Fish & Shellfish Immunology 46 (2015) 501e515
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Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella) Ling-Mei Wen a, Wei-Dan Jiang a, b, Yang Liu a, b, Pei Wu a, b, Juan Zhao a, Jun Jiang a, b, Sheng-Yao Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, *, Lin Feng a, b, c, ** a
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China d Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China e Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2014 Received in revised form 29 June 2015 Accepted 1 July 2015 Available online 6 July 2015
Our study explored the effect of dietary thiamin on growth and immunity (intestine, head kidney, spleen and liver) of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing six graded levels of thiamin at 0.12e2.04 mg/kg diet for 8 weeks. The percentage weight gain (PWG), feed intake and feed efficiency were lower in fish fed the 0.12 mg/kg diet. Thiamin deficiency decreased complement 3 content, lysozyme (LA) and acid phosphatase activities, mRNA levels of hepcidin and interleukin (IL) 10, elevated mRNA levels of interferon g2, tumor necrosis factor a, IL-1b and IL-8 in intestine, head kidney, spleen and liver. The mRNA levels of inhibitor protein-kBa, target of rapamycin (TOR) and NF-E2-related factor 2 (Nrf2), the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase were down-regulated, mRNA levels of myosin light-chain kinase (MLCK), IkB kinases (IKKb and IKKg), nuclear factor kB P65 (NF-kB P65) and Kelch-like-ECH-associated protein 1a (Keap1a) were up-regulated in the intestine of fish fed the thiamin-deficient diet. Additionally, thiamin deficiency decreased claudin b, c and 3, ZO-1 and occludin mRNA levels in each intestinal segment, increased claudin 12 and claudin 15a mRNA levels in distal intestine. In conclusion, thiamin deficiency decreased fish growth and immunity of intestine, head kidney, spleen and liver. The dietary thiamin requirement of young grass carp (242e742 g) based on intestinal LA activity or PWG were determined to be 1.15 or 0.90 mg/kg diet, respectively. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Thiamin deficiency Grass carp Immune response Tight junction Antioxidant capacity
1. Introduction The fish intestine is highly susceptible to pathogenic invasion [1]. To prevent pathogenic invasion, fish have developed intestinal mucosal immune system [2]. It had been demonstrated that disturbance the fish intestinal mucosal immune system resulted in an impaired immune response leading to poor growth performance
* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. ** Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. E-mail addresses: [email protected] (X.-Q. Zhou), [email protected] (L. Feng). http://dx.doi.org/10.1016/j.fsi.2015.07.001 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
[3] and pathogenic invasion [2]. Therefore, maintaining a wellfunctioning intestinal mucosal immune system is of vital importance for fish [2]. Limited study had shown that nutrients deficiency, such as tryptophan [4] and valine [3] deficiency impaired the intestinal mucosal immune of fish. Thiamin is an essential vitamin for fish [5]. To date, no study has paid attention to the effect of thiamin on fish intestinal mucosal immune system. Wellers et al. [6] demonstrated that dietary thiamin deficiency resulted in decreasing the absorption of valine in rat intestine. Moreover, study from our laboratory had been revealed that dietary valine limitation impaired the intestinal mucosal immunity of grass carp [3]. The above data indicating that there may be a possible correlation between thiamin and fish intestinal mucosal immune response, which is worthy of investigation.
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Fish intestinal mucosal immune system is broadly consisted of immune barrier and physical barrier, and the fish intestinal immune barrier mostly relies on its immune response [2]. Fish immune response is closely related to complement [7], lysozyme (LA) [8], acid phosphatase [9], antimicrobial peptides (such as hepcidin and liver expressed antimicrobial peptides 2) and cytokines including interferon g (IFN-g), tumor necrosis factor a (TNF-a), transforming growth factor-b (TGF-b), interleukin 1 (IL-1), interleukin 8 (IL-8) and interleukin 10 (IL-10) [10]. To date, studies had explored the effect of thiamin on fish growth [5], reproduction activity [11] and in vitro immune functions [12]. Additionally, in gold fish (Carassius auratus), study demonstrated that thiamin can prevent the hyperplasia of macrophages [13]. Moreover, macrophages can act to release LA [14] in fish. The above information indicating that thiamin may have effect on fish intestinal immune response, which is valuable for investigation. Furthermore, the expression of cytokines could be involved in nuclear factor kB (NFkB) and target of rapamycin (TOR) signaling pathway in activated mouse microglial cells [15]. Studies from our laboratory demonstrated that nutrients, such as valine [3] and tryptophan [4] regulated the mRNA levels of cytokines partly by affecting the expression of NF-kB and TOR in fish. However, no information has concerned about whether thiamin could modulate cytokines expression through NF-kB and TOR in fish. Langlais and Zhang [16] reported that thiamin deficiency can affect the glutamate level in rats thalamus. Moreover, glutamate can regulate the NF-kB expression level in the rat brain astrocytes [17] and the expression of mTOR in chick glia cells [18]. These observations lead to the idea that thiamin may modulate cytokines expression through NF-kB and TOR in fish, which need to be investigated. Except for the intestinal immune barrier, fish also developed physical barrier in the intestinal mucosal immune system, which is made up of intestinal epithelial cells and tight junctions (TJs) [2]. TJs is mainly composed of zonula occludens (ZO), the transmembrane proteins occludin and claudin in fish [19]. TJs play a vital role in preventing pathogen invasion in fish intestine [20]. However, there is no available information about the relationship between thiamin and TJs in fish. In rats brain, thiamin deficiency increased the endothelial nitric oxide synthase (eNOS) expression level [21]. Moreover, Beauchesne et al. [22] demonstrated that eNOS affected the expression of ZO-1 and occludin in the medial thalamus of mice. The above information suggesting that thiamin may have effect on TJs in fish, which warrants further investigation. Additionally, study had shown that the disruption of TJs is closely related to oxidative damage in human colonic Caco-2 cells [23]. Antioxidant system (including non-enzymatic compounds and antioxidant enzymes) play an important role in preventing oxidative damage [24]. The antioxidant enzymes activities are partly related to their mRNA levels in Japanese eel (Anguilla japonica) [25]. Studies revealed that NF-E2-related factor 2 (Nrf2) and its cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1) play a critical role in regulating antioxidant enzymes mRNA expression in vertebrate [26,27]. Our previous studies showed that nutrient, such as tryptophan [4] regulated the mRNA levels of antioxidant enzymes partly by affecting the expression of Nrf2 and Keap1 in fish. However, no reports at present have concerned about whether thiamin could affect the mRNA expression of antioxidant enzymes via Keap1/Nrf2 signaling pathway in animal. In human brain, thiamin can affect the formation of prostaglandins [28]. Furthermore, prostaglandins up-regulated the expression of Nrf2 in multiple myeloma cells [29]. These information indicating that thiamin may partly through modulating Keap1/Nrf2 signaling pathway to affect the antioxidant capacity of fish, which is valuable for investigation. In addition to intestine, fish head kidney, spleen and liver were
also important for its immune defense [30]. To date, there is no available information about the relationship between thiamin and fish head kidney, spleen and liver immunity. Previous study showed that optimal thiamin can improve the immunity of lake trout (Salvelinus namaycush) kidney leukocytes [12]. Moreover, Wu et al. [31] revealed that the effect of choline on tissue immune response of Jian carp was vary in each tissue. Accordingly, thiamin may have effect on the immunity of fish head kidney, spleen and liver, and the effect of thiamin on their immune response may vary with tissues. However, this hypothesis awaits further investigation. Grass carp is the third biggest contributor to the world's aquaculture production [32]. Nowadays, the culture of fish depends on formulated feed which relies on the nutrients requirement of this species [33]. To date, study only investigated the dietary thiamin requirement of juvenile grass carp [5], the dietary thiamin requirement of young grass carp has not been determined. Furthermore, the nutrients requirement of fish may vary with growth stages [3] and different sensitive indices [34]. Hence, it is valuable to investigate the dietary thiamin requirement of young grass carp based on immune index and growth performance. Thus, the present study was firstly conducted to evaluate the impact of dietary thiamin deficiency on young grass carp intestinal immunity.
2. Materials and methods 2.1. Experimental design and diets The composition of the basal diet is given in Table 1. Fish meal, casein and gelatin were used as the main dietary protein sources, which are found to be limiting in thiamin. The dietary protein level was fixed at 30% according to Khan et al. [33]. The experimental diets were supplemented with thiamin nitrate (Sigma, St Louis, MO, USA) to provide graded levels of thiamin. According to the method
Table 1 Nutrients composition of the basal diet. Ingredients
g kg1
Nutrients contenta
g kg1
Fish meal Casein Gelatin DL-Methionine (99%) a-Starch Corn starch Fish oil Soybean oil Cellulose Ca(H2PO4)2 Vitamin premix (thiamin free)b Trace mineral premixc Thiamin premixd Choline chloride (60%) Ethoxyquin (30%)
37.50 248.10 75.00 1.40 240.00 231.20 25.00 18.90 50.00 22.40 10.00 20.00 15.00 5.00 0.50
Crude protein Crude lipid n-3 n-6 Available phosphorus
293.20 45.40 10.00 10.00 6.00
a Crude protein and crude fat were measured value. Available phosphorus, n-3 and n-6 contents were calculated according to NRC (2011). b Per kilogram of thiamin-free vitamin premix (g/kg): retinyl acetate (500,000 IU/ g), 2.40 g; cholecalciferol (500,000 IU/g), 0.40 g; DL-a-tocopherol acetate (50%), 12.54 g; menadione (23%), 0.79 g; cyanocobalamin (1%), 0.81 g; D-biotin (2%), 4.91 g; folic acid (96%), 0.40 g; ascorbyl acetate (93%), 7.16 g; niacin (99%), 2.17 g; mesoinositol (99%), 19.19 g; calcium-D-pantothenate (98%), 2.43 g; riboflavin (80%), 0.55 g; pyridoxine hydrochloride (98%), 0.59 g. All ingredients were diluted with corn starch to 1 kg. c Per kilogram of trace mineral premix (g/kg): MnSO4$H2O, 1.65 g; MgSO4$H2O, 56.20 g; FeSO4$H2O, 22.90 g; ZnSO4$H2O, 0.63 g; CuSO4$5H2O, 0.02 g; KI, 0.07 g; NaSeO3, 0.004 g. All ingredients were diluted with corn starch to 1 kg. d Thiamin premix: thiamin nitrate was added to obtain graded levels of thiamin. The final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg thiamin/kg diet, respectively.
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described by Klejdus et al. [35], the final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg/kg diet. All ingredients were mixed, pelleted and stored at 20 C until use as described by Jiang et al. [5]. 2.2. Fish management and feeding All experimental procedures were approved by the Animal Care Advisory Committee of Sichuan Agricultural University. After a four-week adaption period as described by Lin et al. [36], grass carp were fed with the thiamin-deficient diet for 2 weeks to reduce the body storage of thiamin according to Huang et al. [37]. At the beginning of the experiment, a total of 540 young grass carp with an initial weight of 243.24 ± 1.27 g (average body length of 24.07 ± 1.86 cm) were randomly distributed into 18 experimental cages (1.4 1.4 1.4 m3) and resulted in 30 fish in each cage. Each cage was equipped with a 100 cm diameter disc in the bottom to collect the uneaten feed according to the method of Wu et al. [38]. During the experimental period, each cage was under natural light and dark cycle. Dissolved oxygen was maintained higher than 6.0 mg/L throughout the experimental period. The water temperature was averaged at 26 ± 2 C, pH value was maintained at 7.0 ± 0.5. Fish in each cage were fed with corresponding diet for four times daily to apparent satiation for 8 weeks. The uneaten feed was collected, dried and weighed to calculate the feed intake (FI) as described by Wu et al. [38].
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280 nm. Then, cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. Primers for each genes were designed according to the sequences of grass carp. The primer sequences and optimal annealing temperatures are shown in Table 2. The mRNA levels of all the genes were performed on the CFX96™ Real-Time PCR Detection System (Bio-Rad, Laboratories, Inc.) according to standard protocols of the primers. Primer amplification efficiencies were approximately 100%. According to the results of our preliminary experiment about the stability of internal control genes (data not shown), b-actin was selected to normalize the mRNA levels of all the genes. The 2DDCT method was used to calculate the mRNA levels of all the genes according to Livak and Schmittgen [49]. 2.5. Data analysis All results were presented as means ± standard deviation (SD). All data were subjected to one-way analysis of variance (ANOVA), and then followed by Duncan's multiple-range test to determine significant differences at the level of P < 0.05 among treatments using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Parameters with significant differences were subjected to a second-degree polynomial regression analysis with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). 3. Results
2.3. Sample collection and intestinal parameters assay
3.1. Growth performance
Fish from each cage were weighted at the beginning and the end of the feeding trial. 12 h after the last feeding, 18 fish were randomly selected from each treatment, anaesthetized in a benzocaine bath according to Luo et al. [3]. Then sacrificed, quickly obtained the intestine, head kidney, spleen and liver. Frozen in liquid nitrogen and then stored at 80 C until analysis as described by Huang et al. [39]. The intestine, head kidney, spleen and liver samples were homogenized on ice in 10 volumes (w v1) of ice-cold physiological saline and centrifuged at 6000 g at 4 C for 20 min, then the collected supernatant was stored as described by Safari et al. [40] for the related parameters analysis. The complement 3 (C3) content and LA activity were measured according to Chi et al. [7] and Liu et al. [41], respectively. Acid phosphatase activity was determined as described by Chi et al. [9]. The reactive oxygen species (ROS) production and malondialdehyde (MDA) content were determined according to Ko et al. [42] and Yang et al. [43], respectively. Protein carbonyl (PC) and reduced glutathione (GSH) contents, catalase (CAT) activity, anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were measured as described by Kuang et al. [44]. The total superoxide dismutase (SOD), CuZnSOD and MnSOD activities were determined as described by Lu et al. [45]. The activity of glutathione peroxidase (GPx) was analyzed according to Nugroho and Fotedar [46]. Glutathione-Stransferase (GST) and glutathione reductase (GR) activities were determined as described by Gorbi et al. [24]. The lactate and adenosine triphosphate (ATP) contents were measured by the method described by Zhou et al. [47] and Wang et al. [48], respectively.
As shown in Table 3, final body weight (FBW), percentage weight gain (PWG), feed intake (FI), feed efficiency (FE) and specific growth rate (SGR) were improved (722.11, 197.02, 605.65, 0.79 and 1.94, respectively) with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued (P > 0.05). Furthermore, using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on PWG was estimated to be 0.90 mg/kg diet (Fig. 1).
2.4. Real-time quantitative PCR The total RNA of intestine, head kidney, spleen and liver were extracted using RNAiso Plus Kit (Takara, Dalian, China) according to the manufacturer's instructions followed by DNase I treatment. The quantity and quality of RNA were assessed by agarose gel electrophoresis at 1% and by spectrophotometric analysis at 260 and
3.2. The immune response related parameters in intestine The C3 contents (Table 4) in proximal intestine (PI), midintestine (MI) and distal intestine (DI) were enhanced and IFN-g2 (Fig. 4A), TNF-a (Fig. 4B), IL-1b (Fig. 4C), NF-kB P65 (Fig. 5A) and IkB kinases b (IKKb) (Fig. 5D) mRNA levels were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. The LA activities and hepcidin (Fig. 3A) mRNA levels in PI, MI and DI were elevated with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. The acid phosphatase activities in PI, MI and DI were increased (56.28, 70.33 and 89.56, respectively) with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. Using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on LA activity in DI is 1.15 mg/ kg diet (Fig. 2). The liver expressed antimicrobial peptides 2 (LEAP2) (Fig. 3B), TGF-b1 (Fig. 4E) and TOR (Fig. 5F) mRNA levels in PI, MI and DI were enhanced with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of IL-8 (Fig. 4D) and IKKg (Fig. 5E) were decreased, IL-10 (Fig. 4F) and inhibitor protein-kBa (IkBa) (Fig. 5B) mRNA levels were increased with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter. Thiamin had no significant effect on IKKa (Fig. 5C) mRNA levels in PI, MI and DI of fish (P > 0.05). The lactate contents (Fig. 6A) in PI, MI and DI were decreased with dietary thiamin levels up to 1.25, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ATP
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Table 2 Primer sequences, thermocycling conditions and accession numbers of genes selected for analyzing by real-time PCR. Gene Hepcidin Forward Reverse LEAP-2 Forward Reverse IFN-g2 Forward Reverse TNF-a Forward Reverse IL-1b Forward Reverse IL-8 Forward Reverse TGF-b1 Forward Reverse IL-10 Forward Reverse NF-kB P65 Forward Reverse IkBa Forward Reverse IKKa Forward Reverse IKKb Forward Reverse IKKg Forward Reverse TOR Forward Reverse Claudin b Forward Reverse Claudin c Forward Reverse Claudin 3 Forward Reverse Claudin 12 Forward Reverse Claudin 15a Forward Reverse ZO-1 Forward Reverse Occludin Forward Reverse MLCK Forward Reverse CuZnSOD Forward Reverse MnSOD Forward Reverse CAT Forward Reverse
Sequences of primers
Thermocycling conditions
Accession number
50 -AGCAGGAGCAGGATGAGC-30 50 -GCCAGGGGATTTGTTTGT-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.3 C 30 s and 72 C 30 s
JQ246442
50 -TGCCTACTGCCAGAACCA-30 50 -AATCGGTTGGCTGTAGGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.3 C 30 s and 72 C 30 s
FJ390415
50 -TGTTTGATGACTTTGGGATG-30 50 -TCAGGACCCGCAGGAAGAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.4 C 30 s and 72 C 30 s
FJ766439
50 -CGCTGCTGTCTGCTTCAC-30 50 -CCTGGTCCTGGTTCACTC-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
HQ696609
50 -AGAGTTTGGTGAAGAAGAGG-30 50 -TTATTGTGGTTACGCTGGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.1 C 30 s and 72 C 30 s
JQ692172
50 -ATGAGTCTTAGAGGTCTGGGT-30 50 -ACAGTGAGGGCTAGGAGGG-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
JN663841
50 -TTGGGACTTGTGCTCTAT-30 50 -AGTTCTGCTGGGATGTTT-30
95 C 30 s, 40 cycles of 95 C 5 s, 55.9 C 30 s and 72 C 30 s
EU099588
50 -AATCCCTTTGATTTTGCC-30 50 -GTGCCTTATCCTACAGTATGTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
HQ388294
50 -GAAGAAGGATGTGGGAGATG-30 50 -TGTTGTCGTAGATGGGCTGAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.3 C 30 s and 72 C 30 s
KJ526214
50 -TCTTGCCATTATTCACGAGG-30 50 -TGTTACCACAGTCATCCACCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.3 C 30 s and 72 C 30 s
KJ125069
50 -GGCTACGCCAAAGACCTG-30 50 -CGGACCTCGCCATTCATA-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
KM279718
50 - GTGGCGGTGGATTATTGG-30 50 - GCACGGGTTGCCAGTTTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
KP125491
50 -AGAGGCTCGTCATAGTGG-30 50 -CTGTGATTGGCTTGCTTT-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
KM079079
50 -TCCCACTTTCCACCAACT-30 50 -ACACCTCCACCTTCTCCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
JX854449
50 -GAGGGAATCTGGATGAGC-30 50 -ATGGCAATGATGGTGAGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.0 C 30 s and 72 C 30 s
KF193860
50 -GAGGGAATCTGGATGAGC-30 50 -CTGTTATGAAAGCGGCAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KF193859
50 -ATCACTCGGGACTTCTA-30 50 -CAGCAAACCCAATGTAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.0 C 30 s and 72 C 30 s
KF193858
50 -CCCTGAAGTGCCCACAA-30 50 -GCGTATGTCACGGGAGAA-30
95 C 30 s, 40 cycles of 95 C 5 s, 55.4 C 30 s and 72 C 30 s
KF998571
50 -TGCTTTATTTCTTGGCTTTC-30 50 -CTCGTACAGGGTTGAGGTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.0 C 30 s and 72 C 30 s
KF193857
50 -CGGTGTCTTCGTAGTCGG-30 50 -CAGTTGGTTTGGGTTTCAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KJ000055
50 -TATCTGTATCACTACTGCGTCG-30 50 -CATTCACCCAATCCTCCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KF193855
50 -GAAGGTCAGGGCATCTCA-30 50 -GGGTCGGGCTTATCTACT-30
95 C 30 s, 40 cycles of 95 C 5 s, 53.0 C 30 s and 72 C 30 s
KM279719
50 -CGCACTTCAACCCTTACA-30 50 -ACTTTCCTCATTGCCTCC-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.5 C 30 s and 72 C 30 s
GU901214
50 -ACGACCCAAGTCTCCCTA-30 50 -ACCCTGTGGTTCTCCTCC-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.4 C 30 s and 72 C 30 s
GU218534
50 -GAAGTTCTACACCGATGAGG-30 50 -CCAGAAATCCCAAACCAT-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.7 C 30 s and 72 C 30 s
FJ560431
L.-M. Wen et al. / Fish & Shellfish Immunology 46 (2015) 501e515
505
Table 2 (continued ) Gene GPx Forward Reverse GST Forward Reverse GR Forward Reverse Nrf2 Forward Reverse Keap1a Forward Reverse Keap1b Forward Reverse b-Actin Forward Reverse
Sequences of primers
Thermocycling conditions
Accession number
50 -GGGCTGGTTATTCTGGGC-30 50 -AGGCGATGTCATTCCTGTTC-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.5 C 30 s and 72 C 30 s
EU828796
50 -TCTCAAGGAACCCGTCTG-30 50 -CCAAGTATCCGTCCCACA-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
EU107283
50 -GTGTCCAACTTCTCCTGTG-30 50 -ACTCTGGGGTCCAAAACG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
JX854448
50 -CTGGACGAGGAGACTGGA-30 50 -ATCTGTGGTAGGTGGAAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.5 C 30 s and 72 C 30 s
KF733814
50 -TTCCACGCCCTCCTCAA-30 50 -TGTACCCTCCCGCTATG-30
95 C 30 s, 40 cycles of 95 C 5 s, 63.0 C 30 s and 72 C 30 s
KF811013
50 -TCTGCTGTATGCGGTGGGC-30 50 -CTCCTCCATTCATCTTTCTCG-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.9 C 30 s and 72 C 30 s
KJ729125
50 -GGCTGTGCTGTCCCTGTA-30 50 -GGGCATAACCCTCGTAGAT-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
M25013
Table 3 Treatment effects on growth performance factors of young grass carp. All data are expressed as mean ± SD (n ¼ 3). Mean values within the same row with different superscripts are significantly different (P < 0.05). IBW: initial body weight (g fish1); FBW: final body weight (g fish1); PWG: percent weight gain (%); FI: feed intake (g fish1); FE: feed efficiency; SGR: specific growth rate (% day1). PWG ¼ 100% [FBW IBW]/IBW), FE ¼ [FBW IBW]/FI, SGR ¼ 100% [ln FBW ln IBW]/days. Dietary thiamin levels (mg kg1 diet) 0.12 IBW FBW PWG FI FE SGR
243.11 575.00 136.50 459.83 0.72 1.54
0.43 ± ± ± ± ± ±
1.84 12.99a 3.58a 1.63a 0.03a 0.03a
243.78 621.67 155.03 501.41 0.75 1.67
0.83 ± ± ± ± ± ±
1.54 4.48b 3.20b 1.15b 0.01ab 0.02b
243.11 722.11 197.02 605.65 0.79 1.94
1.25 ± ± ± ± ± ±
1.02 11.97c 3.83c 1.35c 0.02bc 0.02c
242.44 741.67 205.93 604.85 0.83 2.00
1.62 ± ± ± ± ± ±
0.96 23.69c 10.38c 1.35c 0.04c 0.06c
243.56 733.78 201.30 604.98 0.81 1.97
2.04 ± ± ± ± ± ±
1.64 12.28c 6.31c 1.03c 0.02c 0.04c
243.44 725.56 198.04 603.62 0.80 1.95
± ± ± ± ± ±
1.26 4.29c 0.52c 1.57c 0.01c 0.03c
Regressions YPWG ¼ 38.440x2 þ 115.463x þ 120.434 YFI ¼ 81.937x2 þ 250.262x þ 427.169 YFE ¼ 0.055x2 þ 0.161x þ 0.700 YSGR ¼ 0.255x2 þ 0.765x þ 1.435
contents (Fig. 6B) were increased with dietary thiamin levels up to 1.25, 0.83 and 1.25 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter.
R2 R2 R2 R2
¼ ¼ ¼ ¼
0.935 0.937 0.761 0.943
P P P P
< < <
0.05), whereas the mRNA levels of these genes in DI were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The occludin (Fig. 7G) mRNA levels in PI, MI and DI were improved with dietary thiamin levels up to 0.43, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued.
3.4. Antioxidant-related parameters in intestine Fig. 1. Broken-line regression analysis of percentage weight gain (PWG) of young grass carp fed graded levels of thiamin.
As shown in Fig. 8 and Table 5, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents in
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Table 4 Treatment effects on complement 3 content (C3), lysozyme activity (LA) and acid phosphatase activity in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). C3: mg/g protein; LA: U/mg protein; acid phosphatase: U/mg protein. Dietary thiamin levels (mg kg1 diet) 0.12 C3 PI 3.75 MI 3.29 DI 4.78 LA PI 22.04 MI 25.03 DI 19.44 Acid phosphatase PI 46.37 MI 49.02 DI 72.59
0.43
0.83
1.25
1.62 4.33 ± 0.20b 4.32 ± 0.16c 6.11 ± 0.44bc
2.04
± 0.15a ± 0.20a ± 0.39a
3.82 ± 0.13a 3.65 ± 0.21b 5.75 ± 0.20b
4.32 ± 0.21b 4.19 ± 0.31c 6.42 ± 0.26c
4.37 ± 0.30b 4.33 ± 0.13c 6.21 ± 0.27c
4.32 ± 0.29b 4.29 ± 0.20c 6.18 ± 0.30c
± 1.95a ± 1.99a ± 1.32a
27.03 ± 2.43b 28.52 ± 1.58b 33.53 ± 3.14b
31.77 ± 1.96c 30.09 ± 2.76bc 36.82 ± 3.57b
30.66 ± 2.45c 31.34 ± 2.40bc 45.58 ± 4.06c
32.85 ± 2.95c 31.74 ± 2.88c 43.75 ± 3.87c
30.11 ± 2.41c 30.42 ± 2.54bc 43.40 ± 3.90c
± 1.91a ± 2.76a ± 3.88a
51.06 ± 2.69b 56.64 ± 3.06b 78.76 ± 6.41b
56.28 ± 3.01c 57.11 ± 2.63b 89.56 ± 6.60c
55.77 ± 4.06c 70.33 ± 2.70c 90.26 ± 4.73c
58.60 ± 2.39c 70.08 ± 4.57c 89.62 ± 4.78c
58.19 ± 5.15c 69.02 ± 4.32c 91.60 ± 3.49c
Regressions YC3 in MI ¼ 0.545x2 þ 1.687x þ 3.082 YLA in DI ¼ 11.635x2 þ 36.452x þ 16.784 Yacid phosphatase in MI ¼ 6.350x2 þ 24.841x þ 45.768
R2 ¼ 0.800 R2 ¼ 0.849 R2 ¼ 0.788
P < 0.01 P < 0.01 P < 0.01
thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. CuZnSOD (Fig. 9A) and Nrf2 (Fig. 10A) mRNA levels were increased with dietary thiamin levels up to 0.43 mg/kg diet in fish intestine (P < 0.05), and plateaued thereafter. The mRNA level of CAT (Fig. 9A) was the maximum in fish fed the 1.25 mg/kg diet (P < 0.05). The MnSOD (Fig. 9A), GPx, GST and GR (Fig. 9B) mRNA levels were improved and Keap1a (Fig. 10B) mRNA level was decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. However, thiamin had no significant effect on Keap1b (Fig. 10B) mRNA level in fish intestine (P > 0.05). 3.5. Immune response-related parameters in head kidney, spleen and liver Fig. 2. Broken-line regression analysis of lysozyme (LA) activity in the distal intestine of young grass carp fed graded levels of thiamin.
intestine were decreased with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ASA capacity, GSH content, MnSOD and CAT activities in intestine were increased with dietary thiamin levels up to 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The AHR capacity, CuZnSOD, GPx, GST and GR activities in intestine were increased with dietary
As shown in Table 6, the C3 contents in head kidney, spleen and liver were enhanced with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. LA activities in head kidney, spleen and liver were improved with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. Acid phosphatase activities were increased in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 1.25 and 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The mRNA levels of hepcidin (Fig. 11A) in head kidney, spleen and liver were up-regulated with dietary thiamin levels up to 0.43, 0.83 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The LEAP-2 (Fig. 11B) mRNA levels in
Fig. 3. Effect of dietary thiamin on hepcidin (A) and liver-expressed antimicrobial peptide 2 (LEAP-2) (B) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
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Fig. 4. Effect of dietary thiamin on IFN-g2 (A), TNF-a (B), IL-1b (C), IL-8 (D), TGF-b1 (E) and IL-10 (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). IFN-g2: interferon g2; TNF-a: tumor necrosis factor a; IL-1b: interleukin 1b; IL-8: interleukin 8; TGF-b1: transforming growth factor b1; IL-10: interleukin 10.
spleen and liver were up-regulated with dietary thiamin levels up to 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of LEAP-2 in head kidney (P > 0.05). The mRNA levels of IFN-g2 (Fig. 12A) in head kidney, spleen and liver were down-regulated with dietary thiamin levels up to 0.83, 0.43 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of TNF-a (Fig. 12B) and IL-8 (Fig. 12D) in head kidney, spleen and liver were downregulated with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The IL-1b (Fig. 12C) mRNA levels were decreased and IL-10 (Fig. 12F) mRNA levels were elevated in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The TGF-b1 (Fig. 12E) mRNA levels in head kidney and spleen were up-regulated with dietary thiamin levels up to 0.83 and 0.43 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of TGF-b1 in liver (P > 0.05) (Table 7). 4. Discussion Our study firstly revealed that dietary thiamin deficiency resulted in poor PWG, FI and FE of young grass carp, whereas the
PWG, FI and FE were increased with the supplementation of thiamin. Study indicated that the improvement of fish growth performance was due to the elevated FI and FE [3]. Correlation analysis showed that PWG was positively correlated with FI (r ¼ þ0.994, P < 0.01) and FE (r ¼ þ0.974, P < 0.01), indicating that the improvement of young grass carp growth may be partly due to the fact that appropriate dietary thiamin level elevated the FI and FE. In vertebrate, growth performance mostly depends on intestinal health status, which is closely associated with the intestinal mucosal immune [3]. Thus, we further investigated the effect of thiamin on intestinal immunity of young grass carp. In the current study, compared with optimal thiamin level, thiamin deficiency led to decrease C3 content, LA and acid phosphatase activities, mRNA levels of hepcidin, LEAP-2, TGF-b1 and IL10, and increase the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 in young grass carp intestine, suggesting that dietary thiamindeficient impaired the intestinal immune response of fish. Moreover, thiamin-regulated the mRNA levels of cytokines may be associated with lactate. In activated human macrophages, lactate induced up-regulation of IL-1b and IL-8 mRNA levels [50] and down-regulation of IL-10 mRNA level in mouse macrophage-like cells [51]. In our study, the lactate content was higher in PI, MI and DI of fish fed the thiamin limitation diet. Correlation analysis
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Fig. 5. Effect of dietary thiamin on NF-kB P65 (A), IkBa (B), IKKa (C), IKKb (D), IKKg (E) and TOR (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). NF-kB P65: nuclear factor kB P65; IkBa: inhibitor protein-kBa; IKKa: IkB kinases a; IKKb: IkB kinases b; IKKg: IkB kinases g; TOR: target of rapamycin.
Fig. 6. Effect of dietary thiamin on lactate content (A) and adenosine triphosphate (ATP) content (B) in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
revealed that the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 were positively correlated with lactate content, and TGF-b1 and IL-10 mRNA levels were negatively correlated with lactate content in PI, MI and DI of young grass carp, suggesting that thiamin limitation may elevate lactate production to affect pro-inflammatory
cytokines and anti-inflammatory cytokines expression in fish. The expression of pro-inflammatory cytokines is related to NFkB signaling molecule in activated mouse microglial cells [15]. In the current study, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 in PI, MI and DI of young grass carp, whereas NF-kB
L.-M. Wen et al. / Fish & Shellfish Immunology 46 (2015) 501e515
509
Fig. 7. Effect of dietary thiamin on claudin b (A), claudin c (B), claudin 3 (C), claudin 12 (D), claudin 15a (E), ZO-1 (F), occludin (G) and myosin light-chain kinase (MLCK) (H) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
P65 mRNA levels were decreased with the supplementation of thiamin. Correlation analysis revealed that the mRNA levels of IFNg2, TNF-a, IL-1b and IL-8 were positively correlated with NF-kB P65 mRNA levels in each intestinal segment of young grass carp, suggesting that thiamin deficiency elevated the mRNA levels of proinflammatory cytokines may be partly related to increase the mRNA expression of NF-kB P65 in fish. Furthermore, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 may be partly involved in its upstream signaling molecules, such as TOR. Zhong et al. [15] reported that up-regulation of TOR suppressed the
mRNA expression of NF-kB P65 in activated mouse microglial cells. In the present study, thiamin deficiency decreased the mRNA levels of TOR in all intestinal segments, whereas the mRNA levels of TOR were elevated by optimal thiamin level. Correlation analysis demonstrated that the mRNA levels of NF-kB P65 were negatively correlated with TOR mRNA levels in young grass carp intestine, indicating that dietary thiamin limitation resulted in up-regulating the NF-kB P65 mRNA level partly by down-regulating the mRNA level of TOR in fish. Thiamin-affected the TOR mRNA level in fish may be partly related to adenosine triphosphate (ATP). Hu et al.
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Fig. 8. Effect of dietary thiamin on reactive oxygen species (ROS) production in young grass carp intestine. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
whereas the ATP contents were increased by optimal thiamin level. Correlation analysis indicated that the TOR mRNA levels were positively correlated with the ATP contents in young grass carp intestine, suggesting that thiamin limitation resulted in downregulation of TOR expression may be partly contributed to decrease the ATP content in fish. Besides, the IkBa-mediated nuclear translocation of NF-kB P65 also plays an important role in regulating pro-inflammatory cytokines mRNA expression. In human epithelial cells, down-regulation the expression of IkBa promoted NF-kB p65 nuclear translocation, thereby up-regulating the mRNA levels of TNF-a, IL-1b and IL-8 [53]. In the present study, thiamin limitation led to down-regulate the mRNA level of IkBa in all intestinal segments of young grass carp, indicating that dietary thiamin limitation may promote NF-kB P65 nuclear translocation to elevate the mRNA levels of pro-inflammatory cytokines by downregulating the IkBa mRNA level in fish. Additionally, thiamin deficiency down-regulated the IkBa mRNA expression may be partly
Table 5 Treatment effects on antioxidant-related parameters in young grass carp intestine. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). MDA (malondialdehyde) and PC (protein carbonyl): nmol/mg protein; ASA (anti-superoxide anion) and GR (glutathione reductase): U/g protein; AHR (anti-hydroxyl radical), CuZnSOD (copper/zinc superoxide dismutase), MnSOD (manganese superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase) and GST (glutathione-S-transferase): U/mg protein; GSH (glutathione): mg/g protein. Dietary thiamin levels (mg kg1 diet) 0.12 MDA PC ASA AHR GSH CuZnSOD MnSOD CAT GPx GST GR
6.82 7.01 141.79 120.43 4.50 4.32 2.03 0.90 99.56 42.66 34.14
0.43 ± ± ± ± ± ± ± ± ± ± ±
0.26d 0.39c 6.69a 8.53a 0.20a 0.12a 0.15a 0.05a 7.63a 1.59a 2.43a
5.83 5.35 148.36 132.11 5.40 4.83 2.12 1.02 108.40 45.49 36.67
0.83 ± ± ± ± ± ± ± ± ± ± ±
0.29c 0.33b 9.41ab 5.25b 0.51b 0.36b 0.17a 0.06b 4.25b 2.65ab 2.34ab
4.91 4.25 149.18 144.68 6.98 5.49 2.88 1.32 119.12 46.87 40.14
1.25 ± ± ± ± ± ± ± ± ± ± ±
0.25a 0.31a 6.95ab 3.48c 0.28c 0.18c 0.23b 0.07c 4.39c 1.01b 2.46c
5.15 4.17 152.07 144.54 7.93 5.58 3.38 1.47 118.32 47.77 37.95
1.62 ± ± ± ± ± ± ± ± ± ± ±
0.12ab 0.25a 7.04b 4.91c 0.53d 0.34c 0.28c 0.05d 8.17c 2.60b 2.22bc
5.14 4.36 152.55 143.32 7.43 5.26 3.48 1.48 115.74 48.59 38.26
2.04 ± ± ± ± ± ± ± ± ± ± ±
0.39ab 0.23a 9.00b 2.93c 0.68cd 0.13c 0.19c 0.04d 8.67bc 3.31b 3.31bc
5.27 4.56 152.83 143.16 7.87 5.32 3.40 1.51 116.45 48.21 38.53
± ± ± ± ± ± ± ± ± ± ±
0.13b 0.29a 5.40b 5.01c 0.20d 0.30c 0.26c 0.04d 8.34bc 3.11b 2.97bc
P P P P P P P
< < < < < <
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Full length article
Evaluation the effect of thiamin deficiency on intestinal immunity of young grass carp (Ctenopharyngodon idella) Ling-Mei Wen a, Wei-Dan Jiang a, b, Yang Liu a, b, Pei Wu a, b, Juan Zhao a, Jun Jiang a, b, Sheng-Yao Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, *, Lin Feng a, b, c, ** a
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China d Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China e Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 December 2014 Received in revised form 29 June 2015 Accepted 1 July 2015 Available online 6 July 2015
Our study explored the effect of dietary thiamin on growth and immunity (intestine, head kidney, spleen and liver) of young grass carp (Ctenopharyngodon idella). Fish were fed diets containing six graded levels of thiamin at 0.12e2.04 mg/kg diet for 8 weeks. The percentage weight gain (PWG), feed intake and feed efficiency were lower in fish fed the 0.12 mg/kg diet. Thiamin deficiency decreased complement 3 content, lysozyme (LA) and acid phosphatase activities, mRNA levels of hepcidin and interleukin (IL) 10, elevated mRNA levels of interferon g2, tumor necrosis factor a, IL-1b and IL-8 in intestine, head kidney, spleen and liver. The mRNA levels of inhibitor protein-kBa, target of rapamycin (TOR) and NF-E2-related factor 2 (Nrf2), the activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase were down-regulated, mRNA levels of myosin light-chain kinase (MLCK), IkB kinases (IKKb and IKKg), nuclear factor kB P65 (NF-kB P65) and Kelch-like-ECH-associated protein 1a (Keap1a) were up-regulated in the intestine of fish fed the thiamin-deficient diet. Additionally, thiamin deficiency decreased claudin b, c and 3, ZO-1 and occludin mRNA levels in each intestinal segment, increased claudin 12 and claudin 15a mRNA levels in distal intestine. In conclusion, thiamin deficiency decreased fish growth and immunity of intestine, head kidney, spleen and liver. The dietary thiamin requirement of young grass carp (242e742 g) based on intestinal LA activity or PWG were determined to be 1.15 or 0.90 mg/kg diet, respectively. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Thiamin deficiency Grass carp Immune response Tight junction Antioxidant capacity
1. Introduction The fish intestine is highly susceptible to pathogenic invasion [1]. To prevent pathogenic invasion, fish have developed intestinal mucosal immune system [2]. It had been demonstrated that disturbance the fish intestinal mucosal immune system resulted in an impaired immune response leading to poor growth performance
* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. ** Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. E-mail addresses: [email protected] (X.-Q. Zhou), [email protected] (L. Feng). http://dx.doi.org/10.1016/j.fsi.2015.07.001 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
[3] and pathogenic invasion [2]. Therefore, maintaining a wellfunctioning intestinal mucosal immune system is of vital importance for fish [2]. Limited study had shown that nutrients deficiency, such as tryptophan [4] and valine [3] deficiency impaired the intestinal mucosal immune of fish. Thiamin is an essential vitamin for fish [5]. To date, no study has paid attention to the effect of thiamin on fish intestinal mucosal immune system. Wellers et al. [6] demonstrated that dietary thiamin deficiency resulted in decreasing the absorption of valine in rat intestine. Moreover, study from our laboratory had been revealed that dietary valine limitation impaired the intestinal mucosal immunity of grass carp [3]. The above data indicating that there may be a possible correlation between thiamin and fish intestinal mucosal immune response, which is worthy of investigation.
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Fish intestinal mucosal immune system is broadly consisted of immune barrier and physical barrier, and the fish intestinal immune barrier mostly relies on its immune response [2]. Fish immune response is closely related to complement [7], lysozyme (LA) [8], acid phosphatase [9], antimicrobial peptides (such as hepcidin and liver expressed antimicrobial peptides 2) and cytokines including interferon g (IFN-g), tumor necrosis factor a (TNF-a), transforming growth factor-b (TGF-b), interleukin 1 (IL-1), interleukin 8 (IL-8) and interleukin 10 (IL-10) [10]. To date, studies had explored the effect of thiamin on fish growth [5], reproduction activity [11] and in vitro immune functions [12]. Additionally, in gold fish (Carassius auratus), study demonstrated that thiamin can prevent the hyperplasia of macrophages [13]. Moreover, macrophages can act to release LA [14] in fish. The above information indicating that thiamin may have effect on fish intestinal immune response, which is valuable for investigation. Furthermore, the expression of cytokines could be involved in nuclear factor kB (NFkB) and target of rapamycin (TOR) signaling pathway in activated mouse microglial cells [15]. Studies from our laboratory demonstrated that nutrients, such as valine [3] and tryptophan [4] regulated the mRNA levels of cytokines partly by affecting the expression of NF-kB and TOR in fish. However, no information has concerned about whether thiamin could modulate cytokines expression through NF-kB and TOR in fish. Langlais and Zhang [16] reported that thiamin deficiency can affect the glutamate level in rats thalamus. Moreover, glutamate can regulate the NF-kB expression level in the rat brain astrocytes [17] and the expression of mTOR in chick glia cells [18]. These observations lead to the idea that thiamin may modulate cytokines expression through NF-kB and TOR in fish, which need to be investigated. Except for the intestinal immune barrier, fish also developed physical barrier in the intestinal mucosal immune system, which is made up of intestinal epithelial cells and tight junctions (TJs) [2]. TJs is mainly composed of zonula occludens (ZO), the transmembrane proteins occludin and claudin in fish [19]. TJs play a vital role in preventing pathogen invasion in fish intestine [20]. However, there is no available information about the relationship between thiamin and TJs in fish. In rats brain, thiamin deficiency increased the endothelial nitric oxide synthase (eNOS) expression level [21]. Moreover, Beauchesne et al. [22] demonstrated that eNOS affected the expression of ZO-1 and occludin in the medial thalamus of mice. The above information suggesting that thiamin may have effect on TJs in fish, which warrants further investigation. Additionally, study had shown that the disruption of TJs is closely related to oxidative damage in human colonic Caco-2 cells [23]. Antioxidant system (including non-enzymatic compounds and antioxidant enzymes) play an important role in preventing oxidative damage [24]. The antioxidant enzymes activities are partly related to their mRNA levels in Japanese eel (Anguilla japonica) [25]. Studies revealed that NF-E2-related factor 2 (Nrf2) and its cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1) play a critical role in regulating antioxidant enzymes mRNA expression in vertebrate [26,27]. Our previous studies showed that nutrient, such as tryptophan [4] regulated the mRNA levels of antioxidant enzymes partly by affecting the expression of Nrf2 and Keap1 in fish. However, no reports at present have concerned about whether thiamin could affect the mRNA expression of antioxidant enzymes via Keap1/Nrf2 signaling pathway in animal. In human brain, thiamin can affect the formation of prostaglandins [28]. Furthermore, prostaglandins up-regulated the expression of Nrf2 in multiple myeloma cells [29]. These information indicating that thiamin may partly through modulating Keap1/Nrf2 signaling pathway to affect the antioxidant capacity of fish, which is valuable for investigation. In addition to intestine, fish head kidney, spleen and liver were
also important for its immune defense [30]. To date, there is no available information about the relationship between thiamin and fish head kidney, spleen and liver immunity. Previous study showed that optimal thiamin can improve the immunity of lake trout (Salvelinus namaycush) kidney leukocytes [12]. Moreover, Wu et al. [31] revealed that the effect of choline on tissue immune response of Jian carp was vary in each tissue. Accordingly, thiamin may have effect on the immunity of fish head kidney, spleen and liver, and the effect of thiamin on their immune response may vary with tissues. However, this hypothesis awaits further investigation. Grass carp is the third biggest contributor to the world's aquaculture production [32]. Nowadays, the culture of fish depends on formulated feed which relies on the nutrients requirement of this species [33]. To date, study only investigated the dietary thiamin requirement of juvenile grass carp [5], the dietary thiamin requirement of young grass carp has not been determined. Furthermore, the nutrients requirement of fish may vary with growth stages [3] and different sensitive indices [34]. Hence, it is valuable to investigate the dietary thiamin requirement of young grass carp based on immune index and growth performance. Thus, the present study was firstly conducted to evaluate the impact of dietary thiamin deficiency on young grass carp intestinal immunity.
2. Materials and methods 2.1. Experimental design and diets The composition of the basal diet is given in Table 1. Fish meal, casein and gelatin were used as the main dietary protein sources, which are found to be limiting in thiamin. The dietary protein level was fixed at 30% according to Khan et al. [33]. The experimental diets were supplemented with thiamin nitrate (Sigma, St Louis, MO, USA) to provide graded levels of thiamin. According to the method
Table 1 Nutrients composition of the basal diet. Ingredients
g kg1
Nutrients contenta
g kg1
Fish meal Casein Gelatin DL-Methionine (99%) a-Starch Corn starch Fish oil Soybean oil Cellulose Ca(H2PO4)2 Vitamin premix (thiamin free)b Trace mineral premixc Thiamin premixd Choline chloride (60%) Ethoxyquin (30%)
37.50 248.10 75.00 1.40 240.00 231.20 25.00 18.90 50.00 22.40 10.00 20.00 15.00 5.00 0.50
Crude protein Crude lipid n-3 n-6 Available phosphorus
293.20 45.40 10.00 10.00 6.00
a Crude protein and crude fat were measured value. Available phosphorus, n-3 and n-6 contents were calculated according to NRC (2011). b Per kilogram of thiamin-free vitamin premix (g/kg): retinyl acetate (500,000 IU/ g), 2.40 g; cholecalciferol (500,000 IU/g), 0.40 g; DL-a-tocopherol acetate (50%), 12.54 g; menadione (23%), 0.79 g; cyanocobalamin (1%), 0.81 g; D-biotin (2%), 4.91 g; folic acid (96%), 0.40 g; ascorbyl acetate (93%), 7.16 g; niacin (99%), 2.17 g; mesoinositol (99%), 19.19 g; calcium-D-pantothenate (98%), 2.43 g; riboflavin (80%), 0.55 g; pyridoxine hydrochloride (98%), 0.59 g. All ingredients were diluted with corn starch to 1 kg. c Per kilogram of trace mineral premix (g/kg): MnSO4$H2O, 1.65 g; MgSO4$H2O, 56.20 g; FeSO4$H2O, 22.90 g; ZnSO4$H2O, 0.63 g; CuSO4$5H2O, 0.02 g; KI, 0.07 g; NaSeO3, 0.004 g. All ingredients were diluted with corn starch to 1 kg. d Thiamin premix: thiamin nitrate was added to obtain graded levels of thiamin. The final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg thiamin/kg diet, respectively.
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described by Klejdus et al. [35], the final thiamin concentrations in each experimental diet were determined to be 0.12, 0.43, 0.83, 1.25, 1.62 and 2.04 mg/kg diet. All ingredients were mixed, pelleted and stored at 20 C until use as described by Jiang et al. [5]. 2.2. Fish management and feeding All experimental procedures were approved by the Animal Care Advisory Committee of Sichuan Agricultural University. After a four-week adaption period as described by Lin et al. [36], grass carp were fed with the thiamin-deficient diet for 2 weeks to reduce the body storage of thiamin according to Huang et al. [37]. At the beginning of the experiment, a total of 540 young grass carp with an initial weight of 243.24 ± 1.27 g (average body length of 24.07 ± 1.86 cm) were randomly distributed into 18 experimental cages (1.4 1.4 1.4 m3) and resulted in 30 fish in each cage. Each cage was equipped with a 100 cm diameter disc in the bottom to collect the uneaten feed according to the method of Wu et al. [38]. During the experimental period, each cage was under natural light and dark cycle. Dissolved oxygen was maintained higher than 6.0 mg/L throughout the experimental period. The water temperature was averaged at 26 ± 2 C, pH value was maintained at 7.0 ± 0.5. Fish in each cage were fed with corresponding diet for four times daily to apparent satiation for 8 weeks. The uneaten feed was collected, dried and weighed to calculate the feed intake (FI) as described by Wu et al. [38].
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280 nm. Then, cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions. Primers for each genes were designed according to the sequences of grass carp. The primer sequences and optimal annealing temperatures are shown in Table 2. The mRNA levels of all the genes were performed on the CFX96™ Real-Time PCR Detection System (Bio-Rad, Laboratories, Inc.) according to standard protocols of the primers. Primer amplification efficiencies were approximately 100%. According to the results of our preliminary experiment about the stability of internal control genes (data not shown), b-actin was selected to normalize the mRNA levels of all the genes. The 2DDCT method was used to calculate the mRNA levels of all the genes according to Livak and Schmittgen [49]. 2.5. Data analysis All results were presented as means ± standard deviation (SD). All data were subjected to one-way analysis of variance (ANOVA), and then followed by Duncan's multiple-range test to determine significant differences at the level of P < 0.05 among treatments using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Parameters with significant differences were subjected to a second-degree polynomial regression analysis with SPSS 13.0 (SPSS Inc., Chicago, IL, USA). 3. Results
2.3. Sample collection and intestinal parameters assay
3.1. Growth performance
Fish from each cage were weighted at the beginning and the end of the feeding trial. 12 h after the last feeding, 18 fish were randomly selected from each treatment, anaesthetized in a benzocaine bath according to Luo et al. [3]. Then sacrificed, quickly obtained the intestine, head kidney, spleen and liver. Frozen in liquid nitrogen and then stored at 80 C until analysis as described by Huang et al. [39]. The intestine, head kidney, spleen and liver samples were homogenized on ice in 10 volumes (w v1) of ice-cold physiological saline and centrifuged at 6000 g at 4 C for 20 min, then the collected supernatant was stored as described by Safari et al. [40] for the related parameters analysis. The complement 3 (C3) content and LA activity were measured according to Chi et al. [7] and Liu et al. [41], respectively. Acid phosphatase activity was determined as described by Chi et al. [9]. The reactive oxygen species (ROS) production and malondialdehyde (MDA) content were determined according to Ko et al. [42] and Yang et al. [43], respectively. Protein carbonyl (PC) and reduced glutathione (GSH) contents, catalase (CAT) activity, anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were measured as described by Kuang et al. [44]. The total superoxide dismutase (SOD), CuZnSOD and MnSOD activities were determined as described by Lu et al. [45]. The activity of glutathione peroxidase (GPx) was analyzed according to Nugroho and Fotedar [46]. Glutathione-Stransferase (GST) and glutathione reductase (GR) activities were determined as described by Gorbi et al. [24]. The lactate and adenosine triphosphate (ATP) contents were measured by the method described by Zhou et al. [47] and Wang et al. [48], respectively.
As shown in Table 3, final body weight (FBW), percentage weight gain (PWG), feed intake (FI), feed efficiency (FE) and specific growth rate (SGR) were improved (722.11, 197.02, 605.65, 0.79 and 1.94, respectively) with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued (P > 0.05). Furthermore, using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on PWG was estimated to be 0.90 mg/kg diet (Fig. 1).
2.4. Real-time quantitative PCR The total RNA of intestine, head kidney, spleen and liver were extracted using RNAiso Plus Kit (Takara, Dalian, China) according to the manufacturer's instructions followed by DNase I treatment. The quantity and quality of RNA were assessed by agarose gel electrophoresis at 1% and by spectrophotometric analysis at 260 and
3.2. The immune response related parameters in intestine The C3 contents (Table 4) in proximal intestine (PI), midintestine (MI) and distal intestine (DI) were enhanced and IFN-g2 (Fig. 4A), TNF-a (Fig. 4B), IL-1b (Fig. 4C), NF-kB P65 (Fig. 5A) and IkB kinases b (IKKb) (Fig. 5D) mRNA levels were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. The LA activities and hepcidin (Fig. 3A) mRNA levels in PI, MI and DI were elevated with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. The acid phosphatase activities in PI, MI and DI were increased (56.28, 70.33 and 89.56, respectively) with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. Using the broken-line regression analysis, the dietary thiamin requirement of young grass carp (242e742 g) based on LA activity in DI is 1.15 mg/ kg diet (Fig. 2). The liver expressed antimicrobial peptides 2 (LEAP2) (Fig. 3B), TGF-b1 (Fig. 4E) and TOR (Fig. 5F) mRNA levels in PI, MI and DI were enhanced with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of IL-8 (Fig. 4D) and IKKg (Fig. 5E) were decreased, IL-10 (Fig. 4F) and inhibitor protein-kBa (IkBa) (Fig. 5B) mRNA levels were increased with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter. Thiamin had no significant effect on IKKa (Fig. 5C) mRNA levels in PI, MI and DI of fish (P > 0.05). The lactate contents (Fig. 6A) in PI, MI and DI were decreased with dietary thiamin levels up to 1.25, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ATP
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Table 2 Primer sequences, thermocycling conditions and accession numbers of genes selected for analyzing by real-time PCR. Gene Hepcidin Forward Reverse LEAP-2 Forward Reverse IFN-g2 Forward Reverse TNF-a Forward Reverse IL-1b Forward Reverse IL-8 Forward Reverse TGF-b1 Forward Reverse IL-10 Forward Reverse NF-kB P65 Forward Reverse IkBa Forward Reverse IKKa Forward Reverse IKKb Forward Reverse IKKg Forward Reverse TOR Forward Reverse Claudin b Forward Reverse Claudin c Forward Reverse Claudin 3 Forward Reverse Claudin 12 Forward Reverse Claudin 15a Forward Reverse ZO-1 Forward Reverse Occludin Forward Reverse MLCK Forward Reverse CuZnSOD Forward Reverse MnSOD Forward Reverse CAT Forward Reverse
Sequences of primers
Thermocycling conditions
Accession number
50 -AGCAGGAGCAGGATGAGC-30 50 -GCCAGGGGATTTGTTTGT-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.3 C 30 s and 72 C 30 s
JQ246442
50 -TGCCTACTGCCAGAACCA-30 50 -AATCGGTTGGCTGTAGGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.3 C 30 s and 72 C 30 s
FJ390415
50 -TGTTTGATGACTTTGGGATG-30 50 -TCAGGACCCGCAGGAAGAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.4 C 30 s and 72 C 30 s
FJ766439
50 -CGCTGCTGTCTGCTTCAC-30 50 -CCTGGTCCTGGTTCACTC-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
HQ696609
50 -AGAGTTTGGTGAAGAAGAGG-30 50 -TTATTGTGGTTACGCTGGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.1 C 30 s and 72 C 30 s
JQ692172
50 -ATGAGTCTTAGAGGTCTGGGT-30 50 -ACAGTGAGGGCTAGGAGGG-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
JN663841
50 -TTGGGACTTGTGCTCTAT-30 50 -AGTTCTGCTGGGATGTTT-30
95 C 30 s, 40 cycles of 95 C 5 s, 55.9 C 30 s and 72 C 30 s
EU099588
50 -AATCCCTTTGATTTTGCC-30 50 -GTGCCTTATCCTACAGTATGTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
HQ388294
50 -GAAGAAGGATGTGGGAGATG-30 50 -TGTTGTCGTAGATGGGCTGAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.3 C 30 s and 72 C 30 s
KJ526214
50 -TCTTGCCATTATTCACGAGG-30 50 -TGTTACCACAGTCATCCACCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.3 C 30 s and 72 C 30 s
KJ125069
50 -GGCTACGCCAAAGACCTG-30 50 -CGGACCTCGCCATTCATA-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
KM279718
50 - GTGGCGGTGGATTATTGG-30 50 - GCACGGGTTGCCAGTTTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.3 C 30 s and 72 C 30 s
KP125491
50 -AGAGGCTCGTCATAGTGG-30 50 -CTGTGATTGGCTTGCTTT-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
KM079079
50 -TCCCACTTTCCACCAACT-30 50 -ACACCTCCACCTTCTCCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
JX854449
50 -GAGGGAATCTGGATGAGC-30 50 -ATGGCAATGATGGTGAGA-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.0 C 30 s and 72 C 30 s
KF193860
50 -GAGGGAATCTGGATGAGC-30 50 -CTGTTATGAAAGCGGCAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KF193859
50 -ATCACTCGGGACTTCTA-30 50 -CAGCAAACCCAATGTAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.0 C 30 s and 72 C 30 s
KF193858
50 -CCCTGAAGTGCCCACAA-30 50 -GCGTATGTCACGGGAGAA-30
95 C 30 s, 40 cycles of 95 C 5 s, 55.4 C 30 s and 72 C 30 s
KF998571
50 -TGCTTTATTTCTTGGCTTTC-30 50 -CTCGTACAGGGTTGAGGTG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.0 C 30 s and 72 C 30 s
KF193857
50 -CGGTGTCTTCGTAGTCGG-30 50 -CAGTTGGTTTGGGTTTCAG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KJ000055
50 -TATCTGTATCACTACTGCGTCG-30 50 -CATTCACCCAATCCTCCA-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
KF193855
50 -GAAGGTCAGGGCATCTCA-30 50 -GGGTCGGGCTTATCTACT-30
95 C 30 s, 40 cycles of 95 C 5 s, 53.0 C 30 s and 72 C 30 s
KM279719
50 -CGCACTTCAACCCTTACA-30 50 -ACTTTCCTCATTGCCTCC-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.5 C 30 s and 72 C 30 s
GU901214
50 -ACGACCCAAGTCTCCCTA-30 50 -ACCCTGTGGTTCTCCTCC-30
95 C 30 s, 40 cycles of 95 C 5 s, 60.4 C 30 s and 72 C 30 s
GU218534
50 -GAAGTTCTACACCGATGAGG-30 50 -CCAGAAATCCCAAACCAT-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.7 C 30 s and 72 C 30 s
FJ560431
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505
Table 2 (continued ) Gene GPx Forward Reverse GST Forward Reverse GR Forward Reverse Nrf2 Forward Reverse Keap1a Forward Reverse Keap1b Forward Reverse b-Actin Forward Reverse
Sequences of primers
Thermocycling conditions
Accession number
50 -GGGCTGGTTATTCTGGGC-30 50 -AGGCGATGTCATTCCTGTTC-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.5 C 30 s and 72 C 30 s
EU828796
50 -TCTCAAGGAACCCGTCTG-30 50 -CCAAGTATCCGTCCCACA-30
95 C 30 s, 40 cycles of 95 C 5 s, 58.4 C 30 s and 72 C 30 s
EU107283
50 -GTGTCCAACTTCTCCTGTG-30 50 -ACTCTGGGGTCCAAAACG-30
95 C 30 s, 40 cycles of 95 C 5 s, 59.4 C 30 s and 72 C 30 s
JX854448
50 -CTGGACGAGGAGACTGGA-30 50 -ATCTGTGGTAGGTGGAAC-30
95 C 30 s, 40 cycles of 95 C 5 s, 62.5 C 30 s and 72 C 30 s
KF733814
50 -TTCCACGCCCTCCTCAA-30 50 -TGTACCCTCCCGCTATG-30
95 C 30 s, 40 cycles of 95 C 5 s, 63.0 C 30 s and 72 C 30 s
KF811013
50 -TCTGCTGTATGCGGTGGGC-30 50 -CTCCTCCATTCATCTTTCTCG-30
95 C 30 s, 40 cycles of 95 C 5 s, 57.9 C 30 s and 72 C 30 s
KJ729125
50 -GGCTGTGCTGTCCCTGTA-30 50 -GGGCATAACCCTCGTAGAT-30
95 C 30 s, 40 cycles of 95 C 5 s, 61.4 C 30 s and 72 C 30 s
M25013
Table 3 Treatment effects on growth performance factors of young grass carp. All data are expressed as mean ± SD (n ¼ 3). Mean values within the same row with different superscripts are significantly different (P < 0.05). IBW: initial body weight (g fish1); FBW: final body weight (g fish1); PWG: percent weight gain (%); FI: feed intake (g fish1); FE: feed efficiency; SGR: specific growth rate (% day1). PWG ¼ 100% [FBW IBW]/IBW), FE ¼ [FBW IBW]/FI, SGR ¼ 100% [ln FBW ln IBW]/days. Dietary thiamin levels (mg kg1 diet) 0.12 IBW FBW PWG FI FE SGR
243.11 575.00 136.50 459.83 0.72 1.54
0.43 ± ± ± ± ± ±
1.84 12.99a 3.58a 1.63a 0.03a 0.03a
243.78 621.67 155.03 501.41 0.75 1.67
0.83 ± ± ± ± ± ±
1.54 4.48b 3.20b 1.15b 0.01ab 0.02b
243.11 722.11 197.02 605.65 0.79 1.94
1.25 ± ± ± ± ± ±
1.02 11.97c 3.83c 1.35c 0.02bc 0.02c
242.44 741.67 205.93 604.85 0.83 2.00
1.62 ± ± ± ± ± ±
0.96 23.69c 10.38c 1.35c 0.04c 0.06c
243.56 733.78 201.30 604.98 0.81 1.97
2.04 ± ± ± ± ± ±
1.64 12.28c 6.31c 1.03c 0.02c 0.04c
243.44 725.56 198.04 603.62 0.80 1.95
± ± ± ± ± ±
1.26 4.29c 0.52c 1.57c 0.01c 0.03c
Regressions YPWG ¼ 38.440x2 þ 115.463x þ 120.434 YFI ¼ 81.937x2 þ 250.262x þ 427.169 YFE ¼ 0.055x2 þ 0.161x þ 0.700 YSGR ¼ 0.255x2 þ 0.765x þ 1.435
contents (Fig. 6B) were increased with dietary thiamin levels up to 1.25, 0.83 and 1.25 mg/kg diet in PI, MI and DI (P < 0.05), and plateaued thereafter.
R2 R2 R2 R2
¼ ¼ ¼ ¼
0.935 0.937 0.761 0.943
P P P P
< < <
0.05), whereas the mRNA levels of these genes in DI were decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The occludin (Fig. 7G) mRNA levels in PI, MI and DI were improved with dietary thiamin levels up to 0.43, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued.
3.4. Antioxidant-related parameters in intestine Fig. 1. Broken-line regression analysis of percentage weight gain (PWG) of young grass carp fed graded levels of thiamin.
As shown in Fig. 8 and Table 5, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents in
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Table 4 Treatment effects on complement 3 content (C3), lysozyme activity (LA) and acid phosphatase activity in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) of young grass carp. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). C3: mg/g protein; LA: U/mg protein; acid phosphatase: U/mg protein. Dietary thiamin levels (mg kg1 diet) 0.12 C3 PI 3.75 MI 3.29 DI 4.78 LA PI 22.04 MI 25.03 DI 19.44 Acid phosphatase PI 46.37 MI 49.02 DI 72.59
0.43
0.83
1.25
1.62 4.33 ± 0.20b 4.32 ± 0.16c 6.11 ± 0.44bc
2.04
± 0.15a ± 0.20a ± 0.39a
3.82 ± 0.13a 3.65 ± 0.21b 5.75 ± 0.20b
4.32 ± 0.21b 4.19 ± 0.31c 6.42 ± 0.26c
4.37 ± 0.30b 4.33 ± 0.13c 6.21 ± 0.27c
4.32 ± 0.29b 4.29 ± 0.20c 6.18 ± 0.30c
± 1.95a ± 1.99a ± 1.32a
27.03 ± 2.43b 28.52 ± 1.58b 33.53 ± 3.14b
31.77 ± 1.96c 30.09 ± 2.76bc 36.82 ± 3.57b
30.66 ± 2.45c 31.34 ± 2.40bc 45.58 ± 4.06c
32.85 ± 2.95c 31.74 ± 2.88c 43.75 ± 3.87c
30.11 ± 2.41c 30.42 ± 2.54bc 43.40 ± 3.90c
± 1.91a ± 2.76a ± 3.88a
51.06 ± 2.69b 56.64 ± 3.06b 78.76 ± 6.41b
56.28 ± 3.01c 57.11 ± 2.63b 89.56 ± 6.60c
55.77 ± 4.06c 70.33 ± 2.70c 90.26 ± 4.73c
58.60 ± 2.39c 70.08 ± 4.57c 89.62 ± 4.78c
58.19 ± 5.15c 69.02 ± 4.32c 91.60 ± 3.49c
Regressions YC3 in MI ¼ 0.545x2 þ 1.687x þ 3.082 YLA in DI ¼ 11.635x2 þ 36.452x þ 16.784 Yacid phosphatase in MI ¼ 6.350x2 þ 24.841x þ 45.768
R2 ¼ 0.800 R2 ¼ 0.849 R2 ¼ 0.788
P < 0.01 P < 0.01 P < 0.01
thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. CuZnSOD (Fig. 9A) and Nrf2 (Fig. 10A) mRNA levels were increased with dietary thiamin levels up to 0.43 mg/kg diet in fish intestine (P < 0.05), and plateaued thereafter. The mRNA level of CAT (Fig. 9A) was the maximum in fish fed the 1.25 mg/kg diet (P < 0.05). The MnSOD (Fig. 9A), GPx, GST and GR (Fig. 9B) mRNA levels were improved and Keap1a (Fig. 10B) mRNA level was decreased with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and then plateaued. However, thiamin had no significant effect on Keap1b (Fig. 10B) mRNA level in fish intestine (P > 0.05). 3.5. Immune response-related parameters in head kidney, spleen and liver Fig. 2. Broken-line regression analysis of lysozyme (LA) activity in the distal intestine of young grass carp fed graded levels of thiamin.
intestine were decreased with dietary thiamin levels up to 0.83, 1.25 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The ASA capacity, GSH content, MnSOD and CAT activities in intestine were increased with dietary thiamin levels up to 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The AHR capacity, CuZnSOD, GPx, GST and GR activities in intestine were increased with dietary
As shown in Table 6, the C3 contents in head kidney, spleen and liver were enhanced with dietary thiamin levels up to 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. LA activities in head kidney, spleen and liver were improved with dietary thiamin levels up to 0.83, 0.83 and 1.25 mg/kg diet (P < 0.05), and then plateaued. Acid phosphatase activities were increased in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 1.25 and 1.25 mg/kg diet (P < 0.05), and plateaued thereafter. The mRNA levels of hepcidin (Fig. 11A) in head kidney, spleen and liver were up-regulated with dietary thiamin levels up to 0.43, 0.83 and 0.83 mg/kg diet (P < 0.05), and then plateaued. The LEAP-2 (Fig. 11B) mRNA levels in
Fig. 3. Effect of dietary thiamin on hepcidin (A) and liver-expressed antimicrobial peptide 2 (LEAP-2) (B) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
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Fig. 4. Effect of dietary thiamin on IFN-g2 (A), TNF-a (B), IL-1b (C), IL-8 (D), TGF-b1 (E) and IL-10 (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). IFN-g2: interferon g2; TNF-a: tumor necrosis factor a; IL-1b: interleukin 1b; IL-8: interleukin 8; TGF-b1: transforming growth factor b1; IL-10: interleukin 10.
spleen and liver were up-regulated with dietary thiamin levels up to 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of LEAP-2 in head kidney (P > 0.05). The mRNA levels of IFN-g2 (Fig. 12A) in head kidney, spleen and liver were down-regulated with dietary thiamin levels up to 0.83, 0.43 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of TNF-a (Fig. 12B) and IL-8 (Fig. 12D) in head kidney, spleen and liver were downregulated with dietary thiamin levels up to 0.83, 0.43 and 0.83 mg/kg diet (P < 0.05), and plateaued thereafter. The IL-1b (Fig. 12C) mRNA levels were decreased and IL-10 (Fig. 12F) mRNA levels were elevated in head kidney, spleen and liver with dietary thiamin levels up to 0.83, 0.83 and 0.43 mg/kg diet (P < 0.05), and then plateaued. The TGF-b1 (Fig. 12E) mRNA levels in head kidney and spleen were up-regulated with dietary thiamin levels up to 0.83 and 0.43 mg/kg diet (P < 0.05), and plateaued thereafter. However, thiamin had no significant effect on the mRNA level of TGF-b1 in liver (P > 0.05) (Table 7). 4. Discussion Our study firstly revealed that dietary thiamin deficiency resulted in poor PWG, FI and FE of young grass carp, whereas the
PWG, FI and FE were increased with the supplementation of thiamin. Study indicated that the improvement of fish growth performance was due to the elevated FI and FE [3]. Correlation analysis showed that PWG was positively correlated with FI (r ¼ þ0.994, P < 0.01) and FE (r ¼ þ0.974, P < 0.01), indicating that the improvement of young grass carp growth may be partly due to the fact that appropriate dietary thiamin level elevated the FI and FE. In vertebrate, growth performance mostly depends on intestinal health status, which is closely associated with the intestinal mucosal immune [3]. Thus, we further investigated the effect of thiamin on intestinal immunity of young grass carp. In the current study, compared with optimal thiamin level, thiamin deficiency led to decrease C3 content, LA and acid phosphatase activities, mRNA levels of hepcidin, LEAP-2, TGF-b1 and IL10, and increase the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 in young grass carp intestine, suggesting that dietary thiamindeficient impaired the intestinal immune response of fish. Moreover, thiamin-regulated the mRNA levels of cytokines may be associated with lactate. In activated human macrophages, lactate induced up-regulation of IL-1b and IL-8 mRNA levels [50] and down-regulation of IL-10 mRNA level in mouse macrophage-like cells [51]. In our study, the lactate content was higher in PI, MI and DI of fish fed the thiamin limitation diet. Correlation analysis
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Fig. 5. Effect of dietary thiamin on NF-kB P65 (A), IkBa (B), IKKa (C), IKKb (D), IKKg (E) and TOR (F) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05). NF-kB P65: nuclear factor kB P65; IkBa: inhibitor protein-kBa; IKKa: IkB kinases a; IKKb: IkB kinases b; IKKg: IkB kinases g; TOR: target of rapamycin.
Fig. 6. Effect of dietary thiamin on lactate content (A) and adenosine triphosphate (ATP) content (B) in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
revealed that the mRNA levels of IFN-g2, TNF-a, IL-1b and IL-8 were positively correlated with lactate content, and TGF-b1 and IL-10 mRNA levels were negatively correlated with lactate content in PI, MI and DI of young grass carp, suggesting that thiamin limitation may elevate lactate production to affect pro-inflammatory
cytokines and anti-inflammatory cytokines expression in fish. The expression of pro-inflammatory cytokines is related to NFkB signaling molecule in activated mouse microglial cells [15]. In the current study, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 in PI, MI and DI of young grass carp, whereas NF-kB
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Fig. 7. Effect of dietary thiamin on claudin b (A), claudin c (B), claudin 3 (C), claudin 12 (D), claudin 15a (E), ZO-1 (F), occludin (G) and myosin light-chain kinase (MLCK) (H) mRNA levels in the PI, MI and DI of young grass carp. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
P65 mRNA levels were decreased with the supplementation of thiamin. Correlation analysis revealed that the mRNA levels of IFNg2, TNF-a, IL-1b and IL-8 were positively correlated with NF-kB P65 mRNA levels in each intestinal segment of young grass carp, suggesting that thiamin deficiency elevated the mRNA levels of proinflammatory cytokines may be partly related to increase the mRNA expression of NF-kB P65 in fish. Furthermore, thiamin deficiency up-regulated the mRNA levels of NF-kB P65 may be partly involved in its upstream signaling molecules, such as TOR. Zhong et al. [15] reported that up-regulation of TOR suppressed the
mRNA expression of NF-kB P65 in activated mouse microglial cells. In the present study, thiamin deficiency decreased the mRNA levels of TOR in all intestinal segments, whereas the mRNA levels of TOR were elevated by optimal thiamin level. Correlation analysis demonstrated that the mRNA levels of NF-kB P65 were negatively correlated with TOR mRNA levels in young grass carp intestine, indicating that dietary thiamin limitation resulted in up-regulating the NF-kB P65 mRNA level partly by down-regulating the mRNA level of TOR in fish. Thiamin-affected the TOR mRNA level in fish may be partly related to adenosine triphosphate (ATP). Hu et al.
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Fig. 8. Effect of dietary thiamin on reactive oxygen species (ROS) production in young grass carp intestine. Values are mean ± SD (n ¼ 6). Different letters above a bar denote significant difference (P < 0.05).
whereas the ATP contents were increased by optimal thiamin level. Correlation analysis indicated that the TOR mRNA levels were positively correlated with the ATP contents in young grass carp intestine, suggesting that thiamin limitation resulted in downregulation of TOR expression may be partly contributed to decrease the ATP content in fish. Besides, the IkBa-mediated nuclear translocation of NF-kB P65 also plays an important role in regulating pro-inflammatory cytokines mRNA expression. In human epithelial cells, down-regulation the expression of IkBa promoted NF-kB p65 nuclear translocation, thereby up-regulating the mRNA levels of TNF-a, IL-1b and IL-8 [53]. In the present study, thiamin limitation led to down-regulate the mRNA level of IkBa in all intestinal segments of young grass carp, indicating that dietary thiamin limitation may promote NF-kB P65 nuclear translocation to elevate the mRNA levels of pro-inflammatory cytokines by downregulating the IkBa mRNA level in fish. Additionally, thiamin deficiency down-regulated the IkBa mRNA expression may be partly
Table 5 Treatment effects on antioxidant-related parameters in young grass carp intestine. All data are expressed as mean ± SD (n ¼ 6). Mean values within the same row with different superscripts are significantly different (P < 0.05). MDA (malondialdehyde) and PC (protein carbonyl): nmol/mg protein; ASA (anti-superoxide anion) and GR (glutathione reductase): U/g protein; AHR (anti-hydroxyl radical), CuZnSOD (copper/zinc superoxide dismutase), MnSOD (manganese superoxide dismutase), CAT (catalase), GPx (glutathione peroxidase) and GST (glutathione-S-transferase): U/mg protein; GSH (glutathione): mg/g protein. Dietary thiamin levels (mg kg1 diet) 0.12 MDA PC ASA AHR GSH CuZnSOD MnSOD CAT GPx GST GR
6.82 7.01 141.79 120.43 4.50 4.32 2.03 0.90 99.56 42.66 34.14
0.43 ± ± ± ± ± ± ± ± ± ± ±
0.26d 0.39c 6.69a 8.53a 0.20a 0.12a 0.15a 0.05a 7.63a 1.59a 2.43a
5.83 5.35 148.36 132.11 5.40 4.83 2.12 1.02 108.40 45.49 36.67
0.83 ± ± ± ± ± ± ± ± ± ± ±
0.29c 0.33b 9.41ab 5.25b 0.51b 0.36b 0.17a 0.06b 4.25b 2.65ab 2.34ab
4.91 4.25 149.18 144.68 6.98 5.49 2.88 1.32 119.12 46.87 40.14
1.25 ± ± ± ± ± ± ± ± ± ± ±
0.25a 0.31a 6.95ab 3.48c 0.28c 0.18c 0.23b 0.07c 4.39c 1.01b 2.46c
5.15 4.17 152.07 144.54 7.93 5.58 3.38 1.47 118.32 47.77 37.95
1.62 ± ± ± ± ± ± ± ± ± ± ±
0.12ab 0.25a 7.04b 4.91c 0.53d 0.34c 0.28c 0.05d 8.17c 2.60b 2.22bc
5.14 4.36 152.55 143.32 7.43 5.26 3.48 1.48 115.74 48.59 38.26
2.04 ± ± ± ± ± ± ± ± ± ± ±
0.39ab 0.23a 9.00b 2.93c 0.68cd 0.13c 0.19c 0.04d 8.67bc 3.31b 3.31bc
5.27 4.56 152.83 143.16 7.87 5.32 3.40 1.51 116.45 48.21 38.53
± ± ± ± ± ± ± ± ± ± ±
0.13b 0.29a 5.40b 5.01c 0.20d 0.30c 0.26c 0.04d 8.34bc 3.11b 2.97bc
P P P P P P P
< < < < < <
