Expression of selenoprotein genes in muscle is crucial for the growth of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with selenium yeast

Accepted Manuscript Expression of selenoprotein genes in muscle is crucial for the growth of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with selenium yeast

Li Wang, Xuezhen Zhang, Lei Wu, Qi Liu, Dianfu Zhang, Jiaojiao Yin PII: DOI: Reference:

S0044-8486(17)32291-3 doi:10.1016/j.aquaculture.2018.03.054 AQUA 633155

To appear in:

aquaculture

Received date: Revised date: Accepted date:

20 November 2017 12 March 2018 27 March 2018

Please cite this article as: Li Wang, Xuezhen Zhang, Lei Wu, Qi Liu, Dianfu Zhang, Jiaojiao Yin , Expression of selenoprotein genes in muscle is crucial for the growth of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with selenium yeast. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2018), doi:10.1016/j.aquaculture.2018.03.054

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ACCEPTED MANUSCRIPT

Expression of selenoprotein genes in muscle is crucial for the growth of rainbow trout (Oncorhynchus mykiss) fed diets supplemented with selenium yeast

College of Fisheries, Hubei Provincial Engineering Laboratory for Pond Aquaculture, Huazhong

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Li Wang1 , Xuezhen Zhang1* , Lei Wu1 , Qi Liu1 , Dianfu Zhang1 and Jiaojiao Yin1

Corresponding author:

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*

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Agricultural University, Wuhan 430070, People’s Republic of China

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Xuezhen Zhang, Ph.D.

College of Fisheries, Hubei Provincial Engineering Laboratory for Pond Aquaculture, Huazhong

Phone and fax: +86-27-87282114

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Agricultural University, Shizishan street 1, Wuhan 430070, P.R. China.

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E-mail: [email protected]

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Abstract Selenium (Se) is an essential trace element for fish growth and performs its physiological functions mainly through incorporating into selenoproteins. It is well known that dietary Se regulates fish growth by controlling the synthesis of deiodinase, a kind of selenoproteins. However, recently, as

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many as 41 selenoproteins have been characterized in teleost fish. We propose a hypothesis if other selenoproteins, besides deiodinase, could also involve in the regulation of fish growth. In the present

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study, rainbow trout (Oncorhynchus mykiss) were fed diets supplemented with or without graded

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levels of Se (2, 4 or 6 mg/kg, from selenium yeast, Se-yeast) for 10 weeks. At the end of the feeding trial, fish growth and the expressions of a total of 28 selenoprotein genes in tissues have been

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evaluated. Results showed that dietary Se-yeast supplementation significantly increased fish growth (P < 0.05) without causing oxidative stress in fish tissues. In addition, dietary Se supplementation

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caused a general up-regulation of selenoprotein gene expressions either in liver (10 genes) or in muscle (11 genes) (P < 0.05). Correlation analysis showed that rainbow trout growth was significantly

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and positively correlated to only 4 selenoprotein genes in liver but all the 11 differentially expressed selenoprotein genes in muscle (P < 0.05), and it presented the strongest correlation with the mRNA

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levels of muscle selenoprotein W-like gene (P < 0.05). These results indicate that dietary Se-yeast supplementation is beneficial for rainbow trout growth, and the enhanced growth performance is

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closely correlated to the expressions of muscle selenoprotein genes, particularly the selenoprotein W-like gene. This study reveals the importance of muscle selenoprotein gene expressions, and provides a new concept for the regulatory mechanism of dietary Se on fish growth.

Keywords: Rainbow trout; Dietary selenium supplementation; Organic selenium; Selenium yeast; Selenoproteome.

ACCEPTED MANUSCRIPT 1. Introduction

Selenium (Se) is an essential trace element in fish nutrition (Watanabe et al., 1997; Hamilton, 2004; Khan et al., 2017a). It mainly incorporates into selenoproteins as the 21st amino acid (selenocysteine) to perform its biological functions (Lu and Holmgren, 2009). Dietary Se deficiency has been demonstrated to cause various damages to fish, such as the elevated mortality (Poston et al.,

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1976), depressed growth and feed efficiency (Lin and Shiau, 2005), increased oxidative stress (Bell et

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al., 1987) and hemolytic rates (Khan et al., 2016). Thus, optimal dietary Se level is crucial for fish growth and health.

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Up to now, dietary Se requirements have been studied in a variety of fish species, including

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rainbow trout (Oncorhynchus mykiss) (0.15-0.38 mg/kg diet) (Hilton et al., 1980), channel catfish (Ictalurus punctatus) (0.25 mg/kg diet) (Gatlin and Wilson, 1984), grouper (Epinephelus malabaricus)

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(0.7 mg/kg diet) (Lin and Shiau, 2005), Nile tilapia (Oreochromis niloticus) (1.06-2.06 mg/kg diet) (Lee et al., 2016), blunt snout bream (Megalobrama amblycephala) (0.43 mg/kg diet) (Liu et al., 2017)

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and gibel carp (Carassius auratus gibelio var. CAS III) (0.73-1.19 mg/kg) (Zhu et al., 2017), etc. Nevertheless, many of these studies (such as the studies on rainbow trout and channel catfish

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mentioned above) used inorganic Se (mainly sodium selenite) as Se supplement. There is evidence that inorganic Se is less bioavailable and tolerated at higher concentrations than organic Se (Thiry et al., 2012), such as selenium yeast (Se-yeast). Thus, Se requirements of those fish species based on

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inorganic Se need to be updated when organic forms of Se are supplemented to fish diets (Pacitti et al., 2016a). One evidence is that the growth performance, nutrient utilization and antioxidant capacity of rainbow trout were improved by diets supplemented with 3.5-4.3 mg/kg organic Se (Ozluer-Hunt et al., 2011), which is much higher than the Se requirement of rainbow trout (0.15-0.38 mg/kg) based on sodium selenite (Hilton et al., 1980). Fish growth performance, which determines the yield and economic efficiency, is an important index in the aquaculture industry. Although Se is important for fish growth performance, its regulatory mechanism on fish growth is still unclear. Se performs its biological functions in animals mainly through selenoproteins (Lu and Holmgren, 2009). Several researches reported that dietary Se

ACCEPTED MANUSCRIPT increases fish growth primarily by promoting the synthesis of deiodinase (Khan et al., 2017a), a kind of selenoproteins. Deiodinase regulates the conversion of the inactive thyroid hormone (thyroxine, T4) into a metabolically active thyroid hormone (3,5,3’-triiodothyronine, T3) (Lobanov et al., 2009; Papp et al., 2007). Elevated serum T3 concentration will raise the growth hormone messenger RNA levels in the pituitary cells and promote the synthesis of growth hormone in fish (Moav and McKeown, 1992;

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Farchi-Pisanty et al., 1995), thereby increasing the fish growth (Khan et al., 2016; 2017b). These studies indicate an important role of deiodinase in the regulation of dietary Se on fish growth.

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However, fish owns numerous kinds of selenoproteins. Until now, 41 selenoproteins have been

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characterized in teleost fish (Mariotti et al., 2012). Although most of their functions are still unknown, increasing evidence indicates that they play important roles in various physiological processes

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(Lobanov et al., 2009). Therefore, there is a hypothesis that if other selenoproteins, besides deiodinase, could also involve in the regulation of fish growth. Clarification of this question could help us to

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better understand the regulatory mechanism of Se on fish growth. Rainbow trout (O. mykiss) is cultured widely throughout the world for commercial aquaculture.

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Ozluer-Hunt et al. (2011) reported that its growth performance was increased when fish were fed diets supplemented with Se-yeast at the dietary Se levels of 3.5-4.3 mg/kg. In this study, we supplemented

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rainbow trout diets with 2-6 mg/kg Se (from Se-yeast). Unsurprisingly, fish growth has also been increased by dietary Se supplementation. To figure out which selenoprotein might involve in the

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regulation of rainbow trout growth, we further analyzed the expressions of a total of 28 selenoprotein genes in fish tissues. Additionally, Se accumulations and oxidative status in fish tissues have also been evaluated.

2. Materials and methods

The present experiment was carried out in accordance with the guidelines of the Institutional Animal Care and Use Committees (IACUC) of Huazhong Agricultural University, Wuhan, China.

ACCEPTED MANUSCRIPT 2.1. Experimental diets In the present study, four experimental diets were prepared by supplementing 0, 2, 4 and 6 mg/kg Se from Se-yeast to the basal diet and the measured Se contents (mg/kg diet) in the diets were 1.01; 2.58, 4.36 and 6.26 mg/kg, respectively. Feed ingredients of the experimental diets are presented in Table 1. All ingredients were finely ground, well mixed and pelleted through 3-mm die in a

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commercial pelletizer. Subsequently, diets were air-dried and stored at 4℃ until use. And the

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proximate composition of the experimental diets is presented in Table 1.

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2.2. Experimental design and feeding trial

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Rainbow trout (O. mykiss) were obtained from Enshi Guoxi Fishery Development Co., Ltd. (Hubei, China). And the rearing experiment was conducted in College of Fisheries, Huazhong

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Agriculture University, China. Upon arrival, all fish were kept in a 3500-litre circular tank and fed the basal diet for 2 weeks to acclimatize with the laboratory facilities. The feeding trial was conducted in a flow-through system where each tank was equipped with an

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inlet, outlet and continuous aeration. A flow rate of 1 litre per min was maintained throughout the

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experimental period. After fasted for 24 h, a total of 240 fish (average initial body weight: 144.87 ± 1.71 g) were individually weighed and divided into 4 groups and stocked in 12 plastic tanks (1000 litres). Then, there were 20 fish in each tank. All fish were hand-fed to visual satiation twice daily

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(09.00 and 16.00 hours) with the experimental diets. Water quality parameters were monitored daily during the experiment. Dissolved oxygen and water temperature were maintained at 7.0 ± 0.5 mg/l and 13 ± 1℃, respectively. A photoperiod of 12-h light and 12-h dark were maintained strictly.

2.3. Sample collection After the 10-week feeding trial, fish were fasted for 24 h. The final body weight and length of fish from each tank was individually measured after being anesthetized with tricaine methanesulphonate (MS-222, Western Chemical, Inc., Ferndale, WA, USA). Then, three fish from each replicate tank were packed in low density polypropylene bags (180× 300 cm) for the

ACCEPTED MANUSCRIPT determination of whole body total Se content. The blood was taken from the caudal vein of other three fish in each replicate tank. Serum was collected by keeping the blood at 4℃ for 24 h followed by centrifuging it at 3,000 g for 15 min. Subsequently, viscera along with liver were taken out and weighed to calculate viscerosomatic index (VSI) and hepatosomatic index (HSI), respectively. Then, 2 liver pellets (about 100 mg) were separated for the determination of oxidative/antioxidant parameters

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and mRNA levels, respectively. The remain liver samples were packed in low density polypropylene bags (15×22 cm) for the determination of liver total Se content. 2 dorsal muscle pellets (about 100

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mg) close to the vertebra and in the same location of each fish were sampled for the determination of

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oxidative/antioxidant parameters and mRNA levels, respectively. The remain dorsal muscles were packed in low density polypropylene bags (140×220 cm) for the determination of muscle total Se

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content. After sampled, all samples were immediately snap-frozen in liquid N2 and then stored at -80℃

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until the further analysis.

2.4. Growth and nutrient utilization parameter analysis

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The following variables were evaluated: Survival rate (SR, %) = Nt / No ×100;

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Weight gain rate (WGR, %) = (Wt - Wo ) / Wo ×100; Condition factor (CF, g/cm3 ) = final body weight (g) / final body length3 (cm3 ) ×100;

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Hepatosomatic index (HIS, %) = liver weight (g) / body weight (g) × 100; Viscerosomatic index (VSI, %) = viscera weight (g) / body weight (g) × 100; Feed intake (FI, % body weight/day) = dry feed intake (g) / [1/2 × (Wt + Wo ) × (Tt - To )] × 100; Feed efficiency (FE) = live weight gain (g) /dry feed intake (g). Where; Nt = number of fish at the end of experiment, N o = number of fish at the beginning of experiment, Wt = final weight of fish, Wo = initial weight of fish, Tt = end of experiment (day), To = begin of experiment (day).

ACCEPTED MANUSCRIPT 2.5. Determination of proximate composition and total Se content Proximate composition was analyzed as previously described by our laboratory (Wang et al., 2017). Briefly, dry matter was determined by dried in an oven at 105℃ to constant weight. Crude protein content was determined with an automatic Kjeldahl analyzer (K9860; Jinan Hanon Instruments Co., Ltd, Jinan, China) and the proportion of crude protein was calculated as total

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nitrogen × 6.25. Crude lipid content was extracted after acid hydrolysis according to Soxhlet

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method. Ash was determined by incineration at 550℃ to constant weight.

For the determination of total Se content, samples were subjected to acid digestion using

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concentrated HNO3 and H2O2 , and measured by inductively coupled plasma mass spectrometry

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(Agilent 7500c, Yokogawa Analytical Systems, Tokyo, Japan) according to the method of Fontagné-Dicharry et al. ( 2015).

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2.6. Determination of oxidative status and antioxidant enzyme activity

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Serum samples were directly subjected to the determination of oxidative status and antioxidant enzyme activity after thawed. For liver and muscle tissues, samples were rapidly thawed and

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homogenized in 10 volumes (w/v) of ice-cold normal saline for 3 min, and centrifuged for 15 min at 4000 g. The supernatants were collected for the further analysis. Parameters in terms of the activities of glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and the content of

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malondialdehyde (MDA) were determined by corresponding detection kits purchased from Nanjing Bioengineering

Institute

(Jiangsu,

China)

according

to

the

manufacturer’s

recommendations.

2.7. Gene expression analysis Total RNA samples were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. RNA quantity and quality were determined by spectrophotometry using a NanoDrop ND-1000 (NanoDrop Technologies®, Wilmington, DE, USA) and electrophoresis using 200 ng of total RNA in a 1% (w/v) agarose gel. Subsequently, 1 μg of RNA

ACCEPTED MANUSCRIPT was reverse transcribed in to complementary DNA (cDNA) using PrimeScript™ RT Reagent kit with gDNA Eraser (Takara, Dalian, China). Expression of a total of 28 selenoprotein genes, including iodothyronine deiodinase 1 (Dio1); iodothyronine deiodinase 2 (Dio2); iodothyronine deiodinase 3 (Dio3); fish 15 kDa selenoprotein-like (fep15); glutathione peroxidase 1a (GPX1a); glutathione peroxidase 1b1 (GPX1b1); glutathione

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peroxidase 1b2 (GPX1b2); glutathione peroxidase 4a1 (GPX4a1); glutathione peroxidase 4a2 (GPX4a2); glutathione peroxidase 4b (GPX4b); 15 kDa selenoprotein (SelF); selenoprotein H, I, Ja, K,

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L, N, P and U (SelH, SelI, SelJa, SelK, SelL, SelN, SelP and SelU); selenoprotein M, O, S, T1, T2 and

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W like (SelMl, SelOl, SelSl, SelT1l, SelT2l and SelWl); methionine sulfoxide reductase B 1A-like (MsrB1Al); thioredoxin reductase 3a (TrxR3a) and thioredoxin reductase 3b (TrxR3b), was determined

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by quantitative real-time PCR using specific real-time PCR primers (Table 2). PCR was performed on QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using

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SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (Takara, Dalian, China) in a final volume of 20 μl according to the manufacturer's recommendations. Each PCR run included replicate samples and PCR

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efficiency (E) was measured by the slope of a standard curve using serial dilutions of cDNA of a pool sample. The relative quantification of the target gene was performed using the mathematical model

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described by Pfaffl (2001). The relative expression ratio (R) of a target gene was calculated on the basis of real-time quantitative PCR efficiency and CT deviation (ΔCT) of the average unknown sample versus average control sample, and expressed in comparison to the EF1α reference gene in

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muscle and β-actin reference gene in liver because EF1α and β-actin were the most stable in muscle samples and liver samples, respectively (data not shown). To compare the abundance among different selenoprotein genes, relative abundance of selenoprotein gene mRNA levels was presented as E (–CT target gene) gene

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/E reference gene(–CT reference gene).

2.8. Statistical analysis Relative abundance of selenoprotein gene mRNA levels is presented as means. The other data are expressed as means ± SEM. Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Data were tested for normal distribution with Shapiro-Willks test and homogeneity of the

ACCEPTED MANUSCRIPT variances with Levene’s test. One-way analysis of variance (ANOVA) followed by Duncan’s test were carried out for comparing different treatments when normal variables were analyzed. When data were not normally distributed, they were subjected to nonparametric statistical analysis on ranks (Kruskal-Wallis) followed by Dunn-Bonferroni post-hoc test. Linear regression analysis was performed to plot dietary Se against Se contents of whole body, liver and muscle. Correlations

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between data were examined using Pearson’s correlation test. A probability of P < 0.05 was

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considered significant.

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3.1. Growth performance and nutrient utilization

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3. Results

All experimental diets were well accepted by the experimental rainbow trout and no death was

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observed in all treatments (survival rate: 100%; Table 3). Dietary Se-yeast supplementation enhanced growth performance and somatic parameter of rainbow trout. Fish fed diets supplemented with 2, 4

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and 6 mg/kg Se presented significantly higher final body weight, WGR and CF compared with fish fed the basal diet (P < 0.05), while HSI and VSI showed no significant difference among treatments

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(Table 3). Moreover, the highest numerical final body weight, WGR and CF were found in fish fed diet supplemented with 4 mg/kg Se (Table 3). Nutrient utilization was also influenced by dietary Se-yeast supplementation. FI increased with increasing dietary Se levels and was significantly higher

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in fish fed diets supplemented with 6 mg/kg Se when compared with fish fed the basal diet (P < 0.05, Table 3). Compared to the basal diet, diets supplemented with Se-yeast significantly increased FE (P < 0.05, Table 3). In addition, the highest numerical FE existed in fish fed diet supplemented with 4 mg/kg Se (Table 3).

3.2. Total Se contents in whole body, liver and muscle

Total Se contents in whole body, liver and muscle are presented in Fig. 1. They were all significantly increased (whole body, from 0.38 to 1.06 mg Se per kg wet weight; liver, from 0.48 to 1.48 mg Se per kg wet weight; muscle, from 0.25 to 1.12 mg Se per kg wet weight) as dietary Se

ACCEPTED MANUSCRIPT levels increased from 1.01 to 6.26 mg/kg (P < 0.05). Regression analysis showed that total Se contents in whole body, liver and muscle all follow the linear dose responses to dietary Se levels (whole body, R2 =9169, P < 0.001; liver, R2 =0.8697, P < 0.001 and muscle, R2 =0.9290, P < 0.001).

3.3. Oxidative status and antioxidant enzyme activity

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Dietary Se-yeast supplementation caused no oxidative stress for rainbow trout. Serum and liver MDA contents were not affected when fish were fed diets supplemented up to 6 mg/kg Se (Table 4).

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Meanwhile, the activities of the selenium-dependent enzyme (GPX) and 2 selenium-independent

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antioxidant enzymes (CAT and SOD) also presented no changes in serum and liver (Table 4). It is noteworthy that muscle oxidative status has been improved. MDA content in muscle significantly

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decreased in fish fed diets supplemented with 4 or 6 mg/kg Se (P < 0.05, Table 4). Furthermore, GPX activity significantly increased in muscle with the increasing dietary Se level (P < 0.05, Table 4).

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However, the activities of CAT and SOD showed significant lower in muscle of fish fed diet supplemented up to 6 mg/kg Se (P < 0.05, Table 4).

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3.4. Expressions of selenoprotein genes in liver and muscle

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In the present study, mRNA levels of 28 selenoprotein genes were determined in the liver and muscle of rainbow trout. There was a great difference in the abundance of selenoprotein gene mRNA

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levels between the two tissues. In liver, SelP showed a much higher abundance than the others (Fig. 2a), while in muscle, fep 15, GPX1b2, GPX4a1, GPX4b, SelH, SelJa, SelK, SelMl, SelP, SelT2l, SelU and TrxR3b all highly expressed (Fig. 2b). Elevated dietary Se levels increased the mRNA levels of 10 (GPX1a, GPX1b2, SelK, SelMl, SelP, SelT1l, SelT2l, SelU, SelWl and MsrB1Al, Table 5) and 11 (GPX1a, GPX1b2, GPX4b, SelK, SelP, SelT1l, SelT2l, SelU, SelWl, TrxR3b, MsrB1Al, Table 5) selenoprotein genes in liver and muscle, respectively (P < 0.05). A remarkable phenomenon was observed that the kinds of the differentially expressed selenoprotein genes were highly similar in liver and muscle. These genes were GPX1a, GPX1b2, SelK, SelP, SelT1l, SelT2l, SelU, SelWl and MsrB1Al. However, the trends of mRNA expressions of the differentially expressed selenoprotein genes were different in liver and muscle. The mRNA levels of the differentially expressed selenoprotein genes

ACCEPTED MANUSCRIPT (except SelP and SelT1l) in muscle were increased with increasing dietary Se levels and the highest expressions were found in fish fed diets supplemented with 6 mg Se per kg diet while, in liver, the highest expressions were found in fish fed diet supplemented with 2 or 4 mg Se per kg diet (Table 5).

3.5. Correlation between growth performance and mRNA levels of selenoprotein genes

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Correlation analysis showed that rainbow trout WGR was significantly and positively correlated to the mRNA levels of SelP, SelU, SelWl and MsrB1Al in liver (P < 0.05, Table 6), and the mRNA

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levels of GPX1a, GPX1b2, GPX4b, SelK, SelP, SelT1l, SelT2l, SelU, SelWl, TrxR3b and MsrB1Al in

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muscle (P < 0.05, Table 7). In addition, SelWl mRNA in muscle showed the strongest correlation with

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fish growth performance (Table 7).

4 Discussion

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In fish, organic Se accumulates in tissues at a level directly proportional to dietary intake (Rider et al., 2009). As an organic Se source, Se-yeast contains 70-90% selenomethionine in its

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selenocompounds (Block et al., 2004; Ip et al., 2000). Pacitti et al. (2015) reported that Se-yeast is an excellent Se supplement in rainbow trout feed to deliver the Se accumulation in tissues. Be in line

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with this perspective, the positive relationships between Se accumulations and dietary Se levels have been reported in whole body (Rider et al., 2009) and tissues (Ozluer-Hunt et al., 2011; KüçÜkbay et

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al., 2009; Pacitti et al., 2015, 2016a) when rainbow trout were fed elevated levels of dietary Se-yeast. In the present study, there is also a linear increase of total Se contents in rainbow trout whole body, liver and muscle responding to the increasing dietary Se levels. Additionally, a similar phenomenon has also been found in carp (Cyprinus carpio) (Ashouri et al., 2015; Elia et al., 2011) and blunt snout bream (M. amblycephala) (Liu et al., 2017). Se primarily accumulates as selenomethionine and selenocysteine in rainbow trout when fish are fed diets supplemented with Se-yeast (Godin et al., 2015). However, excessive accumulation of selenoamino acids could also cause oxidative stress for rainbow trout during the metabolic process of selenoamino acids (Palace et al., 2004; Misra et al., 2012a). In the present study, diets supplemented

ACCEPTED MANUSCRIPT with Se-yeast did not increased the content of MDA, a product of lipid peroxidation and frequently measured as a marker of oxidative stress (Esterbauer et al., 1991), in trout serum, liver and muscle. It indicates that supplementing up to 6 mg/kg Se from Se-yeast (dietary Se level: 6.26 mg/kg) causes no oxidative stress in rainbow trout tissues. This is in line with the results of Rider et al. (2009) that liver MDA content was not affected when rainbow trout were fed diets containing up to 7.4 mg/kg organic

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Se for 10 weeks. Previously, Rider et al. (2009) and Pacitti et al. (2015; 2016b) reported that growth performance

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of rainbow trout showed no changes when fish were fed diets supplemented with 0.25-8 mg/kg Se

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from Se-yeast. However, in the present study, diets supplemented with 2, 4 and 6 mg/kg Se (from Se-yeast) significantly increased fish growth performance. This difference in fish growth performance

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might be contributed to the different fish stages, diet composition, rearing conditions, feeding strategies or other factors. Similar to the present result, the improved growth performance of rainbow

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trout has also been observed by Ozluer-Hunt et al. (2011) when fish were fed diets supplemented with 3 or 4 mg/kg Se from Se-yeast.

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Se serves its physiological functions mainly through selenoproteins (Lu and Holmgren, 2009, Wrobel et al., 2015). Considering the improvement of trout growth by dietary Se supplementation, we

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have further investigated the expressions of a total of 28 selenoprotein genes in trout liver and muscle (2 tissues mainly involve in metabolism and growth, respectively) to reveal the potential regulatory

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mechanism of dietary Se on rainbow trout growth performance. Relative abundance among selenoprotein gene expressions was different in liver and muscle. In liver, SelP exhibited a much higher abundance than the other selenoprotein genes. Selenoprotein P is primarily synthesized in liver and secreted to plasma to function as the transport and delivery of Se to remote tissues (Papp et al., 2007). Thus, the much high abundance of SelP in liver might be attributed to the high accumulation of Se in this tissue. However, in muscle, many selenoprotein genes, including fep 15, GPX1b2, GPX4a1, GPX4b, SelH, SelJa, SelK, SelMl, SelP, SelT2l, SelU, SelWl and TrxR3b exhibited the high abundances. This difference between liver and muscle indicates that tissues should keep the high expressions of the specific selenoproteins to maintain their homeostasis (Huang et al., 2016).

ACCEPTED MANUSCRIPT Dietary Se level is crucial for mRNA levels of selenoprotein genes in animals (Sunde et al., 2009; Sunde and Raines, 2011). In the present study, more than 1/3 of the determined selenoprotein genes, either in liver or in muscle, have been up-regulated by various Se supplementation levels. More up-regulated selenoprotein genes were observed in muscle (11 genes) than liver (10 genes). It is in consonance with a similar research in broiler that the most up-regulated selenoprotein genes were

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detected in muscle when broilers were fed diets supplemented with exogenous Se for 6 weeks (Huang et al., 2016). In addition, the trends of differentially expressed selenoprotein genes responding to

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increasing dietary Se levels were also different in liver and muscle. In muscle, most of them (except

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SelP and SelT1l) increased following a dose response to dietary Se levels, while in liver, they increased at low Se supplemental levels, subsequently decreased with more Se supplementation. The

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different expression patterns of selenoprotein genes in liver and muscle might be attributed to the tissue specific and hierarchy expression of selenoprotein genes (Sunde and Raines, 2011). Although

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muscle selenoprotein genes seemed not satisfied with the present dietary Se levels, excessive dietary Se was adverse to maintain the high expressions of liver selenoprotein genes. This is similar to the

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result of a research conducted in chickens that high level of dietary Se declined the expressions of several selenoproteins in the liver (Huang et al., 2016). In addition to the different expression patterns

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of selenoprotein genes in tissues, a noteworthy phenomenon was observed that most of the differentially expressed selenoprotein genes were the same kinds in liver and muscle. They were

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GPX1a, GPX1b2, SelK, SelP, SelT1l, SelT2l, SelU, SelWl and MsrB1Al. This result indicates the more important roles of these selenoproteins than the others in rainbow trout physiological processes. We further investigated the correlations between rainbow trout growth performance and the mRNA levels of the differentially expressed selenoprotein genes in liver and muscle. Results showed that fish WGR was significantly and positively correlated to only 4 selenoprotein genes (SelP, SelU, SelWl and MsrB1Al) in liver but all the 11 differentially expressed selenoprotein genes (GPX1a, GPX1b2, GPX4b, SelK, SelP, SelT1l, SelT2l, SelU, SelWl, TrxR3b and MsrB1Al) in muscle. Although liver is a primary organ in animal metabolism, it seems that dietary Se-yeast improved rainbow trout growth performance mainly through regulating the selenoprotein gene expressions in muscle, the main tissue directly involving in fish growth (Johnston et al., 2010).

ACCEPTED MANUSCRIPT It was noteworthy that, among the differentially expressed selenoprotein genes in muscle, SelWl showed the highest up-regulated fold at each dietary Se level and the strongest correlation with fish growth. Selenoprotein W is a cytosolic protein and highly expresses in animal skeletal muscle (Vendeland et al., 1993; Whanger, 2009). It was reported that declined expression of selenoprotein W could lead to a variety of diseases, such as Keshan Disease in human (Gu, 1983), white muscle

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disease in lambs (Whanger, 2000) and selenium-responsive myopathy in livestock (Whanger, 2000). Although the most functions of selenoprotein W are still unknown, selenoprotein W has been reported

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to inhibit the binding of 14-3-3 protein to the transcriptional co-activator with PDZ-binding motif

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(TAZ) (Jeon et al., 2014) and cell division cycle 25 (CDC25) (Park et al., 2012), thereby promoting the proliferation and differentiation of skeletal muscle cells. Thus, we inferred that the expression of

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selenoprotein W in the muscle is much important for rainbow trout growth. In summary, Se-yeast is an excellent Se supplement in rainbow trout feed to deliver the Se

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accumulation in fish tissues. Supplementing with 2-6 mg/kg Se from Se-yeast increased rainbow trout growth without causing oxidative stress in fish tissues. Expressions of selenoprotein genes in the liver

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and muscle were extensively up-regulated by elevated dietary Se levels. However, much high level of dietary Se was adverse to maintain the high expression of selenoprotein genes in trout liver.

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Correlations between fish growth and selenoprotein gene expressions reveal that selenoproteins in muscle are more important for rainbow trout growth than those in liver. In addition, among the

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selenoprotein genes in the muscle, SelWl showed the most relevance to fish growth. The results of the present study reveal a potential link between fish growth and muscle selenoproteins, and provide a new direction for the further researches on the regulatory mechanism of dietary Se on fish growth.

Acknowledgements

This work was supported by the Fundamental Reaearch Funds for the Central Universities [grant number 2662015PY024] and Da Bei Nong Group Promoted Project for Young Scholar of HZAU [grant number 2017DBN018].

ACCEPTED MANUSCRIPT

References

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Fig. 1

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Fig. 2

ACCEPTED MANUSCRIPT Table 1 Formulation and composition of the experimental diets (g/kg dry diet).

Poultry meal Corn gluten

a

a

Soybean meal

a

Soy protein concentrate Wheat gluten

a

Pregelatinized starch b

Soy lecithin Betaine

a

c

50.0

50.0

50.0

40.0

40.0

40.0

40.0

152.5

152.5

152.5

152.5

50.0

50.0

50.0

50.0

60.0

60.0

60.0

60.0

50.0

50.0

50.0

50.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

40.0

130.0

130.0

130.0

130.0

5.0

5.0

5.0

5.0

5.0

5.0

4.0

4.0

4.0

4.0

50.0

50.0

50.0

50.0

1.0

1.0

1.0

1.0

-

0.5

1

1.5

91.87

92.66

92.44

91.97

49.56

50.03

50.34

49.75

16.93

17.33

17.59

17.91

9.70

9.78

9.36

9.35

1.01

2.58

4.36

6.26

5.0

Vitamin premix

e

Mineral premix

f

Choline chloride

g

h

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Analytical composition Dry matter Crude protein Crude lipid

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Ash

Total Se content (mg/kg) a

50.0

5.0

d

Se-yeast

6 334.0

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Fish oil

a

4 334.0

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Krill meal

a

2 334.0

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Blood meal a

0 334.0

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Fishmeal

a

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Se supplementation (mg/kg diet) Ingredient

Fish meal (crude protein 678.2 g/kg, crude lipid 80.1 g/kg), blood meal (crude protein 898.6 g/kg,

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crude lipid 14.1 g/kg), krill meal (crude protein 507.4 g/kg, crude lipid 31.7 g/kg), poultry meal (crude protein 681.0 g/kg, crude lipid 198.2 g/kg), corn gluten (crude protein 637.4 g/kg, crude lipid 22.3 g/kg), soybean meal (crude protein 485.2 g/kg, crude lipid 8.9 g/kg), soy protein concentrate(crude protein 857.2 g/kg, crude lipid 34.1 g/kg), wheat gluten (crude protein 838.8 g/kg, crude lipid 102.2 g/kg) and pregelatinized starch were purchased from Hubei Haida feed Co., Ltd (Hubei, China). b

Dalian, China.

c

Soy lecithin (≥ 70%, Aladdin, Shanghai, China).

d

Betaine (98%, Aladdin, Shanghai, China).

e

Vitamin premix (g/kg product): retinol acetate, 60, thiamine, 8; lactoflavine, 12; nicotinic acid, 80;

ACCEPTED MANUSCRIPT D-calcium panthothenate, 24; pyridoxine-HCl, 12; D-biotin, 0.4; cyanocobalamin, 0.02; L-ascorbic acid, 600; cholecalciferol, 0.04; DL-a-tocopherol acetate, 96; menadione, 8; folic acid, 4; α-cellulose, 95.54. f

Mineral premix (g/kg product): Calcium Lactate, 350; K2 HPO4 , 250; Ca(H2 PO4 )2 .H2 O, 175; NaCl, 75;

KI, 0.125; CuSO4 .5H2 O, 37.5; MnSO4 .H2 O, 2.5; ZnSO4 .H2 O, 14.05; K2 CO3 , 35; α-cellulose, 60.825. Choline chloride (99%, Aladdin, Shanghai, China).

h

Se-yeast (total Se content: 4 g/kg, Angel Yeast Co., Ltd, Hubei, China).

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ACCEPTED MANUSCRIPT Table 2 Oligonucleotide primers used in the quantitative real-time PCR assays Ge ne

Forward primer (5’-3’)

Reverse primer (5’-3’)

name

Amplic

Annealin

PCR

Accession

on size

g

e fficien

No.

(bp)

temperat

cy

ure (℃) Dio1

GGT GGT CTACATTGCCGAGG

CAT CCACTACGATGGGGCAC

149

56

2.11

Dio2

AT TTTGTATGCCGAT GCACATG

TACGGCGCTAACCT CTGTTT

200

56

1.85

XM_0216033 51.1 NM_0011242 68.1

T GGT CAT GGCAAACCCTACT

194

fep15

T GCT GGACT CTCTGGGT TTC

T T GTGCT CACAACTCGTCCC

107

GPX1

AT AAACCGT CCAGGCAGAGC

AGCCGT T GGT ACT GGT GAAG

162

ACT AACCCGT CCCAGT GTTG

GACAT T TTCCAAGGT GTGCCC

GCGGGGAGT GACAT T T ACCA

GT AAT CCCTGGT GGT CGTGC

AAGCT GT GGACT CGT TCTTGT

56

PT

AGGCCTAT CCCAAAGGT TGC

1.94

XM_0215747 97.1

56

1.87

RI

Dio3

56

1.99

HE687021.1

100

56

2.03

HE687022.1

163

56

1.87

HE687023.1

AGT T TACCGGGGT TTTCCCTC

200

56

1.96

HE687024.1

AGAAAT ACAGGGGCGACGT T

GCAT CT CCGCAAACT GAGAG

90

56

2.01

HE687025.1

ACGCT GACAAAGGT T TACGC

AGT ACCCT TTCCCTTGGGCT

200

56

1.94

HE687026.1

SelF

T GCCAGCAGGAGGT CCATAT

ACACTATCCGT GTTCCACTTG

242

60

1.98

NM_0011244

SelH

AAGCT GACGAGT GTATGGGC

T T TTACTCCGGGGCT T CTGG

107

56

1.94

SelI

CACT CCCACCACGAGGAT T C

AT CAGCAGCCAAGGAACCAA

161

56

2.03

SelJa

CAGCT CAACGACCCAAACAG

CT CATTGT CAGGACCCCGT G

182

56

1.82

SelK

AT TTTTGT GTAGGCGT GGGT T

GAGGGGT CAGAAACAGGAAG

198

56

1.96

127

56

2.03

GPX1

SC

a

b1 GPX1 GPX4

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b2

GPX4

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a1 a2 GPX4 b

78.1

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AC C

G

GAGGAT CGACGCT GAAGACG

SelMl

GGT T T GCAGAATAGGT GAGC G

SelN

ED

AG

SelL

T GCAACAGAGT AGCGGAT AG

54.3

CCCGAAT GCGT GT T CCCTAT

XM_0216113 60.1 NM_0011935 04.2 XM_0216156 79.1 NM_0011934 56.1

CACCT T CGCTTTTGCGAGAC

182

56

1.88

XM_0216216 17.1

T GCT CTCGCCCCT ACACAAA

139

56

1.94

T SelOl

XM_0215839 86.1

C CAGCT CAGT GT CTATGCGGT

NM_0011957

XM_0215747 46.1

ACCCACCCAAGT AAAGCGAG

182

56

1.89

XM_0215743 33.1

SelP

a

CACCGGAAGGGAGACAACT C

AGCCCCT CAGT GT CATGTTG

186

56

1.94

XM_0216065 22.1 XM_0216065 23.1

MsrB1 Al

CCAAAGCAAGCACAAGT C

ACCT CACCACCGAAGAAG

155

58

2.03

XM_0215586 59.1

ACCEPTED MANUSCRIPT SelSl

CT CGT GT GTGACTGGT GT CTT

GCT ACT TTTCCATGCTCCGT G

129

56

1.93

SelT1l

CAACCGCAACAGCCAT T TTC

T T AAAGACCTGT GATCTGGAA

188

56

1.99

SelT2l

CCAGT T TGTAGCCGCT TGC

GCCGT CAAGAT ACCCGT TTG

156

56

2.05

SelU

T ACT GTCCTGTCTTCGAGGT G

CCT T GGT CAGACACAGGT CA

121

56

2.02

XM_0215867 93.1

GGG

XM_0215897 58.1 XM_0215835 61.1 NM_0011935

SelWl

GGGT CGCT CCGCCT T ATTTA

GAACT T GGGCCT GT ACCCTC

174

56

1.99

TrxR3

AGT CAACCCCAAGAACGGT A

CAGAAGAGACT GT GGT ACAC

297

56

2.05

HF969246.1

a

AGG

CT CCAA

TrxR3

CAAAGT CAACCCCAAGAATG

CAGAAGAGACT GT GGT ACAC

56

1.82

HF969247.1

b

GTAAGA

CT CCAG

GAPD

T T CACCACTACAACCCAATCA

CACAAT CAGCTTCCCGT CC

2.08

NM_0011242

H

AC

RI

46.1 XM_0215913

β-actin

GAT GGGCCAGAAAGACAGCT

105

1.99

ACCCGAGGGACAT CCT GT G

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T CCT CTTGGT CGTTTCGCTG

234

SC

T CGT CCCAGT TGGT GACGAT

A EF1α

300

159

PT

17.1

56 56

46.1 NM_0011242 35.1

56

1.94

NM_0011243 39.1

Dio, iodothyronine deiodinase; fep15, fish 15 kDa selenoprotein-like; GPX, glutathione peroxidase;

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Sel, selenoprotein; SelF, 15 kDa selenoprotein; SelMl, selenoprotein M-like; SelOl, selenoprotein O-like; MsrB1Al, methionine sulfoxide reductase B 1A-like; SelSl, selenoprotein S-like, SelT1l, selenoprotein T1-like; SelT2l, selenoprotein T2-like; SelWl, selenoprotein W-like; TrxR, thioredoxin

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reductase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EF1α, eukaryotic translation elongation factor 1 alpha; β-actin, actin beta. a

mRNA level of SelP was the sum of SelP transcript variant X1(XM_021606522.1) mRNA level and

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transcript variant X2(XM_021606523.1) mRNA level.

ACCEPTED MANUSCRIPT Table 3 Growth performance, nutrient utilization and somatic parameter of rainbow trout fed experimental diets for 10 weeks. Se supplementation (g/kg diet) 2

4

6

Initial weight (g)

144.14 ± 1.21

145.43 ± 0.63

144.15 ± 0.77

144.05 ± 0.76

Final weight (g)

252.49 ± 6.13a

286.50 ± 3.07b

291.47 ± 3.82b

289.43 ± 4.64b

WGR (%)

75.20 ± 5.38a

94.82 ± 2.54b

102.18 ± 1.79b

100.90 ± 2.46b

FI (% day -1 )

1.12 ± 0.04a

1.18 ±0.03ab

1.19 ± 0.01ab

1.26 ± 0.01b

FE

0.66 ± 0.03a

0.73 ± 0.02b

0.79 ± 0.03b

0.75 ± 0.01b

CF (g/cm3 )

1.75 ± 0.06a

1.82 ± 0.06ab

1.89 ± 0.10b

1.84 ± 0.08ab

HSI (%)

1.25 ± 0.05

1.27 ± 0.04

1.33 ± 0.12

1.28 ± 0.09

VSI (%)

12.29 ± 1.03

12.87 ± 2.06

12.70 ± 1.67

12.70 ± 2.16

SR (%)

100

100

100

100

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Parameters

WGR, weight gain rate; FI, feed intake; FE, Feed conversion; CF, condition factor; HIS,

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hepatosomatic index; VSI, viscerasomatic index; SR, survival rate. Values are represented as mean ± SEM (n = 3 tanks). In each line, different superscript letters indicate

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significant differences between treatments (P < 0.05).

ACCEPTED MANUSCRIPT Table 4 MDA content and activities of GPX, CAT and SOD in serum, liver and muscle of rainbow trout fed experimental diets for 10 weeks. Se supplementation (g/kg diet) Parameters

0

2

4

6

GPX activity (mU/ml)

267.88 ± 9.88

285.68 ± 6.97

251.79 ± 15.88 279.92 ± 27.07

CAT activity (U/ml)

625.83 ± 97.35

435.72 ± 29.19 546.88 ± 93.22 418.11 ± 101.57

SOD activity (U/ml)

20.55 ± 0.63

20.39 ± 0.25

20.67 ± 0.73

19.15 ± 0.18

MDA (nmol/ml)

14.40 ± 1.20

15.28 ± 1.82

19.96 ± 2.12

17.74 ± 0.34

GPX activity (mU/mg protein)

21.15 ± 2.03

21.34 ± 3.49

CAT activity (U/mg protein)

391.21 ± 21.54

354.69 ± 14.71 320.65 ± 11.59 343.27 ± 10.26

SOD activity (U/mg protein)

5.14 ± 0.34

4.64 ± 0.24

3.89 ± 0.21

4.29 ±0.18

MDA (nmol/mg protein)

0.95 ± 0.06

0.75 ± 0.11

1.25 ± 0.12

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22.93 ± 2.49

24.47 ± 1.90

1.19 ± 0.18

GPX activity (mU/mg protein)

23.68 ± 1.86a

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Serum

27.39 ± 3.17a

39.83 ± 2.37b

54.60 ± 3.93c

CAT activity (U/mg protein)

81.27 ± 10.13a

97.10 ± 8.60a

82.15 ± 7.99a

33.86 ± 6.01b

SOD activity (U/mg protein)

8.53 ± 0.09a

8.62 ± 0.18a

7.62 ± 0.50ab

5.68 ± 0.08b

MDA (nmol/mg protein)

1.33 ± 0.03a

1.00 ± 0.09ab

0.83 ± 0.02c

0.97 ± 0.03bc

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GPX, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; MDA, malondialdehyde.

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Values are represented as mean ± SEM (n = 9 fish per treatment). In each line, different superscript

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letters indicate significant differences between treatments (P < 0.05).

ACCEPTED MANUSCRIPT Table 5 Selenoprotein gene expressions in the liver and muscle of rainbow trout fed experimental diets for 10 weeks. Liver

0 mg/kg 2 mg/kg 4 mg/kg 6 mg/kg Se Se Se Se

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± 1.27 0.08 ± 0.90 0.17 ± 1.36 0.32 ± 0.84 0.04 ± 1.99 0.22 b ± 0.81 0.14 ± 3.12 0.40 b ± 1.30 0.16 ± 1.47 0.19 ± 1.47 0.13 c ± 1.17 0.18 ± 1.04 0.07 ± 1.08 0.08 ± 1.14 0.14 ± 1.50 b 0.12 ± 1.13 0.21 ± 1.03 0.11 ± 1.01 0.10 ± 1.12 0.15 ± 1.72 0.14 b ± 2.04 0.33 b ± 1.60 0.24 ± 3.50 0.51 c

PT

±

1.06 0.08 1.69 0.65 1.30 0.26 1.07 0.14 1.56 0.17 ab 1.19 0.17 2.90 0.30 b 1.17 0.11 1.23 0.15 1.28 0.14 bc 1.09 0.12 1.10 0.07 1.25 0.16 1.00 0.25 1.17 a 0.08 1.46 0.25 0.95 0.09 0.96 0.10 1.03 0.11 1.41 0.14 b 1.83 0.34 ab 1.49 0.39 2.22 0.41 ab

RI

±

SC

1.13 0.16 1.35 0.40 1.05 0.22 1.05 0.11 1.03 0.08 a 1.08 0.13 1.09 0.15 a 1.03 0.10 1.12 0.22 1.04 0.10 a 1.05 0.11 1.04 0.11 1.06 0.14 1.07 0.15 1.01 a 0.05 1.11 0.17 1.05 0.12 1.03 0.08 1.01 0.06 1.01 0.06 a 1.06 0.11 a 1.02 0.17 1.11 0.12 a

NU

6 mg/kg Se 1.15 ± 0.18 1.03 ± 0.19 1.36 ± 0.44 1.42 ± 0.64 1.38 ± 0.10 b 1.74 ± 0.27 1.57 ± 0.16 b 1.02 ± 0.08 0.96 ± 0.14 0.95 ± 0.09 0.91 ± 0.18 0.79 ± 0.11 0.95 ± 0.12 1.57 ± 0.21 1.06 ± 0.07 ab 1.45 ± 0.08 0.94 ± 0.05 a 0.85 ± 0.07 0.96 ± 0.05 1.38 ± 0.13 b 1.37 ± 0.16 ab 1.19 ± 0.35 1.59 ± 0.12 ab

MA

4 mg/kg Se 1.21 ± 0.15 1.77 ± 0.22 0.89 ± 0.10 0.75 ± 0.12 1.66 ± 0.09 b 1.97 ± 0.68 1.71 ± 0.09 b 1.12 ± 0.05 2.89 ± 0.76 1.02 ± 0.08 1.16 ± 0.21 0.78 ± 0.06 0.82 ± 0.08 1.63 ± 0.15 1.21 ± 0.06 b 1.33 ± 0.13 1.31 ± 0.07 c 0.89 ± 0.06 0.96 ± 0.06 1.54 ± 0.08 b 1.60 ± 0.12 bc 1.45 ± 0.27 3.50 ± 0.51 c

Muscle

ED

2 mg/kg Se 1.30 ± 0.14 1.81 ± 0.28 0.72 ± 0.09 0.71 ± 0.07 1.45 ± 0.13 b 2.57 ± 0.53 1.41 ± 0.16 ab 1.01 ± 0.07 2.20 ± 0.37 1.07 ± 0.06 1.17 ± 0.11 0.83 ± 0.09 1.07 ± 0.10 1.33 ± 0.16 0.92 ± 0.05 a 1.26 ± 0.12 1.21 ± 0.02 bc 0.81 ± 0.06 0.94 ± 0.05 1.37 ± 0.11 b 1.86 ± 0.16 c 1.18 ± 0.28 2.22 ± 0.41 b

AC C

0 mg/kg Se Dio1 1.09 ± 0.15 Dio2 1.22 ± 0.11 Dio3 1.22 ± 0.42 fep15 1.05 ± 0.10 GPX1a 1.02 ± 0.07 a GPX1b1 1.53 ± 0.42 GPX1b2 1.08 ± 0.16 a GPX4a1 1.01 ± 0.05 GPX4a2 1.20 ± 0.24 GPX4b 1.03 ± 0.08 SelF 1.05 ± 0.11 SelH 1.05 ± 0.12 SelI 1.07 ± 0.14 SelJa 1.08 ± 0.14 SelK 1.01 ± 0.06 a SelL 1.07 ± 0.14 SelMl 1.02 ± 0.08 ab SelN 1.01 ± 0.05 SelOl 1.01 ± 0.04 SelP 1.01 ± 0.05 a MsrB1Al 1.05 ± 0.12 a SelSl 1.36 ± 0.31 SelT1l 1.11 ± 0.12 a

EP T

Gene name

± 1.18 0.06 ± 0.44 0.05 ± 1.84 0.33 ± 0.92 0.09 ± 2.29 0.44 b ± 1.18 0.13 ± 4.19 1.14 b ± 1.33 0.13 ± 1.22 0.27 ± 1.57 0.16 c ± 1.05 0.18 ± 1.12 0.06 ± 1.22 0.11 ± 1.08 0.09 ± 1.57 b 0.14 ± 1.31 0.32 ± 0.94 0.04 ± 0.96 0.05 ± 1.18 0.13 ± 1.59 0.15 b ± 2.37 0.27 b ± 1.27 0.25 ± 2.52 0.40 bc

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

ACCEPTED MANUSCRIPT 1.12 ± 1.16 ± 0.05 ab 0.08 b SelU 1.02 ± 1.36 ± 0.08a 0.06 b SelWl 1.04 ± 1.83 ± 0.05 a 0.12 c TrxR3a 1.06 ± 0.92 ± 0.12 0.16 TrxR3b 1.02 ± 0.93 ± 0.07 0.08 Dio, iodothyronine deiodinase;

0.98 ± 0.06 ab 1.58 ± 0.07 c 1.99 ± 0.12 c 0.63 ± 0.07 0.80 ± 0.06 fep15, fish

0.88 ± 1.01 ± 1.21 ± 1.30 ± 1.51 ± 0.07 a 0.05 a 0.08 ab 0.11 ab 0.14 b 1.21 ± 1.01 ± 1.26 ± 1.56 ± 1.57 ± 0.08 ab 0.05 a 0.14 ab 0.16 b 0.17 b 1.48 ± 1.27 ± 2.94 ± 4.20 ± 4.64 ± 0.10 b 0.07 a 0.46 b 0.34 c 0.43 c 0.81 ± 1.12 ± 1.15 ± 1.20 ± 1.41 ± 0.13 0.18 0.23 0.36 0.17 0.81 ± 1.07 ± 1.91 ± 2.07 ± 2.36 ± a ab b b 0.12 0.15 0.30 0.27 0.36 15 kDa selenoprotein-like; GPX, glutathione peroxidase;

PT

SelT2l

Sel, selenoprotein; SelF, 15 kDa selenoprotein; SelMl, selenoprotein M-like; SelOl, selenoprotein

RI

O-like; MsrB1Al, methionine sulfoxide reductase B 1A-like; SelSl, selenoprotein S-like, SelT1l,

SC

selenoprotein T1-like; SelT2l, selenoprotein T2-like; SelWl, selenoprotein W-like; TrxR, thioredoxin reductase.

NU

Values are represented as mean ± SEM (n = 9 fish per treatment). In each tissue, different superscript

AC C

EP T

ED

MA

letters in each line indicate significant differences among treatments (P < 0.05).

ACCEPTED MANUSCRIPT Table 6 Correlation between growth performance and the expressions of differentially expressed selenoprotein genes in the liver of rainbow trout fed experimental diets for 10 weeks. Genes

WGR P value

GPX1a

0.557

0.060

GPX1b2

0.479

0.115

SelK

0.092

0.776

SelMl

0.188

0.559

SelP

0.666

0.018

SelT1l

0.495

0.102

SelT2l

-0.097

SelU

0.578

SelWl

0.703

MsrB1Al

0.652

RI

PT

r

0.764

SC

0.049 0.011

NU

0.022

WGR, weight gain rate; r, Pearson correlation coefficient; GPX1a, glutathione peroxidase 1a; GPX1b2, glutathione peroxidase 1b2; SelK, selenoprotein K; SelMl, selenoprotein M-like; SelP,

MA

selenoprotein P; SelT1l, selenoprotein T1-like; SelT2l, selenoprotein T2-like; SelU, selenoprotein U;

AC C

EP T

ED

SelWl, selenoprotein W-like; MsrB1Al, methionine-R-sulphoxide reductase B 1A-like.

ACCEPTED MANUSCRIPT Table 7 Correlation between growth performance and the expressions of differentially expressed selenoprotein genes in the muscle of rainbow trout fed experimental diets for 10 weeks. WGR

Genes

P value

GPX1a

0.628

0.029

GPX1b2

0.687

0.014

GPX4b

0.672

0.017

SelK

0.696

0.012

SelP

0.685

0.014

SelT1l

0.628

0.029

SelT2l

0.608

SelU

0.589

SelWl

0.819

TrxR3b

0.684

MsrB1Al

0.670

RI

PT

r

0.036

SC

0.044 0.001

NU

0.014 0.017

WGR, weight gain rate; r, Pearson correlation coefficient; GPX1a, glutathione peroxidase 1a;

MA

GPX1b2, glutathione peroxidase 1b2; GPX4b, glutathione peroxidase 4b; SelK, selenoprotein K; SelP, selenoprotein P; SelT1l, selenoprotein T1-like; SelT2l, selenoprotein T2-like; SelU, selenoprotein U; SelWl, selenoprotein W-like; TrxR3b, thioredoxin reductase 3b; MsrB1Al, methionine-R-sulphoxide

AC C

EP T

ED

reductase B 1A-like.

ACCEPTED MANUSCRIPT Highlights: 

Response of selenoproteome to supra-nutritional dietary selenium-yeast has been systematically investigated in rainbow trout;



A total of 28 selenoprotein genes have been analyzed in trout liver and muscle;



Diet supplemented with selenium-yeast generally changed the expression of trout selenoprotein

Rainbow trout growth was highly correlated to the expression of muscle selenoprotein genes,

EP T

ED

MA

NU

SC

RI

especially selenoprotein W-like gene.

AC C



PT

genes;