were randomly divided into six groups and fed six separate diets with graded

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dietary levels of selenium (0.025-1.049 mg/kg diet) for 80 days. Results showed that selenium deficiency (1)

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caused oxidative damage in part by reducing the activities of antioxidant enzymes (such as SOD, CAT, GPx,

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GST and GR) and glutathione (GSH) content, down-regulating the transcript abundances of antioxidant

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enzymes (except GSTp1) partly related to Kelch-like-ECH-associated protein 1a (Keap1a) / NF-E2-related

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factor 2 (Nrf2) signalling; (2) aggravated apoptosis in part by up-regulating the mRNA levels of caspase-2,

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-3, -7, -8 and -9, which were partially related to p38MAPK/FasL/caspase-8 signalling and JNK/(BAX, Bcl-2,

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Mcl-1b, IAP)/(Apaf1, caspase-9) signalling; (3) damaged the tight junctions in part by down-regulating the

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mRNA levels of ZO-1 (except spleen), ZO-2 (except spleen), claudin-c, -f, -7, -11 and claudin-15, and

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up-regulating the mRNA levels of claudin-12, which were partially related to myosin light chain kinase

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(MLCK) signalling. Interesting, selenium deficiency failed to affect the expression of GSTp1, Keap1a,

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occludin, claudin-b, claudin-3c, ZO-1 (spleen only) and ZO-2 (spleen only) in the head kidney, spleen and

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skin of grass carp. Finally, based on the activities of glutathione peroxidase (GPx) and reactive oxygen

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species (ROS) content in the head kidney, spleen and skin, the dietary selenium requirements for young

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grass carp were estimated to be 0.558-0.588 mg/kg diet.

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Keywords: Selenium; Antioxidation; Apoptosis; Tight junctions; Grass carp (Ctenopharyngodon idella)

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1. Introduction In recent years, high density aquaculture has MANUSCRIPT caused fish to be more susceptible to stress and disease ACCEPTED

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emergence, which may disturb fish health [1]. Evidences have shown that fish health is closely related to the

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immune function and structural integrity of immune organs [2, 3]. In fish, the head kidney and spleen are the

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major immune organs [4]. Skin, as an important mucosal immune organ, is the first line of defence for fish

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[5]. Previous studies from our laboratory have shown that mineral deficiencies (such as iron and phosphorus)

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impaired the immune function and structural integrity of these immune organs in young grass carp

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(Ctenopharyngodon idella) [3, 6]. Selenium is an essential mineral element for the growth of fish [7-9],

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which can regulate the amino acid metabolism [10], lipid metabolism [11] and glycometabolism [12]. Our

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previous study showed that selenium deficiency reduced the growth performance and impaired the immune

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function of the immune organs (such as head kidney, spleen and skin) in young grass carp [13]. However, so

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far, the relationship between selenium deficiency and the structural integrity of fish immune organs is

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unclear. Bell et al. reported that selenium deficiency reduced liver vitamin E level in rainbow trout

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(Oncorhynchus mykiss) [14]. Previous study from our laboratory observed that vitamin E deficiency resulted

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in impairment of structural integrity in the immune organs of grass carp [15]. Consequently, there is a

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potential connection between selenium deficiency and structural integrity of the immune organs in fish,

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which was worth for investigating.

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It is well known that the cellular structure integrity plays an important role in maintaining the structural

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integrity of fish immune organs, which is related to antioxidants (e.g. MnSOD) and cell apoptosis (e.g.

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caspase-8) [2]. In human, studies indicated that the antioxidants could be regulated by NF-E2-related factor

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2 (Nrf2) [16], and the cell apoptosis could be regulated by p38 mitogen-activated protein kinase (p38MAPK)

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[17] and c-jun N-terminal kinase (JNK) [18]. However, so far, no studies have investigated the impacts of

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selenium deficiency on antioxidant and apoptosis as well as the possible signalling pathways in fish immune

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organs. In human, studies demonstrated that selenium supplement elevated the expression of estrogen

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receptor β (ER β) in MDA-MB231 cells [19], and increasing ER β level could up-regulate the mRNA level

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of MnSOD in MRC5 cells [20]. Additionally, studies on human also discovered that selenium deficiency

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up-regulated the mRNA level of Vascular Endothelial Growth Factor (VEGF) in PC3 cells [21], and

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upregulation of VEGF expression could activate the Nrf2 signalling pathway in BeWo cells [22]. Besides,

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Yu et al. reported that selenium deficiency promoted the serum TNF-α level in mice [23]. TNF-α could

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induce the activation of caspase-8 in adult ventricular myocytes [24], and activate the p38MAPK signalling

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in murine C2C12 cells [25] and JNK signalling in human liver [26]. These evidences revealed that there

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might be an association between selenium deficiency and cellular structure integrity referring to antioxidant

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and cell apoptosis, as well as their related signalling pathways in fish immune organs, which is a valuable

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topic for investigation.

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As a report, the tight junctions (e.g. ZO-1, occludin) is an important intercellular junctional structure

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[27], which could be regulated by myosin light chain kinase (MLCK) [28]. Unfortunately, so far, no studies

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have investigated the impacts of selenium deficiency on tight junctions and the possible signalling pathways

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of fish immune organs. Studies on mice showed that selenium deficiency elevated the activity of pancreas

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inducible nitric oxide synthase (iNOS) [29] which could down-regulate the expression of ZO-1 [30]. In

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addition, Zhou et al. reported that selenium deficient elevated the serum interleukin-1β (IL-1β) level in rats

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[31]. In human Caco-2 cells, IL-1β caused downregulation of occludin expression [32] and upregulation of

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MLCK expression [33]. Hence, all the data above suggested a possible relationship between selenium

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deficiency and TJs as well as the possible regulation mechanisms in the immune organs of fish, which is a

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subject worthy of investigation.

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In this study, we used the same growth trial as our previous study [13], which is a part of a larger study

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conducted to investigate the effects of selenium on fish growth and health status. As we know, fish growth is

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closely related to the immune function of immune organs which rely on its structural integrity [3, 6]. Our

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previous study showed that selenium deficiency reduced the growth performance and impaired the immune

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function of immune organs in young grass carp [13]. Thus, in this study, we are for first time to explore the

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effects of selenium deficiency on antioxidant, apoptosis and tight junctions, as well as the possible regulation

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mechanisms in the head kidney, spleen and skin of grass carp. These results provide a reference for

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formulating commercial feed of grass carp, and provide partial theoretical evidence for the research of

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defense mechanism in fish immune organs.

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2. Materials and methods

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2.1. Experimental diets preparation

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The present study used the same growth trial as our previous study [13]. The proximate compositions of

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basal diet were analyzed and shown in Table 1 according to AOAC (2005) [34]. In this study, organic 4

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selenium (selenium yeast) was added to the basal diet to provide graded concentrations of 0 (no supplement),

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0.2, 0.4, 0.6, 0.8 and 1.0 mg Se/kg diet. The measured selenium concentrations of six diets by inductively

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coupled plasma mass spectrometry (ICP-MS) [35] were 0.025 (basal diet), 0.216, 0.387, 0.579, 0.795 and

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1.049 mg/kg diet, respectively. Whereafter, these diets were sufficient mixed and were made into pellets as

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we’ve described earlier [13]. Lastly, the prepared diets were sealed in a plastic bag and then stored at −20 °C

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as recommended by Ashouri et al. [36] and the diets were thawed in a refrigerator at 4 °C for 24 h before

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feeding as per Wang et al. [37].

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(Table 1 inserted here)

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2.2. Experimental process and sample collection

All experiments were designed according to the University of Sichuan Agricultural Animal Care

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Advisory Committee. Grass carp were obtained from a local fishery (Sichuan, China). Prior to feeding trial,

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fish were acclimated for 2 weeks and fed with the basal diet [36]. Subsequently, 540 healthy fish (mean

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weight 226.48 ± 0.68 g) were randomly assigned to 18 experimental cages (1.4 L × 1.4 W × 1.4 H m),

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resulting in 30 fish per cage. Furthermore, each cage was equipped with a disc (100 cm diameter) in the

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bottom to collect the uneaten feed as described by Zheng et al. [13]. Next, the fish from the six groups were

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fed with their respective diets four times per day for 80 days. Thirty minutes after feeding, the uneaten feed

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was collected, dried and weighed to calculate the feed intake (FI) as we’ve described earlier [13]. During the

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experiment, the rearing water was partially (halfway) changed once every 4 days and the water quality was

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determined daily according to the procedures of Zheng et al. [13]. The dissolved oxygen content was greater

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than 6.0 mg/L. The pH value and water temperature were measured to be 7.5 ± 0.3 and 28.5 ± 2.0 °C,

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respectively. The selenium concentration in rearing water was determined to be 1.204 ± 0.040 µg/L as

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described by Pacitti et al. [35]. Moreover, the experiment was conducted under a natural light and dark cycle,

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which was similar to a previous from our lab [38]. After the growth trial, using the prevalent pathogens to

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impair the structural integrity of fish immune organs is a common approach to evaluate the nutritional

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protection on the structural integrity of fish immune organs [3, 6]. To our knowledge, A. hydrophila is a

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popular pathogen which could impair the structural integrity of fish immune organ [39]. After an 80 days

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growth trial, fifteen similar body weight fish from each treatment group were intraperitoneally injected with

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A. hydrophila for 14 days as described by Pan et al [2].

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At the end of stress test, fish were anaesthetized in a benzocaine bath as described by Tang et al. [40].

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Next, the head kidney, spleen and skin samples were quickly removed, small portions frozen in liquid

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nitrogen and then stored at −80 °C until analysis as described by Guo et al.[3].

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2.4. Biochemical analysis

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Tissue homogenates of head kidney, spleen, and skin were prepared with 10 multiple (w/v) ice-cold

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saline and centrifuged at 6000 g at 4ºC for 20 min, and then the supernatant were stored until used for the

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analysis of related parameters as described by Pan et al. [2]. The reactive oxygen species (ROS) content was

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assayed by the fluorescent probe DCFH-DA method as described by Gozali et al. [41]. The malondialdehyde

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(MDA) content was determined through measuring the pink color produced by the reaction of thiobarbituric

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acid (TBA) with MDA at 90–100 °C as described by Esterbauer et al.[42]. The protein carbonyl (PC)

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content was evaluated by 2, 4-dinitrophenylhydrazine (DNPH) method as described by Lund et al.[43]. The

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anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacities were determined by the method

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described by Jiang et al.[44]. The activities of total superoxide dismutase (SOD) and copper/zinc superoxide

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dismutase (CuZnSOD) were analyzed based on the enzymes' ability to inhibit the oxidation of

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hydroxylamine catalyzed by the xanthine–xanthine oxidase system according to Reyes-Becerril et al. [45]

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and Feng et al.[46], respectively. The activity of manganese superoxide dismutase (MnSOD) was calculated

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by deducting CuZnSOD from total SOD. The activities of CAT and glutathione peroxidase (GPx) were

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analyzed according to the method described by Chen et al. [47]. The activities of glutathione reductase (GR)

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and glutathione S-transferase (GST) were analyzed according to the method described by Wang et al. [48].

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The GSH content was measured according to the method described by Rhee et al. [49].

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2.5. Analysis of DNA fragmentation

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Fragmented DNA of the head kidney and spleen tissue was isolated as described by Wang et al.[48].

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The DNA fragmentation was analyzed by electrophoresis for 1.5 h at 80 V using 2% agarose gel and the

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same amount of DNA for each sample. Lastly, the gel was examined and photographed using a Gene Genius

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Bio-Imaging system (Syngene, Frederick, MD, USA).

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2.6. Real-time polymerase chain reaction (PCR) analysis

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Total RNA samples were isolated from the head kidney, spleen and skin using RNAiso Plus Kit

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(Takara, Dalian, China). The quality and quantity of RNA were determined by electrophoresis on 1% 6

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agarose gels [50] and determined by spectrophotometric at 260 and 280 nm [51, 52], respectively. Then,

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single-stranded cDNA was prepared from total RNA by reverse transcription using a PrimeScript™ RT

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reagent Kit (TaKaRa, Dalian, China) according to the manufacturer’s protocols. PCR specific primers were

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designed according to the sequences that cloned in our laboratory and the published in the gene bank of

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grass carp (Table S1) for quantitative real-time PCR. The target and housekeeping gene amplification

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efficiency were calculated according to the specific gene standard curves generated from 10-fold serial

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dilutions and the primers amplified with an efficiency of approximately 100%. To confirm the specificity

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and purity of all PCR products, melt curve analysis was carried out after amplification. According to the

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results of our preliminary experiment concerning the evaluation of internal control genes (data not shown),

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β-actin and GADPH were used as reference gene to normalize cDNA loading as described by Vandesompele

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et al. [53]. Results of gene expression were analyzed using the 2−∆∆CT method according to Schmittgen et al.

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[54].

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(Table S1 inserted here) 2.7. Western blot

Protein homogenates were prepared from the head kidney, spleen and skin samples; antibodies were

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used and western blotting was performed as in our previous study [55]. Briefly, the protein concentrations

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were determined by a BCA assay kit (Beyotime Biotechnology Inc., China). Subsequently, the sample

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protein was separated by SDS-PAGE and then transferred to a PVDF membrane for the western blot

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analysis. After transfer, the membrane was blocked for 1.5 h with 0.5% BSA at room temperature and then

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incubated with primary antibody overnight at 4 °C. The antibody of Nrf2 (ab31163, 1:1000 dilution) was the

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same as in our previous study [56] and was purchased from Abcam (Cambridge, UK). The antibodies of

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lamin B1 (AF5161, 1:1000 dilution) and β-actin (AF7018, 1:3000 dilution) were the same as those in our

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previous study [13] and were purchased from Affinity BioReagents (Golden, Colorado, USA). In this study,

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β-actin and lamin B1 were used as control proteins for total protein and nuclear protein. The blots were

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washed three times and followed by a 1.5 h incubation with goat anti-rabbit horseradish

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peroxidase-conjugated secondary antibody (A0208, 1:8000 dilution, Beyotime Biotechnology, Shanghai,

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China) in TBST. Lastly, the immune complexes were visualized using ECL reagents (Affinity Biosciences

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Inc., America). The western bands were quantified using NIH Image 1.63 software (National Institutes of

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Mental Health, Bethesda, USA). Different treatments were expressed relative to the level of control group.

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This experiment was repeated at least three times, and similar results were obtained each time.

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2.8. Statistical analysis

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The results are represented with the means ± SD. Data were subjected to one-way ANOVA followed by

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the Duncan's multiple-range test to determine significant differences among six treatment groups using SPSS

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18.0 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to be statistically significant. Quadratic

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regression model was used to estimate the optimal level of dietary selenium for young grass carp according

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to Wang et al. [57].

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3. Results

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3.1 Antioxidant-related parameters in the head kidney, spleen and skin of young grass carp

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3.1.1 Oxidative statuses and antioxidant responses in the head kidney, spleen and skin of young grass carp

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As shown in Table 2. In the head kidney, the content of MDA, PC and ROS significantly decreased

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with increasing selenium levels up to 0.579 mg/kg diet (P < 0.05) and then increased substantially (P < 0.05).

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The activities of AHR and CuZnSOD gradually rose as selenium levels added up to 0.579 mg/kg diet, then

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gradually descended. The activities of CAT, GPx and GST gradually rose as selenium levels added up to

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0.387 mg/kg diet, then gradually descended. The activities of MnSOD and GR significantly increased as

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selenium levels rose to 0.579 mg/kg diet (P < 0.05) and then significantly decreased (P < 0.05). The content

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of GSH significantly rose with increasing selenium levels up to 0.387 mg/kg diet (P < 0.05) and then

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gradually decreased. The activity of ASA gradually increased as selenium levels rose to 0.579 mg/kg diet

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and then significantly decreased (P < 0.05). In the spleen, the content of MDA and PC gradually decreased

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as selenium levels rose to 0.579 mg/kg diet and then increased significantly (P < 0.05). The content of ROS

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significantly decreased as selenium levels added up to 0.579 mg/kg diet (P < 0.05) and then obvious elevated

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(P < 0.05). The activities of CAT and GPx were gradually rose with increased selenium levels up to 0.387,

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mg/kg diet, and were then decreased gradually. The GSH content was gradually rose with increased

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selenium level up to 0.579 mg/kg diet, and was then decreased gradually. The activity of CuZnSOD

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significantly increased as selenium levels rose to 0.579 mg/kg diet (P < 0.05) and then significantly

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decreased (P < 0.05). The activities of AHR, GST and GR significantly rose with increasing selenium levels

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up to 0.387 mg/kg diet (P < 0.05) and then gradually decreased. The activities of ASA and MnSOD

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gradually increased as selenium levels rose to 0.579 mg/kg diet and then significantly decreased (P < 0.05).

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In the skin, the content of ROS significantly decreased with increasing selenium levels up to 0.579 mg/kg

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diet (P < 0.05) and then rose substantially (P < 0.05). The content of MDA and PC significantly decreased

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with increasing selenium levels up to 0.387 mg/kg diet (P < 0.05) and then gradually increased. The

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activities of CuZnSOD, MnSOD, GPx and GST gradually increased as selenium levels rose to 0.579 mg/kg

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diet, then decreased gradually. The activity of CAT gradually increased as selenium level rose to 0.387

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mg/kg diet, then decreased gradually. The activity of ASA and GSH content significantly increased as

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selenium levels rose to 0.579 mg/kg diet (P < 0.05) and then significantly decreased (P < 0.05). The

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activities of AHR and GR significantly increased with increasing selenium levels up to 0.387 mg/kg diet (P

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< 0.05) and then gradually decreased.

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(Table 2 inserted here)

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3.1.2 Relative mRNA levels of antioxidant enzymes and related signalling molecules in the head kidney,

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spleen and skin of young grass carp

As shown in Fig. 1. In the head kidney, the mRNA levels of CuZnSOD, MnSOD and GR gradually

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rose with increasing selenium levels up to 0.387 mg/kg diet and then decreased slowly. The mRNA levels of

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CAT, GPx1a, GPx1b, GPx4a, GSTp2, GSTo2 and Nrf2 gradually increased as selenium levels rose to 0.579

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mg/kg diet and then decreased slowly. The mRNA level of GPx4b significantly up-regulated with increasing

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selenium levels up to 0.387 mg/kg diet (P < 0.05) and then down-regulated gradually. The mRNA levels of

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GSTR and GSTo1 significantly increased as selenium levels added up to 0.216 mg/kg diet (P < 0.05) and

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then plateaued. The mRNA level of Keap1a gradually depressed as selenium levels rose to 0.579 mg/kg diet

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and then slowly increased. In the spleen, the mRNA levels of CuZnSOD, MnSOD, CAT, GPx1a, GSTR,

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GSTp2, GSTo2, GR and Nrf2 all gradually rose with increasing selenium levels up to 0.579 mg/kg diet and

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then decreased gradually. The mRNA level of GPx1b gradually increased as selenium levels rose to 0.387

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mg/kg diet and then decreased gradually. The mRNA levels of GPx4a and GSTo1 significantly increased as

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selenium levels rose to 0.387 mg/kg diet (P < 0.05), then depressed gradually. The mRNA level of GPx4b

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significantly increased as selenium level rose to 0.579 mg/kg diet (P < 0.05), then depressed gradually. The

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mRNA level of Keap1a significantly depressed as selenium levels rose to 0.387 mg/kg diet (P < 0.05) and

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then gradually increased. In the skin, the mRNA levels of CuZnSOD, GPx1b, GSTo2 and Nrf2 gradually

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up-regulated with increasing selenium levels up to 0.579 mg/kg diet, then down-regulated gradually. The

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mRNA levels of GPx1a and GPx4b gradually up-regulated with increasing selenium levels up to 0.387

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mg/kg diet, then down-regulated gradually. The mRNA levels of MnSOD, CAT, GPx4a, GSTR, GSTp2,

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GSTo1 and GR significantly increased as selenium levels rose to 0.387 mg/kg diet (P < 0.05) and then

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decreased gradually. The mRNA level of Keap1a gradually depressed with increasing selenium levels up to

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0.579 mg/kg diet and then gradually increased.

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(Fig. 1 inserted here)

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3.1.3 Protein levels of Nrf2 in the head kidney, spleen and skin of young grass carp

As shown in Fig. 2. In the head kidney, the protein level of nuclear Nrf2 significantly increased as

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selenium levels rose to 0.387 mg/kg diet (P < 0.05) and then gradually decreased. The protein level of total

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Nrf2 gradually increased as selenium levels rose to 0.579 mg/kg diet and then gradually decreased. In the

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spleen, the protein level of nuclear Nrf2 significantly increased with increasing selenium levels up to 0.387

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mg/kg diet (P < 0.05) and then gradually depressed. The protein level of total Nrf2 gradually increased as

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selenium levels rose to 0.579 mg/kg diet and then gradually decreased. In the skin, the protein level of

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nuclear Nrf2 gradually increased with increasing selenium levels up to 0.579 mg/kg diet and then gradually

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decreased. The protein level of total Nrf2 significantly increased as selenium levels rose to 0.387 mg/kg diet

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(P < 0.05) and then gradually depressed.

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3.2 Apoptosis-related parameters in the head kidney, spleen and skin of young grass carp As shown in Fig. 3. In the head kidney, the obvious ladder-like pattern of DNA were observed at 0.025,

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0.795 and 1.049 mg/kg diet. Similar results were obtained in the spleen. As shown in Fig. 4. In the head

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kidney, the mRNA levels of cysteinyl aspartic acid-protease (caspase)-2, -3, -7, apoptotic protease

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activating factor-1 (Apaf-1), B-cell lymphoma protein 2 associated X protein (Bax) and c-Jun N-terminal

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kinase (JNK) all gradually decreased as selenium levels rose to 0.579 mg/kg diet and then increased slowly.

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The mRNA level of Fas ligand (FasL) significantly down-regulated with increasing selenium levels up to

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0.387 mg/kg diet (P < 0.05) and then up-regulated gradually. The mRNA levels of caspase-8 and -9

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significantly decreased as selenium levels rose to 0.216 mg/kg diet (P < 0.05) and then plateaued. The

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mRNA level of p38 mitogen-activated protein kinase (p38MAPK) gradually decreased as selenium levels

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increased up to 0.579 mg/kg diet and then increased substantially (P < 0.05). The mRNA levels of B-cell

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lymphoma-2 (Bcl-2) and IAP increased gradually with increasing selenium levels up to 0.579 mg/kg diet and

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then gradually depressed. The mRNA level of myeloid cell leukemia-1b (Mcl-1b) speedy increased as

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selenium levels rose to 0.387 mg/kg diet (P < 0.05) and then gradually decreased. In the spleen, the mRNA

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levels of caspase-2, -7, -8, -9, FasL, Apaf-1and Bax speedy reduced as selenium levels rose to 0.387 mg/kg

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diet (P < 0.05) and then increased gradually. The mRNA levels of caspase-3 and JNK gradually

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down-regulated with increasing selenium levels up to 0.579 mg/kg diet and then gradually up-regulated. The

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mRNA level of p38MAPK gradually decreased as selenium levels rose to 0.579 mg/kg diet and then

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increased substantially (P < 0.05). The mRNA levels of Bcl-2, IAP and Mcl-1b all gradually up-regulated as

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selenium levels rose to 0.579 mg/kg and then gradually down-regulated. In the skin, the mRNA levels of

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caspase-2, -3, -7, -8, -9, FasL, Apaf-1, BAX and JNK gradually down-regulated with increasing selenium

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levels up to 0.579 mg/kg and then up-regulated gradually. The mRNA level of p38MAPK significantly

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depressed as selenium levels rose to 0.579 mg/kg diet (P < 0.05) and then obvious increased (P < 0.05). The

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mRNA levels of Bcl-2 and IAP gradually up-regulated as selenium levels added up to 0.579 mg/kg diet and

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then down-regulated gradually. The mRNA level of Mcl-1b significantly rose with increasing selenium

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levels up to 0.387 mg/kg diet (P < 0.05) and then decreased gradually.

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(Fig. 3, 4 inserted here)

3.4 TJs related parameters in the head kidney, spleen and skin of young grass carp

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As shown in Fig. 5. In the head kidney, the mRNA levels of zonula occluden 2b (ZO-2b), claudin-c,

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-7b and -15a gradually increased as selenium levels rose to 0.579 mg/kg diet, then decreased slowly. The

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mRNA levels of claudin-7a and -11 increased as selenium levels rose to 0.387 mg/kg diet, then decreased

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slowly. The mRNA levels of ZO-1 and claudin15b all significantly rose with increasing selenium levels up

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to 0.216 mg/kg diet and then plateaued. The mRNA level of claudin-f significantly up-regulated as selenium

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levels added up to 0.387 mg/kg diet (P < 0.05) and then down-regulated gradually. The mRNA levels of

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claudin-12 and myosin light chain kinase (MLCK) significantly decreased as selenium level rose to 0.579

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mg/kg (P < 0.05) and then increased gradually. In the spleen, the mRNA levels of claudin-c, -f, -7a, -7b, -11,

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-15a and -15b all gradually up-regulated as selenium levels rose to 0.579 mg/kg diet and then

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down-regulated gradually. The mRNA levels of claudin-12 and MLCK gradually decreased with increasing

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selenium levels up to 0.579 mg/kg and then increased gradually. In the skin, the mRNA levels of ZO-1,

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claudin-f and -15b all gradually rose with increasing selenium levels up to 0.579 mg/kg diet and then

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gradually decreased. The mRNA levels of ZO-2b, claudin-c, -7b, -11 and -15a all significantly increased as

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selenium levels rose to 0.387 mg/kg diet (P < 0.05) and gradually decreased. The mRNA level of claudin-7a

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significantly rose with increasing selenium levels up to 0.216 mg/kg diet (P < 0.05) and then plateaued. The

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mRNA levels of claudin-12 and MLCK gradually decreased as selenium levels rose to 0.579 mg/kg and then

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increased slowly.

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(Fig. 5 inserted here) 4. Discussion

In this study, we used the same animal trial as our previous study [13]. Reportedly, fish growth is

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closely associated with the immune function and structural integrity of its immune organs [3, 6]. Our

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previous study has shown that selenium deficiency reduced the growth performance and impaired the

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immune function of the immune organs in young grass carp [13]. However, so far, the relationship between

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selenium deficiency and structural integrity of the immune organs is unclear. Thus, in the present study, for

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the first time, we next investigated the effects of selenium deficiency on the structural integrity and the

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related signalling pathways in fish immune organs.

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4.1 Selenium deficiency induced oxidative damage partially relating to Nrf2 signalling in the head kidney,

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spleen and skin of fish

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Studies have shown that ROS is a marker of oxidative stress and can damage to lipids and proteins [64].

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It is well known that MDA and PC are biochemical markers of lipid and protein peroxidation, respectively

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[65]. In this study, our data showed that selenium deficiency aggrandized the content of ROS, MDA and PC

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resulting in oxidative damage in the head kidney, spleen and skin of grass carp. In fish, the antioxidant

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system protects fish from oxidative damage, which mainly consists of antioxidant enzymes and

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non-enzymatic antioxidants [3, 66, 67]. In this study, we discovered that selenium deficiency reduced the

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activities of antioxidant enzymes (such as SOD, CAT, GPx, GST and GR) and GSH content, indicated that

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selenium deficiency impaired the antioxidant ability in the head kidney, spleen and skin of fish. According

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to a report, the activities of antioxidant enzymes are related to their corresponding mRNA levels in rats [68].

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Our data showed that selenium deficiency reduced the mRNA levels of antioxidants (except GSTp1) in the

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head kidney, spleen and skin of grass carp. Further correlation analysis showed that the activities of

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CuZnSOD, MnSOD, CAT, GPx, GST and GR were positively correlated with their mRNA levels (except

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GSTp1) (Table 4). Interestingly, our results indicated that selenium deficiency only down-regulated GSTp2

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(rather than GSTp1) mRNA levels in the head kidney, spleen and skin of grass carp, which might be partly

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related to Keap1a. Taguchi et al. reported that Keap1 inhibited the gene expression of GSTp2 (rather than

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GSTp1) in mice [69]. In our study, selenium deficiency up-regulated Keap1a gene expression, which

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validated our assumptions.

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In fish, the gene expression of antioxidant enzymes is related to Keap1/Nrf2 signalling pathway [55,

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65]. Interestingly, we discovered that selenium deficiency only up-regulated the mRNA levels of Keap1a

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(rather than Keap1b) in the head kidney, spleen and skin, which might be partly related to phospholipid.

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Study reported that selenium deficiency reduced the phospholipid level in shrimp intestine [70]. Previous

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study from our laboratory showed that lower phospholipid level up-regulated keap1a (rather than keap1b)

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mRNA levels in the intestines of grass carp [71]. Thus, we hypothesize that selenium deficiency resulted in

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decreased phospholipid level to up-regulated Keap1a (rather than Keap1b) gene expression, which requires

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further investigation. Furthermore, it has been reported that nuclear Nrf2 protein level has been used to

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monitor the activation of Nrf2 signalling [72]. In this study, selenium deficiency reduced the protein levels

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of nuclear Nrf2 in the head kidney, spleen and skin, indicated that selenium deficiency inhibited the Nrf2

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signalling. Additionally, correlation analysis showed that the mRNA levels of antioxidant enzymes (such as

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SOD, CAT, GPx, GST and GR) were positively related to the protein levels of nuclear Nrf2 and were

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negatively related to the mRNA levels of Keap1a (Table S2). The above data indicated that selenium

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deficiency down-regulated the mRNA levels of antioxidant enzyme in the head kidney, spleen and skin of

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fish, which were regulated by Keap1a/Nrf2 signalling. In addition, Itoh et al. reported that Nrf2 turns over

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rapidly in macrophages and that new protein synthesis is required for the nuclear accumulation of Nrf2 [73].

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In our research, the mRNA levels and nuclear protein levels of Nrf2 were all reduced in selenium deficiency

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group, supporting this view. In recent years, some studies have shown that oxidative stress could cause

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cellular apoptosis in human cell [74, 75]. Thus, we next investigated the relationship between selenium

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deficiency and apoptosis as well as the related signalling in the head kidney, spleen and skin of grass carp.

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4.2 Selenium deficiency aggravated apoptosis partially relating to p38MAPK and JNK signalling in the

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head kidney, spleen and skin of fish

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Admittedly, DNA fragmentation is one of the hallmarks of apoptosis [76]. In this study, our data

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showed that selenium deficiency aggravated the DNA fragmentation in the head kidney, spleen and skin of

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grass carp. As we know, caspases are also closely associated with apoptosis [77] and divided into apoptotic

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initiator caspases (including caspase-8 and -9) and apoptotic executioner caspases (including caspase-3 and

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-7) according to their function in apoptosis [78]. Currently, in mammalian cells, two major apoptosis

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pathways have been defined, the death receptor pathway (FasL/caspase-8) and the mitochondria pathway

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[(Bcl-2, Mcl-1b and Bax)/(Apaf-1, caspase-9)] [79]. It was reported that in human, Fas/FasL gene

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expression were regulated by p38MAPK in leukemia U937 Cells [80] and Bax/Bcl-2 gene expression were

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regulated by JNK in leukemia K562 cells [81]. In this study, our data showed that selenium deficiency

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exacerbates apoptosis in head kidney, spleen and skin of grass carp. Further correlation analysis showed that

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the mRNA levels of FasL and caspase-8 were positively related to p38MAPK (Table S2); the mRNA levels

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of caspase-2, -3, -7 and -9 were positively related to Bax and Apaf-1 and were negatively related to Bcl-2,

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Mcl-1b and IAP (Table S2). The above results indicate that selenium deficiency aggravated apoptosis partly

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relating to the activation of death receptor and mitochondria apoptotic pathways in the head kidney, spleen

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and skin of grass carp.

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4.3 Selenium deficiency disturbed TJs partially relating to MLCK signalling in the head kidney, spleen

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and skin of fish

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Tight junctions (TJ) plays a vital role in maintaining intercellular integrity [82], which includes barrier

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forming and pore-forming [6]. Study reported that the TJs could be regulated by MLCK in human Caco-2

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cells [83]. In this study, our data suggested that selenium deficiency impaired the tight junction in the head

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kidney, spleen and skin of fish. Surprisingly, we found that selenium deficiency failed to affect the mRNA

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levels of claudin-b and -3c in the head kidney, spleen and skin of grass carp, which might be all correlated

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with cortisol. It has been reported that selenium deficiency promoted the blood cortisol levels in finishing

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lambs [84]. Cortisol could down-regulated the mRNA levels of tight junction protein (such as ZO-1,

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claudin-c, -h, -12) but not claudin-b in goldfish gill epithelia [85] and claudin-3c in puffer fish gill epithelia

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[86]. In addition, our data also showed that selenium deficiency failed to affect the expression of ZO-1 and

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-2b only in spleen, which might related to methionine. It has been reported that selenium deficiency reduced

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the methionine content in rats liver [87]. However, Pan et al. reported that methionine deficiency only

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down-regulated the expression of ZO-1 and ZO-2b in head kidney and skin (rather than in spleen) [2],

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supporting our assumptions. Lastly, selenium deficiency failed to affect the expression of occludin in the

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head kidney, spleen and skin might be related to IL-10. Oshima et al. reported that IL-10 increased the

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expression of occludin in mice [88]. Our previous study found that selenium deficiency did not affect the

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expression of IL-10 in head kidney, spleen and skin, supporting our hypothesis. Further correlation analysis

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showed the mRNA levels of ZO-1, ZO-2b, occludin, claudin-c, -f, -7a, -7b, -11, -15a and -15b were

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negatively related to the mRNA levels of MLCK in the head kidney, spleen and skin of grass carp (Table S2).

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The above results indicate that selenium deficiency damaged the TJs partly relating to the activation of

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MLCK signalling in the head kidney, spleen and skin of fish.

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4.4. Selenium excess impaired structural integrity of the head kidney, spleen and skin in fish

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It has been reported that selenium has a narrow range between nutritive requirements and toxicity [35].

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Our data showed that selenium excess (1.049 mg/kg diet) had adverse effects on the structural integrity

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(except GSTR, GSTo1, caspase-8 and caspase-9 in head kidney, claudin-11 in spleen, claudin-7a, -15a and

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ZO-2b in skin) of the immune organs in grass carp. The potential reasons might be related to the production

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of free radicals. Some studies reported that suitable level of selenium plays an important role in scavenging

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free radicals, but high doses promotes the production of free radicals [89, 90]. Our data showed that

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selenium excess promoted the production of ROS in the head kidney, spleen and skin of grass carp. As a

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report, the disadvantages of ROS are cause damage to lipids, proteins and DNA [64]. Meanwhile, ROS

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could impair the activities of antioxidant enzymes in fish [91, 92]. In human leukemia U937 cells, ROS

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evoked p38 MAPK activation and up-regulated the expression of FasL [80]. In addition, previous researches

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form our laboratory found that the production of ROS induced damage to the TJs of gill in grass carp [91,

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92]. These above results might partly revealed that the potential reasons of selenium excess damaged the

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structural integrity of fish immune organs. Certainly, the details of these potential mechanisms require

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further investigation.

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4.5. Selenium requirements based on structural integrity of the head kidney, spleen and skin in young

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grass carp

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In intensive aquaculture, fish are vulnerable to oxidative stress induced by extrinsic factors [93-95],

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which seriously threatens the health of fish [93]. It has been reported that oxidative stress refers to elevated

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intracellular levels of ROS that cause damage to lipids, proteins and DNA [64]. In addition, Rotruck et al. 15

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reported that selenium is a component of the enzyme glutathione peroxidase (GPx) [96] which removes the

404

excess of potentially damaging radicals produced during oxidative stress [97]. Thus, in our study, based on

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the content of ROS and the activities of GPx in the head kidney, spleen and skin, the selenium requirements

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for young grass carp were estimated to be 0.578, 0.588, 0586, 0.558, 0.577 and 0.581 mg/kg diets (Table 3),

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respectively, which were similar to the estimate based on growth performance (0.546 mg/kg diet) [13]. The

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potential reason might be related to the biological function of selenium. Selenium has dual functions of

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nutrition and toxicity [8, 35]. It has been reported that higher dose of selenium induced the production of

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ROS in the blood cells of juvenile yellow catfish (Pelteobagrus fulvidraco) [90], which damaged the

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structural integrity of fish organs [91, 98]. Similarly, the requirements of trace elements (such as iron [3] and

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zine [55]) based on antioxidant-related indices were also close to that on the growth requirement of young

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grass carp. These observations indicate that trace elements should be carefully administered for fish health.

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5. Conclusions

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In summary (Fig. 6), selenium deficiency damaged the structural integrity of the head kidney, spleen

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and skin in young grass carp, as displayed in the following aspects. Selenium deficiency (1) caused oxidative

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damage in part by down-regulating the activities of antioxidant enzymes (SOD, CAT, GPx, GSTR, GR) and

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their mRNA levels which were partially related to Keap1a (rather than Keap1b)/Nrf2 signalling; (2)

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exacerbated apoptosis in part by activating p38MAPK/FasL/caspase-8/(caspase-3 and -7) and JNK/(Bax,

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Bcl-2, Mcl-1b and IAP)/(Apaf-1, caspase-9)/(caspase-3 and -7) signalling pathways; (3) damaged the tight

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junctions in part by down-regulating the expression of tight junctions (except occludin, claudin-b and -3c in

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the head kidney, spleen and skin; ZO-1 and ZO-2 in spleen), which were regulated by MLCK. Finally, based

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on the ROS content and GPx activities in the head kidney, spleen and skin, the dietary selenium

424

requirements for young grass carp were estimated to be 0.558–0.588 mg/kg diet.

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Acknowledgements

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This research was financially supported by the National Natural Science Foundation of China

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(31672664), National Department Public Benefit Research Foundation (Agriculture) of China (201003020),

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National Basic Research Program of China (973 Program) (2014CB138600), The Earmarked Fund for China

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Agriculture Research System (CARS-45), Outstanding Talents and Innovative Team of Agricultural

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Scientific Research (Ministry of Agriculture), Science and Technology Support Program of Sichuan 16

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Province of China (2014NZ0003), Major Scientific and Technological Achievement Transformation Project

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of Sichuan Province of China (2013NC0045), The Demonstration of Major Scientific and Technological

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Achievement Transformation Project of Sichuan Province of China (2015CC0011) and The Modern

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Agricultural Industry Technology System of Sichuan Freshwater Fish Innovation Team. The authors would

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like to thank the personnel of these teams for their kind assistance.

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564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

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ACCEPTED MANUSCRIPT

AC C

692 693 694 695 696

22

Table 1 Composition and nutrients content of the basal diet. Ingredients

ACCEPTED MANUSCRIPT Nutrients content

%

Casein

14.50

Soybean protein isolate

Crude protein

13.00

1

Crude lipid

5.90

n-3

Fish oil

2.94

n-67

α-starch

24.00

Corn starch

26.30

Cellulose

5. 00

Ca(H2PO4)2

1.04 0.96 8

Available phosphorus

1.50 2

Vitamin premix

1.00 3

Selenium-free Mineral premix

2.00

Selenium premix4

1.00 5

Choline chloride premix

1.00

Ethoxyquin (30%)

0.05

4.43

0.40

RI PT

1.81

28.81

6

7

Amino acid mixture

Soybean oil

%

6

SC

697 698

699

1

700

367.7. All ingredients were diluted with corn starch to 1 kg.

701

2

702

L-a-tocopherol acetate (50%), 23.23; menadione (22.9%), 0.83; thiamine nitrate (98%), 0.09; calcium-d-pantothenate (98%),

703

3.85; pyridoxine hydrochloride (98%), 0.62; cyanocobalamin (1%), 0.94; niacin (99%), 4.04; D-biotin (2%), 0.75;

704

meso-inositol (98%), 19.39; folic acid (95%), 0.42; riboflavin (80%), 0.73; ascorhyl acetate (95%), 9.77. All ingredients

705

were diluted with corn starch to 1 kg.

706

3

707

(30.0% Fe), 12.2500; ZnSO4.H2O (34.5% Zn), 8.2460; CuSO4.5H2O (25.0% Cu), 0.9560; KI (76.9% I), 0.0650. All

708

ingredients were diluted with corn starch to 1 kg.

709

4

Selenium premix: premix was added to obtain graded levels of selenium with selenium yeast from diet 1 to 6.

710

5

Choline chloride premix (g/kg premix): Choline chloride premix (50%), 261.90 g; All ingredients were diluted with corn

711

starch to 1 kg.

712

6

Crude protein and crude lipid content were measured value.

713

7

n-3 and n-6 content were calculated according to Zeng et al.2015 [99].

714

8

Available phosphorus were calculated according to Wen et al.2015 [100].

M AN U

TE D

Vitamin premix (g/kg premix): retinyl acetate (500,000 IU/g), 0.39; cholecalciferol (500,000 IU/g), 0.40; D,

EP

Mineral premix (g/kg premix): MnSO4.H2O (31.8% Mn), 2.6590; MgSO4.H2O (15.0% Mg), 200.0000; FeSO4.H2O

AC C

715

Amino acid premix (g/kg premix): Met 78.0, Arg 44.1, His 52.6, Trp 13.6, Ile 11.9, Cys 27.2, Thr 23.8, Glu 381.1 and Gly

23

716

Table 2

717

Effects of dietary selenium levels on antioxidant related parameters in the head kidney, spleen and skin of young grass carp.

ACCEPTED MANUSCRIPT

Dietary selenium level (mg/kg diet) Se 0.025

Se 0.216

Se 0.387

Se 0.579

Se 0.795

Se 1.049

Head kidney MDA

51.58±2.11d

47.79±2.77c

44.21±2.14b

40.77±1.51a

44.05±1.66b

47.87±1.10c

PC

6.14±0.52e

4.92±0.15c

4.39±0.28b

3.64±0.27a

4.68±0.45bc

5.51±0.43d

ROS

100.00±4.81f

53.06±3.94a

71.87±3.51c

86.23±3.06e

d

c

30.38±1.63b

ASA

28.20±1.41

AHR

49.71±2.50a

32.32±1.70

5.17±0.48

MnSOD

2.42±0.20a

61.56±3.18b

b

36.57±2.49

52.84±2.81b

a

CuZnSOD

78.16±2.71d

6.07±0.55

54.78±1.36bc

bc

6.33±0.38

3.74±0.37b

a

1.95±0.06

CAT

1.87±0.15

GPx

145.54±11.41a a

cd

ab

ab

6.52±0.53

5.95±0.56d c

2.10±0.05 c

6.14±0.45 2.01±0.07

178.33±6.63c

GR

16.76±0.88a

19.27±1.40b

21.03±1.34c

24.16±1.73d

GSH

3.49±0.34a

3.90±0.11b

4.76±0.47d

4.50±0.24d

63.12±2.84d

56.41±3.07bc

53.51±2.42b

48.66±1.51a

47.21±1.68

d

PC

8.95±0.79

ROS

100.00±6.12e

6.63±0.35

b

5.78±0.27

83.28±5.31d

ASA

94.36±5.14

a

103.72±6.14

AHR

53.12±3.52a

56.54±2.18b

a

CuZnSOD

7.01±0.51

MnSOD

2.19±0.20a

5.35±0.28

67.32±4.69b b

122.71±9.24

3.91±0.25b

8.12±0.48c

113.94±6.60

61.06±2.15c

60.72±1.93c

57.54±1.13b

d

e

c

2.91±0.06b

3.58±0.34d

3.78±0.28d

3.30±0.17c

b

b

b

2.28±0.10

2.41±0.06

2.41±0.04

57.47±3.73c

b

74.50±5.91c

131.36±10.84

12.55±0.78

44.45±4.31bc

4.20±0.40bc

6.30±0.30

d

158.30±12.93ab 18.88±1.45b

11.39±0.75

ab

bc

53.58±1.01b

a

56.97±3.70a cd

1.94±0.13ab

22.54±1.08c

b

9.79±0.49

a

a

M AN U

MDA

3.48±0.19b

bc

45.96±4.35

SC

Spleen

5.80±0.24b

162.80±15.85b

bc

39.41±3.52

48.58±4.68

52.97±2.22bc

bc

4.52±0.33c

GST

43.10±3.51

54.09±1.81bc

c

c

181.54±10.74c

35.62±1.75

55.56±0.86c

4.77±0.37c 2.12±0.14

168.71±11.08bc

bc

38.56±0.97

RI PT

a

10.46±0.24 2.35±0.16

86.79±3.85d c

104.79±7.05b 55.96±2.51b 9.31±0.31b 2.80±0.16b 2.27±0.08ab

CAT

2.19±0.21

GPx

86.02±5.89a

93.53±5.97b

107.76±6.24c

106.34±6.23c

98.27±4.06b

95.01±8.62b

GST

44.17±4.32a

50.38±3.35b

57.25±4.67c

55.17±4.85bc

53.66±4.49bc

51.21±4.69b

a

b

c

GSH

3.88±0.36a

4.33±0.20ab

5.35±0.32c

5.76±0.55c

5.30±0.52c

4.40±0.36b

33.65±0.39d

32.40±0.56b

31.22±0.37a

31.25±0.31a

31.98±0.22b

32.88±0.39c

c

5.56±0.43

PC

6.24±0.56

ROS

100.00±6.99e

ASA

131.40±5.63

a

AHR

129.25±4.29a

CuZnSOD

12.42±0.85a

CAT GPx GST GR GSH

5.76±0.45

143.82±6.63

70.43±4.64

129.08±7.38a 43.24±0.62

a

5.06±0.42a

4.12±0.36

64.18±3.99b

b

156.72±9.97

a

4.37±0.30

52.15±4.15a c

169.90±6.10

a

5.44±0.45b

75.18±4.80c d

153.61±10.99

85.35±2.92d bc

143.99±9.72b

146.43±7.13c

145.56±6.40c

137.93±4.81b

134.09±5.87ab

12.86±1.09ab

13.98±1.37bc

14.49±0.92c

13.56±1.26abc

13.01±0.32ab

b

5.28±0.33ab

a

a

19.50±1.93

136.61±6.31b 6.92±0.68

5.12±0.23a

4.04±0.33

82.56±6.08d

AC C

MnSOD

a

b

EP

MDA

22.93±1.03

17.96±1.21b

15.55±1.13

Skin

23.06±1.67

b

GR

TE D

18.75±0.92

c

81.91±6.74

bc

143.33±11.96b 53.68±1.34 6.95±0.59c

b

7.67±0.76

c

7.84±0.66

5.55±0.24b 83.42±7.95

7.32±0.64

5.53±0.38b c

151.90±13.65bc 68.60±5.20

c

d

7.72±0.31d

89.33±2.76

5.42±0.21ab c

161.80±9.60c 67.33±5.39 8.83±0.64e

bc

d

87.82±7.04

c

147.80±10.84b 58.82±5.43

c

7.38±0.58cd

6.66±0.26b 5.23±0.14ab 74.65±6.45ab 140.74±11.58ab 52.20±4.68b 6.19±0.41b

718

1

719

malondialdehyde (nmol/g tissues); PC, protein carbonyl (nmol/mg protein); ROS, reactive oxygen species (% DCF

720

florescence); AHR, anti-hydroxyl radical (U/mg protein); ASA, anti-superoxide anion (U/g protein); CuZnSOD (U/mg

721

protein); MnSOD (U/mg protein); CAT (U/mg protein); GPx (U/mg protein); GST (U/mg protein); GR (U/g protein); GSH,

722

glutathione (mg/g protein).

Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). MDA,

24

723

Table 3

724

The optimal selenium requirements based on different indices for young grass carp.

ACCEPTED MANUSCRIPT R2

Selenium requirement

P

0.9366

0.578 mg/kg

< 0.05

0.9183

0.588 mg/kg

< 0.01

0.8711

0.586 mg/kg

< 0.01

0.8030

0.558 mg/kg

= 0.087

YGPx in spleen = -67.1830 x + 77.5234 x + 82.6502

0.8097

0.577 mg/kg

= 0.083

YGPx in skin = -59.8263 x2 + 69.4905 x + 68.5057

0.9440

0.581 mg/kg

< 0.05

Regressive equation Y ROS in head kidney = 136.9546 x2 - 158.2018 x + 103.8980 2

Y ROS in spleen = 119.5988 x - 140.7461 x + 104.5702 2

Y ROS in skin = 129.2243 x - 151.5586 x + 104.8636 2

YGPx in head kidney = -102.2854 x + 114.2461 x + 146.3834 2

RI PT

725

AC C

EP

TE D

M AN U

SC

726

25

727

Fig. 1.

AC C

728

EP

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

729

Fig. 1. Relative mRNA levels of antioxidant enzymes and related signalling molecules in the head kidney (A), spleen (B)

730

and skin (C) of young grass carp. Data represent means of six fish in each group, error bars indicate S.D. Values having

731

different letters are significantly different (P < 0.05).

732

26

733

Fig. 2.

TE D

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734

Fig. 2. Western blot analysis of Nrf2 in the head kidney (A), spleen (B) and skin (C) of young grass carp. Data represent

736

means of three fish in each group, error bars indicate S.D. Values having different letters are significantly different (P <

737

0.05).

AC C

738

EP

735

27

739

Fig. 3.

SC

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ACCEPTED MANUSCRIPT

740

Fig. 3. DNA analysis by 2% agarose gel electrophoresis of the genomic DNA extracted from the head kidney and spleen of

742

young grass carp. Lane 1: selenium deficiency, Lane 2- Lane 6: the levels of dietary selenium were 0.216, 0.387, 0.579,

743

0.795 and 1.049 mg/kg, respectively. This experiment was repeated three times with similar results achieved.

M AN U

741

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744

28

745

Fig. 4.

AC C

746

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747

Fig. 4. Relative mRNA levels of caspases and related signalling molecules in the head kidney (A), spleen (B) and skin (C)

748

of young grass carp. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters

749

are significantly different (P < 0.05).

750

29

751

Fig. 5.

AC C

752

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ACCEPTED MANUSCRIPT

753

Fig. 5. Relative mRNA levels of tight junction complexes and MLCK in the head kidney (A), spleen (B) and skin (C) of

754

young grass carp. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are

755

significantly different (P < 0.05).

756

30

757

Fig. 6.

M AN U

SC

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ACCEPTED MANUSCRIPT

758

Fig. 6. The potential pathways about the effects of selenium on the structural integrity in the head kidney, spleen and skin of

760

fish.

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759

31

ACCEPTED MANUSCRIPT Highlights Compared with optimal selenium supplementation: 1、 Selenium deficiency caused oxidative damage in fish immune organs. 2、 Selenium deficiency aggravated apoptosis in fish immune organs.

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3、 Selenium deficiency damaged the tight junctions in fish immune organs.

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4、 Selenium modulated Nrf2, p38MAPK, JNK and MLCK signaling in fish immune organs.