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Accepted Manuscript
Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology
and growth performance of Hybrid sturgeon
Hui Fei, Guo-dong Lin, Cheng-cai Zheng, Meng-meng Huang, Shi-Chao Qian, Zhenju Wu, Cong Sun, Zhen-guang Shi, Jian-you Li, Bing-nan Han
PII:
S1050-4648(18)30506-0
DOI:
10.1016/j.fsi.2018.08.031
Reference:
YFSIM 5489
To appear in:
Fish and Shellfish Immunology
Received Date: 13 July 2018
Revised Date:
14 August 2018
Accepted Date: 15 August 2018
Please cite this article as: Fei H, Lin G-d, Zheng C-c, Huang M-m, Qian S-C, Wu Z-j, Sun C, Shi Z-g,
Li J-y, Han B-n, Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology
and growth performance of Hybrid sturgeon, Fish and Shellfish Immunology (2018), doi: 10.1016/
j.fsi.2018.08.031.
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Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2
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on immunology and growth performance of Hybrid sturgeon
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Hui Fei a,e*,Guo-dong Lin a,e, Cheng-cai Zheng a,e, Meng-meng Huang a,e , Shi-Chao
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Qian b, Zhen-ju Wu b, Cong Sun a,e, Zhen-guang Shi c, Jian-you Li d, Bing-nan Han a,f
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a. College of Life Sciences, Zhejiang Sci-Tech University, 310018, Hangzhou, China
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b. Hangzhou Biopeptide Biotech Co., Ltd. 310012, Hangzhou, China
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c. Yunnan Amur sturgeon group Co., Ltd. 654200, Huize, China
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d. Huize Dian Ze aquaculture Co., Ltd. 654200, Huize, China
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e. Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine,
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Zhejiang Sci-Tech University, Hangzhou 310018, China.
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f. Qingdao Master Biotechnology Co., Ltd. 266000, China.
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Key words : Bacillus amyloliquefaciens; Yarrowia lipolytica lipase 2; Hybrid
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sturgeons; Growth performance; Innate immunity
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*Correspondence to:
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Dr. Hui Fei
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Tel./fax: +86 571 868 48367.
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E-mail: feihui@zju.edu.cn (H, Fei)
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Abstract:
A 12-weeks feeding trial was performed to investigate the possible effects of
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supplementation of Hybrid sturgeon diet with Bacillus amyloliquefaciens (GB-9) and
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Yarrowia lipolytica lipase2 (YLL2) single or combined on immune response and
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growth performance of Hybrid sturgeon (Acipenser schrenkii ♂and Acipenser baeri
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♀). For this aim, Hybrid sturgeons were fed with four experimental diets namely: Diet
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1 (0-control), Diet 2 (5.0g/kg GB-9), Diet 3 (4.0g/kg YLL2), and Diet 4 (5.0g/kg
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GB-9 + 4.0g/kg YLL2), respectively. After fed with varied diets, growth performance,
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mucosal immune response, leukocytes immune response and serum immunological
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response were measured. The results indicated that supplementations of GB-9 +
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YLL2 resulted in a significant increase in final weight, Docosahexaenoic acid (DHA)
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and Eicosapentenoic acid (EPA) concentration, compared with that of control (p <
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0.05).
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leukocytes phagocytosis activity and reactive oxygen species level,and serum
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alternative complement pathway activity, peroxidase and lysozyme activity were
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significantly higher in supplemented groups compared to the control (p < 0.05). The
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highest values were recorded in fish fed both YLL2 and GB-9 with respect to the
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individual application. The present results suggested that the combination of these
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supplementation could be considered as potential feed-additives for aquaculture
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farmed fish.
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For innate immunity, the results showed that skin mucus lysozyme activity,
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1. Introduction
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Sturgeons, especially Hybrid sturgeons (Acipenser schrenkii ♂ and Acipenser baeri
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♀), as a high-quality protein and unsaturated fatty acid resource [1, 2],have been
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large-scale cultured and shown a high market value. Sturgeon aquaculture
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is developing rapidly, China has become the largest sturgeon culture region since
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2000 [3]. However, the available information on sturgeon about dietary lipid is limited,
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which is an important factor in intensive fish farming and successful mass production
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[4]. Hybrid sturgeons are subjected to stress condition that damage fish immune
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systems, leading to increased infection of pathogens and the emergence of diseases
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[5].
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In recent years, dietary administration of functional (biological activity) feed
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additives has been suggested as an environmental friendly alternative approach to
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enhance immune response and increase the growth performance of fish [4-5]. Bacillus
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subtilis, one of the most studied probiotics in fish and shrimp, has been reported to
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have various beneficial properties, including immunostimulant and disease resistance
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substances [6-9]. While most research evaluate the effect of administering probiotics
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individually [7-9], modulation of immune responses using a combination of lipase and
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probiotics has rarely been investigated in fish. Therefore, the present investigation
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was carried out to study the effect of dietary supplementation with the probiotic
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bacterium, Bacillus amyloliquefaciens (GB-9), Yarrowia lipolytica lipase 2 (YLL2)
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and their combination on the immunology and growth performance of hybrid
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sturgeon.
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YLL2 is a typically lipase which has the characteristics of tolerance to the
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gastrointestinal environment, good biological safety, stable at low pH and superior
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temperature performance. YLL2 has been reported to have various beneficial
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properties when supplementing in juvenile fish diets, including reducing the amount
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of fish oil adding in diet, promoting the secretion of digestive enzymes [10-12]. More
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interestingly, YLL2 could efficiently hydrolyze the crude fish oil to produce
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polyunsaturated fatty acids, especially DHA and EPA, which would improve
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fecundity, egg hatchability, cell viability , immune functions and the overall quality of
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the fish [4, 13, 14].
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Probiotics GB-9 are active microorganisms that are beneficial to the host and are
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commonly used as immunomodulators [15]. Several reports suggest that
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supplemented probiotics can improve growth, immune responses and alleviate fish
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resistance [16-19]. The most commonly used probiotics in aquaculture are Bacillus,
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Lactobacillus and Saccharomyces [20, 21]. Bacillus sp. is a non-pathogenic aerobic
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gram-positive bacterium that enhances physical condition and GI microbial
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populations either by oral administration or by oral administration to bacterial water
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of the fish [15, 22]. GB-9 is a non-pathogenic aerobic Gram-positive bacterium
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belonging to the genus B. subtilis, which produces various bacteriostatic substances as
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probiotics including antimicrobial proteins, lipopeptides, antibiotics, macrolides,
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oligopeptidases, polyketides, peptides and polyketides [23-27]. While GB-9 is added
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to fish diet, it plays a beneficial role in the intestine by inhibiting the growth of
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harmful intestine bacteria through producing antimicrobial substances, inhibiting the
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growth of competing bacteria.
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2. Materials and methods
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2.1 Juvenile hybrid sturgeons
2-month juvenile hybrid sturgeons (5.0 g mean body weight) were reared in in-door
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plastic pool at aquatic farm of Yunnan Amur Sturgeon Group Co., Ltd., China. 400
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juvenile fish were randomly divided into four groups of 100 fish per plastic pool.
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Average stocking density was 0.5 kg/m3. The juvenile fish were fed every 3 h to the
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apparent satiation observed from July 2017 to October 2017 under natural
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photoperiod. Water temperature ranged between 10 and 12 ℃ during the experimental
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period.
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2.2 Microorganisms and Lipase
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GB-9 strain (GB-9) was provided by Zhejiang Science and Technology University,
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China, and stored in 20% glycerol. The mixture bacterial liquid was inoculated into
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5.0 mL LB medium (5 g L-1 peptone,10 g L-1 yeast extract,10 g L-1 NaCl in 1.0 L
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sterile water ), inoculation amount of 1%, 37 ℃, 220 rpm cultured for 12 h, then
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transferred to 1.0 L LB medium, inoculated for 12h under the same condition. After
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fermentation, the products was sent to spray drying (input temperature of 170 ℃,
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output temperature of 85 ℃, evaporation capacity 2 L/h) with corn starch as an
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auxiliary material (Wproducts:Wcorninlet =1:10), to harvest GB-9 preparation (2×109
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CFU/g).
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Yarrowia lipolytica lipase 2 (YLL2) was kindly provided by Dr. Jinyong Yan
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(College of Life Science & Technology, Huazhong University of Science and
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Technology, China) [28, 29]. Yarrowia lipolytica was inoculated into 50 mL medium
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(10 g L-1 Yeast Extract , 20 g L-1 Peptone, 50 g L-1 Sucrose) in 500 mL conical flask,
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inoculation amount of 2%, 28 ℃, 250 rpm cultured for 84 h, after centrifugation for 30
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min (5000r/min), the supernatant was sent to spray drying (input temperature of
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150 ℃, output temperature of 80 ℃, evaporation capacity 2 L/h) with corn starch as
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an auxiliary material (Wproducts:Wstarch =1:5), The enzyme activity of YYL2 mixture
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powder was 4125 U/mg, assayed according to published method [11].
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2.3 Diet
For preparation of the experimental diets, the basal diet was supplemented with
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different levels of GB-9 and YLL2 as follows: Control group, (Diet 1-Control);
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Group1: 5.0 g/kg of GB-9 (Diet 2); Group2: 4.0g /kg YLL2 (Diet 3) and Group3: 5.0
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g/kg of GB-9 + 4.0 g/kg YLL2 for Diet 4 (Table 1).The GB-9 concentration (5.0 g/kg,
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107 CFU/g fodder ) employed was based in previous studies carried [30]. The YLL2
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concentration (4.0 g/kg, 16.5 U/g fodder) determined by basal diet composition (13.5g
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crude lipid/100g fodder). At this concentration, the YLL2 could hydrolyze the crude
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lipid with relatively high efficiency and maintain relatively low cost of use.
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Briefly, normal pellet diet was crushed, mixed with tap water (where GB-9 was
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added at the desired concentration) and made again into pellets, while YLL2 was
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mixed with tap water and then added at the desired concentration and incubated for 1
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h in order to help hydrolyze the crude lipid.
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2.4 Growth performance
The growth performance of hybrid sturgeons fed different experimental diets was
calculated at the end of the feeding trial, based on the following formulae:
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2.4.1 Determination of SR, WG, SGR, FCR
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Survival rate (SR, %) = 100 × number of final fish / number of initial fish;
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Weight gain (WG) = final weight (g) - initial weight (g);
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Specific growth rate (SGR, % day−1) = 100 × [ln (final body weight) − ln (initial
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body weight)] / days;
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Total amount of the feed consumed (g) = total amount of the fed food − collected
uneaten feed/leaching loss rate;
Feed conversion ratio (FCR) = feed given (dried weight) / weight gain (wet
weight).
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2.4.2 Concentration of DHA and EPA
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In the total lipid extract, concentration of DHA and EPA of hybrid sturgeon was
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determined. Total lipids were extracted and measured through dichloromethane
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method [31, 32]. Fatty acid methyl esters were prepared by acid-catalyzed
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transmethylation of total lipids using boron trifluoride-methanol [33], and were
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analyzed in a Thermo Scientific Trace GC Ultra gas chromatograph. The
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chromatograph was equipped with a Restek-Rtx-225, 60.0 m × 250 µm capillary
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column. Nitrogen was used as carrier gas (1.4 mL min-1) and the thermal gradient was
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a constant temperature of 100℃ for 13 min, 100-180 ℃ at 10 ℃ min-1, 180-200 ℃ at
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1℃ min-1 and 200-230 ℃ at 4 ℃ min-1. Injector and flame ionization detector
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temperatures were 270 ℃ and 280 ℃, respectively. The fatty acid concentration of
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hybrid sturgeon was determined with artificial fatty acid as a standard.
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2.5 Measurement of innate immune response
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At the end of the feeding trial (12 weeks), 12 randomly selected fish from each
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group were selected for the immunological assays, and skin mucus was collected by
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scrapping the mucus off the fish's skin by clean slides. After processing with PBS
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(0.01 mM, pH 7.4 ) with EDTA and PFSM, the mucus samples were centrifuged to
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get supernatant and stored at -80 ℃ for use [34, 35]. Briefly, whole blood samples
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were obtained from the caudal vein of each specimen with a 23-gauge needle and 10.0
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mL syringe. After clotting at 4 ℃, each sample was centrifuged (5000 g, 25min, 4 ℃)
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and the serum was collected and frozen at -80 ℃ until further analysis. Leukocytes
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were isolated from peripheral blood with 10.0 mL syringe (containing anticoagulant),
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each sample was centrifuged (100 g, 10 min, 4 ℃) to remove erythrocytes. Leukocytes
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were extracted following the method by Van et. al., [34, 35]. The protein
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concentration of skin mucus and serum was assayed with BCA method.
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2.5.1 Fish 50% complement hemolysis (CH50) level
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The CH50 was assayed by using Fish 50% complement hemolysis (CH50) ELISA
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Kit (KeShun. Bio, Shanghai, China). Briefly, 50 µL standard liquid was added to
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standard well, then10 µL testing sample (skin mucus and serum), 40 µL diluent was
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added to the testing sample well, while blank well add nothing. After that, 100 µL
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HRP-conjugate reagent was added to each well, subsequently incubated for 60
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minutes at 37 ℃. After incubation, each well was washed for five times, 50 µL
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chromogen solution A and 50 µL chromogen solution B was added to each well. After
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incubated for 15 minutes at 37 °C, 50 µL terminal solution was added to each well,
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consequently determined at OD450. All above assays were repeated on three occasions.
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2.5.2 Alternative complement pathway activity (ACH50)
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The activity of the alternative complement pathway was assayed using sheep red
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blood cells (300 µL SRBC, Biomedics) as targets [36, 37]. The degree of haemolysis
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and the lysis curve for each specimen were obtained. The volume of serum producing
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50% haemolysis (ACH50) was determined and the number of ACH50 units/mL
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obtained for each experimental group.
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2.5.3 Reactive oxygen species level (ROS level)
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Leukocyte were adjusted to the concentration of 3×106 cells/mL (V=1.0 mL) with
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PBS buffer solution, the activity oxygen fluorescent probe was added until the final
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concentration reached 2.0 µM, incubated on rotary equipment for 50 min at room
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temperature without light [38-40]. After cells were washed three times with PBS, the
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fluorescence intensity was detected on the flow cytometer (BD-FACSAria II). Setting
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the assay with the parameters of PE-Texas Red-A, the average fluorescence intensity
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(MFI) of P1 PE-Texas Red-A was analyzed as the result.
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2.5.4 Lysozyme activity (LZM)
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The method described by Parry et al. was used for determination of lysozyme
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activity [41]. The lysozyme activity of mucus and serum was measured and expressed
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as mg/mL.
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2.5.5 Peroxidase activity (POD)
Peroxidase activity was measured with peroxidase assay kit [42]. The optical
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density was read at 420 nm in a cuvette (optical path=1.0cm). Standard samples
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without serum or skin mucus were used as blanks. Peroxidase activity was defined as
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follows:
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Peroxidase-activity (U/mL)
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=
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OD − OD blank
T otal reaction v olume ( mL)
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÷ Reaction ti me × Diluting factor × 1000
12 × Optical path (1.0cm )
Sample ( mL)
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2.5.6 Phagocytosis activity
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Phagocytic ability was defined as the percentage of cells with one or more ingested
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fluorescent beads within the phagocytic cell population [43, 44]. All samples
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(leukocytes) were analyzed in a flow cytometer (BD-FACSAria II) with an argonion
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laser adjusted to 488 nm. The quantitative study of the flow cytometric results was
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made using the statistical option of the Lysis Software Package (BD-FACSAria II).
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2.6 Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) to test the
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difference between diets. Multiple comparisons among means were conducted using
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Duncan's procedure when significant difference was identified. Difference was
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regarded as significant when p < 0.05. All statistical analyses were performed by
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SPSS 18.0 software package.
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3.Result
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3.1 Growth performance
The growth performances of juvenile hybrid sturgeons fed the diets containing
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alternative supplementation over a period of 12 weeks are showed in Table 3. There
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was a statistically significant increase of Group 3 and Group 2 in the specific growth
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rate (SGR), weight gain (WG), final weight (FW) compared with that of control (p <
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0.05; Table 3). The highest SGR and WG values were observed in fish fed both
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dietary GB-9 and YLL2 (Table 3). However, no significant differences in these
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parameters were observed in fish fed GB-9 alone (p > 0.05; Table 3). The FCR was
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significantly lower in fish fed 5.0 g/kg GB-9 + 1.0 g/kg YLL2 than in other groups,
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while control group presented the highest values (p < 0.05). Survival rate of the fish
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showed significant differences between the Group3 and control group (Table 3).
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3.2 Protein concentration of skin mucus and serum
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In order to investigate the influence of supplemented with different levels of GB-9
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and YLL2, protein concentration was evaluated. The result showed that there were no
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significant differences in skin mucus among the groups, while protein concentration
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of serum was increased compared with that of control (Table. 4).
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3.3 Mucosal immune response
The mucosal immunity of hybrid sturgeon fed on different supplemented diets was
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evaluated after 12 weeks of the treatment. Among the mucosal immune response,
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lysozyme was the most affected by the assayed diets. The result showed that
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lysozyme activity significantly increased following 12 weeks of the feeding trial with
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the GB-9 + YLL2 diets compared to the control diet (Fig. 1). The highest values 10.57
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± 0.53 (µg/mL) was recorded for fish fed both GB-9 and YLL2 supplementations.
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However, no significant difference of peroxidase activity and CH50 was recorded
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between experimental groups (Fig. 2 and Fig. 3). No alternative complement pathway
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activity (ACH50) was observed after assayed for 2.5 h in all groups.
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3.4 Leukocytes immunological response
To elucidate the cellular immune response, phagocytosis rates and ROS levels were
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determined between control group and supplemented groups after 12-weeks feed trail.
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It has been reported that phagocytosis plays an important role in the host-defense
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mechanisms and contributes to inflammation and the immune response.
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Phagocytosis activity was analyzed by flow cytometry, which showed that the
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phagocytic percentage of hybrid sturgeon leukocytes fed on different supplemented
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diets was promoted with varying degrees compared with that of the control (Fig. 4),
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indicating that YL2 and GB-9 enhanced the phagocytosis activity of leukocytes.
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Reactive oxygen species (ROS) is of fundamental importance for host defense and
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cellular signaling [45]. As shown in (Fig. 5), ROS levels showed that hybrid sturgeon
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treated with supplement in diets increased as compared to control.
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3.5 Serum immunological response
To elucidate the serum immunological response, peroxidase activity, 50%
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complement hemolysis (CH50) level, lysozyme activity, and alternative complement
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pathway activity (ACH50) were observed between control group and supplemented
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groups after 12-weeks feed trail.
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Peroxidase may play an important role in increasing host defenses against
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pathogens [46]. lysozyme is likely the most powerful bacteriolytic protein because
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of
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defence against bacterial infections [47]. The alternative complement pathway
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comprises plasma proteins, which represent the major humoral defense mechanism
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against infection. To determine the effect of supplement diet in immunity, peroxidase
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activity, lysozyme activity and alternative complement pathway were performed in
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serum of hybrid sturgeon. Results (Fig. 6, Fig. 7 and Fig. 8) showed that YYL2 and
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GB-9 induced the upregulation of peroxidase, lysozyme and alternative complement
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pathway activity, compared with that of control.
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the ability to cleave the bacterial peptidoglycan and contributes to host
The complement system plays a critical role in the identification and removal of
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foreign substances and immune complexes and in the stimulation of inflammatory
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response. The 50% hemolytic complement (CH50) assay is the most conventional
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method for functional activity of the classical complement pathway. In our data,
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YYL2 and GB-9 as immunostimulant agent, downregulated CH50 level of hybrid
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sturgeon serum compared with that of control.
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4. Discussion
According to the latest statistics, the global aquaculture production reached 73.8
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million metric tons in 2014 [48]. Taking into account this production level and the
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main cost intensive aquaculture (even as high as 84% of the total production cost),
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improving feed efficiency and additives are most beneficial to ensure the profitability
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of the aquaculture industry [49]. Hence, it has become a new trend to add feed
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additives to increase the utilization rate of aquaculture feeds [48]. Probiotics are
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considered as potential feed additives in aquaculture. It is demonstrated that GB-9 can
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improve the immune status and disease resistance of Nile tilapia [50]. Sen Liu et al.
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showed that the supplementation of exogenous lipase can improve the growth, gut
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function, and health status of juvenile grass carp [51]. Aly et al. reported that Bacillus
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B. subtilis increased WG and SGR of Oreochromis O. niloticus [52]. However, the
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study of Bacillus and exogenous lipase supplement in hybrid sturgeon is limited.
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It has been shown that Bacillus strains in the diet can increase disease resistance
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by stimulating cellular and humoral immune functions, including phagocytic activity,
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lysozyme activity and peroxidase activity [53-55]. Peroxidase may play critical role in
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increasing host defenses against pathogens [46]. Lysozyme is an indispensable
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bactericidal cationic enzyme that hydrolyzes the peptidoglycan layers of bacterial cell
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walls
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N-acetylglucosamine and is increased in the sera of fish during infection with various
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variable microorganisms [56, 57]. It activates phagocytosis and the complement
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system and is an important factor in the natural immune response in fish against
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pathogenic infections [58]. In our data, lysozyme activity of skin mucus and serum
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and peroxidase activity of serum are greatly enhanced, when GB-9 is added at 4×107
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CFU/g. Phagocytosis plays an important role in the host-defense mechanisms and
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contributes to innate immunity [44, 59, 60]. During phagocytosis, phagocytic cells
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produce a high degree microbiocidal reactive oxygen specificity (ROS) and attack
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invading pathogens [61]. This ROS-producing activity is known as respiratory burst
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activity and is commonly used as an index to assess the defensive ability of defense
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pathways-causes [62]. Our data showed respiratory burst activity and phagocytosis
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activity of hybrid sturgeon leukocytes, which fed with GB-9 increased significantly
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compared with that of control.
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Al Marzooqi and Leeson reported that supplementing lipase can improve the fat
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digestion of broilers [63]. Fat digestion can produce fatty acids in the intestines of
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mammals and fish [64]. It has been reported that dietary medium-chain fatty acids
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may affect bacterial metabolites and thus affect the intestinal health of weaned piglets
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[65].Therefore, exogenous lipase supplementation may affect the fish's gut by raising
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fat digestion to improve health levels, which requires further investigation [66, 67]. In
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this study, the addition of exogenous lipase YLL2 to diets can significantly improve
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the growth performance of fish, increase the content of DHA and EPA, help maintain
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the physical barrier of the intestine and enhance the immune ability, including
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lysozyme activity of skin and serum, the peroxidase and alternative complement
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pathway activity of serum, the ROS-producing activity and phagocytosis activity of
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leukocytes.
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Taken together, it has been proved that supplementation of probiotics and natural
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substances combined may exhibit higher up-regulating effects on Nile tilapia immune
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parameters and growth performance than that of separately supplementation [47]. In
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our study, supplementation of GB-9 and YLL2 together improved the growth
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performance and promoted the immune stimulation of hybrid sturgeon significantly,
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especially enhanced the lysozyme activity of skin and serum, phagocytosis activity of
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leukocytes and alternative complement pathway activity. This might be due to the
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DHA and EPA hydrolyzed by YYL2 improved the poor establishment of the GB-9 in
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the gastrointestinal tract of hybrid sturgeon and might have promoted the growth of
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GB-9 [68].
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Regarding to the survival rate, group 4 showed the maximum survival rate
342
compared with other experimental groups. The survival rate exhibited some relevance
343
with the ROS level, phagocytosis activity and skin mucus lysozyme activity. It could
344
be inferred that the higher level of skin mucus lysozyme activity could help hybrid
345
sturgeon eliminate the pathogenic bacterium from external environment, the higher
346
level of phagocytosis activity and ROS could promote the leukocyte migration, help
347
eliminate local invading bacteria in hybrid sturgeon [69].
EP
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In addition, the alternative complement pathway comprises plasma proteins,
349
which represent the major humoral defense mechanism against infection. In teleost
350
fish, the complement system plays a key role in the adaptive immune response [62].
351
Panigrahi et al. showed that serum complement C3 increased significantly with the
352
increase of B. licheniformis [70]. In our study, the fact the ACH50 increased after fed
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with lipase, supported previous reports. Sun et al. proved B. pumilusor B. Diets
354
containing C. clausii can activate the complement system during the initial feeding
355
[71]. However, after 12-weeks days of feeding, the levels of C3 in the probiotic group
356
did not differ from those in the control group. In our study, CH50 level showed no
357
significant difference compared with that of control in skin mucus, while
358
downregulated in serum compared with that of control. Our data supported previous
359
reports that persistent activation of complement may lead to host adverse reactions
360
and immunosuppression [55, 72]. However, the impact of probiotics on the
361
complement system and its related immune functions remains to be further
362
investigated.
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353
In short, current research showed that dietary consumption of YLL2 and GB-9 can
364
significantly increase the growth performance, protein concentration and serum
365
immune response of hybrid sturgeon. This study may constitute a new strategy for
366
fish feeding supplementation of exogenous lipase and Bacillus. However, the precise
367
mechanism of how YLL2 and GB-9 stimulate growth and immune system of Hybrid
368
sturgeon is not clarified as yet and further research on this aspect is needed.
370
371
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Acknowledgements
The authors wish to thank Zhejiang Provincial Natural Science Foundation of
372
China
(No.
LQ18B060005);
Zhejiang
Sci-Tech
University
Foundation
373
(No.11612932611702); Special Fund for Agro-scientific Research in the Public
374
Interest of Zhejiang province (LGN18C190011); Project for Jiaozhou Excellent
ACCEPTED MANUSCRIPT
Innovation Team (18-CX-1)
376
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593
Table 1
594
The formulation of experimental groups (g/kg diet).
mealc
Wheat flourd
Soy lecithin
Mineral
c
Premixe
GB-9
YLL2
460
460
460
460
200
200
200
200
260
260
260
50
50
50
30
30
30
/
5.0
/
/
extruded pellets.
b
598
c
599
d
600
e
30
/
5.0
4.0
4.0
YiHai Kerry Investment Company Limited, Shandong, China;
Guchan Group, Beijing, China;
Beijing Enhalor Biotech Ltd. Co. Beijing, China
Table 2
603
Proximate compositions of experimental groups (g/kg diet).
EP
602
Proximate composition
(% dry matter)
Group 1
Group 2
Group 3
Group 4
Dry matter (% diet)
Crude protein
92.7
42.0
93.6
42.4
95.1
42.5
97.8
42.7
Crude lipid
Ash
Lys
13.5
63.5
1.60
13.6
64.6
1.60
13.5
63.7
1.60
13.6
64.8
1.60
AC
C
606
50
Fishmeal were supplied by FuShen Fish meal Co., Qingdao, China
601
605
260
All diets were produced at Yunnan Amur Sturgeon Group Co., Ltd. China, as
597
604
Group 4
M
AN
U
596
a
Group 3
TE
D
595
Group 2
RI
PT
Fish meal
Soybean
Group 1
b
SC
Ingredientsa
ACCEPTED MANUSCRIPT
Table 3
608
Effect of dietary supplementation on growth performance and feed utilization of
609
juvenile hybrid sturgeon, A. schrenkii ♂ and A. baeri ♀, fed experimental diets for 12
610
weeks.
RI
PT
607
Diet 1
Diet 2
Diet 3
Diet 4
IW (g)
5.0 ± 0.05
5.0 ± 0.07
5.0 ± 0.08
5.0 ± 0.03
FW (g)
57.5 ± 1.5c
61.3 ± 1.7b
75.2 ± 1.6b
80.9 ± 1.8a
WG (g)
52.5 ± 1.4c
56.3 ± 1.8b
70.2 ± 2.1b
75.9 ± 1.5a
SR (%)
90
96
SGR (%)
2.91 ± 0.02c
2.98 ± 0.03b
FCR
1.41 ± 0.02a
1.35 ± 0.00b
1.99
β
EPA(%)
1.11
93
97
3.22 ± 0.03b
3.31± 0.02a
1.26 ± 0.00b
1.23 ± 0.02b
2.35
5.95
6.22
1.51
3.01
3.37
M
AN
U
DHA(%)α
SC
Index
IW=initial weigh fish-1; FW=Final weight fish-1; SGR =Specific growth rate fish-1;
612
FCR = Feed conversion ratio; SR = Survival rate;
613
Data are mean ± SEM. Means not bearing the same superscript letters in the same row
614
are significantly different (p < 0.05).
615
DHA (%)α means the concentration of DHA in hybrid sturgeon muscle; EPA(%)β
616
means the concentration of EPA in hybrid sturgeon muscle.
617
EP
TE
D
611
Table.4
619
The protein concentration of skin mucus and serum
620
AC
C
618
Groups
Protein concentration of skin
mucus (mg/mL)
Protein concentration of
serum (mg/mL)
Control group
4.91
15.90
Group 1
Group 2
Group 3
4.65
4.77
4.51
17.12
21.25
23.66
ACCEPTED MANUSCRIPT
Figure legends
622
Fig. 1. Skin mucus lysozyme activity of hybrid sturgeon after 12 weeks of feeding
623
trial fed on different diets (mean ±S.E., n=6): Control Group (0-control), Group 1 (5.0
624
g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2).
625
The bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
626
one-way ANOVA).
RI
PT
621
SC
627
Fig. 2. Skin mucus peroxidase activity of hybrid sturgeon after 12 weeks of feeding
629
trial fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0
630
g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2).
631
The bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
632
one-way ANOVA).
TE
D
M
AN
U
628
633
Fig. 3. Skin mucus CH50 level of hybrid sturgeon after 12 weeks of feeding trial fed
635
different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of
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GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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Fig. 4. Phagocytosis rate of hybrid sturgeon leukocyte after 12 weeks of feeding trial
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fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg
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of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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Fig. 5. ROS levels of hybrid sturgeon leukocyte after 12 weeks of feeding trial fed
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different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of
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GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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Fig. 6. Peroxidase activity of hybrid sturgeon serum after 12 weeks of feeding trial
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fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg
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of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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Fig. 7. Lysozyme activity of hybrid sturgeon serum after 12 weeks of feeding trial fed
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different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of
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GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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Fig. 8. Alternative complement pathway activity of hybrid sturgeon serum after 12
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weeks of feeding trial fed different diets (mean ± S.E., n=6): Control Group
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(0-control), Group 1 (5.0 g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg
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of GB-9+4.0 g/kg YLL2). The bars with different superscripts (a, b, and c) are
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significantly different (p < 0.05, one-way ANOVA).
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Fig. 9. CH50 level of hybrid sturgeon serum after 12 weeks of feeding trial fed
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different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of
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GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The
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bars with different superscripts (a, b, and c) are significantly different (p < 0.05,
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one-way ANOVA).
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1. The supplementations of GB-9 and YLL2 resulted in a significant increase in final
weight, DHA and EPA concentration of Hybrid sturgeon.
2. The skin mucus lysozyme activity, leukocytes phagocytosis activity and reactive
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oxygen species level were significantly higher in supplemented group.
3. The serum alternative complement pathway activity, peroxidase and lysozyme
activity were significantly higher in supplemented group.
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4. The combination of exogenous lipase and Bacillus could be considered as
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potential feed-additives for aquaculture farmed fish.
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