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Supplemental effects of biofloc powder on growth performance, innate immunity, and disease resistance of Pacific white shrimp Litopenaeus vannamei

Supplemental effects of biofloc powder on growth performance, innate immunity, and disease... An 8-week feeding trial was conducted to study the effect of dietary supplementation of a biofloc powder on growth performance and non-specific immune response of Litopenaeus vannamei. Seven experimental diets were prepared with supplementation of graded levels of dried biofloc powder by 0, 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0% (designated as Con, BF0.5, BF1, BF2, BF4, BF6, and BF8, respectively). Triplicate groups of shrimp (1.01 ± 0.01 g) were hand-fed with one of the diets four times a day. At the end of the feeding trial, significantly (P ˂ 0.05) higher growth performance and feed utilization were obtained in BF4 groups compared to those fed the Con diet. The innate immunity of shrimp was improved by the dietary supplementation of biofloc. Dietary inclusion of biofloc at the level of 4.0% significantly increased disease resistance of shrimp against Vibrio harveyi. The results indicate that biofloc might be used as a dietary supplement for growth performance, innate immunity and disease resistance of Pacific white shrimp. Keywords: Biofloc powder, Innate immunity, Growth performance, Disease resistance, Litopenaeus vannamei Background reuse excreted dissolved nitrogen by heterotrophic bacteria Diseases caused by infectious microorganisms are known by controlling carbon and nitrogen ratio in the culture to be one of the major constraints in the shrimp aquacul- water (Avnimelech, 1999). The biofloc in the BFT system is ture industry for the past decades (Ekasari et al., 2014). formed by aggregating many substances such as, other mi- Thus, there is a need to control the disease outbreaks in croorganisms, microalgae, zooplankton, and trapped or- this sector. A concept of a functional feed is an emerging ganic particles or solids from uneaten feeds (De Schryver paradigm in aquaculture industry to develop nutritionally et al., 2008; Crab et al., 2012; Ekasari et al., 2014). It has balanced diets with functions to control the diseases and been demonstrated that the active and condensed microor- culture water system by feed additives (Li and Gatlin ganisms together with suspended organic or inorganic parti- 2004; Lee et al., 2013; Wongsasak et al., 2015). cles tend to from biofloc, which can be consumed The application of biofloc technology (BFT) in shrimp constantly by cultured shrimp as a natural food source aquaculture has gained great attention. BFT offers a prac- (Burford et al., 2004; Wasielesky et al., 2006; Kent et al., tical solution to control culture water quality effectively with 2011). Thus, the biofloc itself provides biomass which is negligible water exchange and improves shrimp growth per- served as nutrients for shrimp especially as a protein source formances in the healthy culture environment (De Schryver or immunostimulants. Previous studies showed that biofloc et al., 2008; Stokstad, 2010; Avnimelech, 2012; Crab et al., had enhanced cellular immune response and antioxidant 2012; Xu and Pan 2013). The mechanism of the BFT is to status of cultured shrimp by its richness in microbes and bioactive compounds (Ju et al., 2008; Xu and Pan 2013). Similar result was found when L. vannamei were fed a for- * Correspondence: kjlee@jejunu.ac.kr Department of Marine Life Sciences, Jeju National University, Jeju 63243, mulated 35% crude protein diet in biofloc-based culture South Korea tanks for a period of 30 days; however, it is not clear if Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 2 of 7 feeding a relatively low protein diet (35%) with the contribu- Table 1 Biofloc chemical composition (dry matter) tion of biofloc could sustain the health status of cultured Parameter Biofloc shrimp in such systems (Xu and Pan 2013). Jang et al. Proximate composition (%) (2011) also reported that the expression of Crude protein 28.7 prophenoloxidase-activating enzyme was significantly in- Crude lipid 2.30 creased in the shrimp reared in a biofloc system. Crude ash 43.0 Pacific white shrimp is one of the most important Amino acid composition (%) shrimp species currently being cultured in many coun- tries. Over the past decade, mass production of L. vanna- Methionine 0.36 mei has been demonstrated in biofloc-based intensive Arginine 1.17 culture systems under high aeration and negligible water Phenylalanine 1.16 exchange (Avnimelech, 2012; Haslun et al., 2012). Re- Leucine 1.81 cently, it was demonstrated that dietary inclusion of bio- Isoleucine 1.02 floc had enhanced growth performance of L. vannamei Lysine 1.11 (Ju et al., 2008; Kuhn et al., 2010; Bauer et al., 2012). There is a lack of information to support the role of biofloc in di- Valine 1.51 ets on growth and immune response in the species. Threonine 1.31 Hence, to investigate the suitability of biofloc as a dietary Histidine 0.41 supplement, we included it in diets at different levels and Trace mineral composition (mg/kg) fed to L. vannamei. The present study aims to evaluate Zinc 521.8 the effect of dietary supplementation of biofloc on growth Aluminum 287.8 performance, non-specific immune response, and suscep- tibility to bacterial infection caused by V. harveyi in L. Manganese 180.7 vannamei juveniles. Iron 146.9 Copper 34.0 Methods Aluminum silicate 3.4 Preparation of biofloc Cadmium 2.7 The biofloc was collected from L. vannamei BFT tanks. A biofloc suspension was prepared in a 300 ton polypropyl- ene tank (45 × 6.5 m) filled with seawater and stocked with South Korea) and pelleted through a pellet machine (SP-50; L. vannamei. The C: N ratio was maintained at 15:1 using Gumgang Engineering, Daegu, Korea). The pelleted diets molasses as a carbon source. Probiotics containing Bacil- were dried at25°Cfor 12 h, andstoredat −24 °C until lus subtilis, Lactobacillus casei and Saccharomyces cerevi- used. Formulation and proximate composition of the ex- siae (Total bacterial account = 1 × 10 CFU/mL) were perimental diets are shown in Table 2. added into the biofloc ponds once a week until enough biofloc sample was collected. Biofloc collection was con- Shrimp and feeding trial tinued for 12 h with enough and strong aeration in the The feeding trial was conducted in indoor shrimp culture BFT tanks. During the aeration, sludges (flocs) were facilities at the Marine Science Institute of the Jeju Na- formed on the water surface. The collected flocs were rap- tional University (Jeju, South Korea). Juvenile L. vannamei idly frozen at −80 °C, and then vacuum freeze-dried. The was obtained from NeoEnBiz shrimp farm (Dangjin, dried flocs were ground into fine powder (<100 μm) and South Korea). Shrimps were fed a commercial diet (35% kept in airtight containers in refrigerator until used in ex- crud protein) twice daily for 10 days to be acclimated to perimental diets. Chemical composition of the biofloc the experimental conditions and facilities. Then, the powder is provided in Table 1. shrimps (initial mean body weight, 1.01 ± 0.01 g) were randomly distributed into twenty one acryl aquaria of Experimental diets and design 96 L capacity at a density of 18 shrimp per aquarium. Each Seven experimental diets were formulated to be isonitro- aquarium was supplied with filtered seawater and aeration −1 genous (400 g kg crude protein) and isocaloric (16 MJ kg to maintain enough dissolved oxygen. Triplicate groups of −1 ). A basal fishmeal based diet was regarded as a control shrimp were hand-fed with one of the test diets four times and supplemented with biofloc at the incremental levels of a day at 08:00, 12.00, 16.00, and 18:00 h for 8 weeks. Daily 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0% (designated as Con, BF0.5, feeding rates were slowly reduced from 10 to 8% of wet BF1, BF2, BF4, BF6, and BF8, respectively) at the expense of body weight during the 8-week feeding period. Water in soybean meal and wheat flour. All dry ingredients were the aquaria was exchanged every 3-day intervals while in- thoroughly mixed in a feed mixer (NVM-16, Gyeonggido, side of the aquaria were cleaned by a sponge to prevent Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 3 of 7 Table 2 Dietary formulation and proximate composition of the (Robertson et al., 1987). The hemolymph-anticoagulant seven experimental diets for L. vannamei (% dry matter) mixture (diluted hemolymph) was placed in five sterile Ingredients Experimental diets eppendorf tubes containing equal volume for the determin- ation of the total hemocyte counts (THC) and respiratory Con BF0.5 BF1 BF2 BF4 BF6 BF8 burst activity. After the abovementioned measurements Brown fishmeal 28.0 28.0 28.0 28.0 28.0 28.0 28.0 with diluted hemolymph, the remained samples was Soybean meal 30.0 30.0 30.0 29.5 28.5 27.5 26.5 centrifuged at 800×g for 20 min using a high-speed refriger- Squid liver meal 3.00 3.00 3.00 3.00 3.00 3.00 3.00 ated microcentrifuge (Micro 17 TR; HanilBioMed Inc., Wheat flour 29.0 28.5 28.0 27.5 26.5 25.5 24.5 Gwangju, Korea) and stored at −70 °C for determination of Starch 5.00 5.00 5.00 5.00 5.00 5.00 5.00 phenoloxidase (PO), superoxide dismutase (SOD) activities, Fish oil 1.00 1.00 1.00 1.00 1.00 1.00 1.00 total immunoglobulin (Ig) level and glutathione peroxidase (GPx) activity. Mineral mix 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Analyses of moisture and ash contents of biofloc Vitamin mix 1.00 1.00 1.00 1.00 1.00 1.00 1.00 powder and diet samples were performed by the Choline chloride 1.00 1.00 1.00 1.00 1.00 1.00 1.00 standard procedures (AOAC, 1995). Crude protein Lecithin 1.00 1.00 1.00 1.00 1.00 1.00 1.00 was measured by using an automatic Kjeltec Analyzer Biofloc 0.00 0.50 1.00 2.00 4.00 6.00 8.00 Unit 2300 (Foss Tecator, Höganäs, Sweden), and Chemical composition (% dry matter) crude lipid was determined using the Soxhlet method with extraction in diethyl ether (Soxhlet Extraction Dry matter 93.2 93.8 94.0 93.2 93.1 93.6 93.5 System C-SH6, Korea). Crude protein 40.8 40.9 40.7 40.6 41.0 41.0 41.0 Crude lipid 6.70 7.50 7.10 7.00 7.60 7.30 7.50 Monitoring of non-specific immune responses Crude Ash 8.00 8.40 8.30 8.60 9.30 10.0 10.8 A drop of the diluted haemolymph was placed in a Energy, MJ/kg diet 16.6 16.6 16.6 16.5 16.4 16.2 16.1 hemocytometer to measure THC using an inverted phase- Mineral premix (g/kg mixture): MgSO .7H O, 80.0; NaH PO .2H O, 370.0; KCl, contrast microscope (Olympus, Model CH30RF200, 4 2 2 4 2 130.0; Ferric citrate, 40.0; ZnSO .7H O, 20.0; Ca-lactate, 356.5; CuCl, 0.2; 4 2 Olympus Optical Co., LTD, Japan). Diluted hemolymph AlCl .6H O, 0.15; Na Se O , 0.01; MnSO .H O, 2.0; CoCl .6H O, 1.0 3 2 2 2 3 4 2 2 2 protein content was measured using a microprotein deter- Vitamin premix (g/kg mixture): L-ascorbic acid, 121.2; DL-tocopheryl acetate, 18.8; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridoxine hydrochloride, 1.8; mination method (C-690; Sigma). Oxidative radical pro- niacin, 36.4; Ca- -pantothenate, 12.7; myo-inositol, 181.8; D-biotin, 0.27; folic duction by hemocytes during respiratory burst was acid, 0.68; p-aminobezoic acid, 18.2; menadione, 1.8; retinyl acetate, 0.73; cholecalficerol, 0.003; cyanocobalamin, 0.003 measured through the nitro blue tetrazolium (NBT) assay described by Dantzler et al. (2001). PO activity was mea- the growth of microflora. A 12:12 h light/dark regime sured spectrophotometrically by recording the formation (08:00–19:00 h, light period) was maintained by timed of dopachrome produced from L-dihydroxyphenylalanine fluorescent lighting. The water temperature was main- (L-DOPA, Sigma) following the procedure of Hernández- tained at 28 ± 1 °C, pH ranged from 7.04 to 8.04, and dis- López et al. (1996). Lysozyme activity was determined fol- −1 solved oxygen was kept above 6.0 mg L and total lowing previously described method (Paglia and Valentine, ammonia nitrogen and nitrite were kept <0.1 and 1967). SOD activity was measured by the percentage reac- −1 0.005 mg L , respectively. Growth of shrimp was mea- tion inhibition rate of enzyme with WST-1 (water-soluble sured with 2-week intervals. Feeding was stopped 16 h tetrazolium dye) substrate and xanthine oxidase using a prior to weighing or hemolymph sampling to minimize SOD Assay Kit (Sigma, 19160) according to the manufac- handling stress on the shrimp. turer’s instructions. Each endpoint assay was monitored by absorbance at 450 nm (the absorbance wavelength for Sample collection and analyses the colored product of WST-1 reaction with superoxide) At the end of the feeding trial, all shrimp in each tank after 20 min of reaction time at 37 °C. The percent inhib- were counted and bulk-weighed for calculation of ition was normalized by mg protein and presented as growth parameters and survival. Five shrimp per tank SOD activity units. Ig level was determined according to (fifteen shrimp per dietary treatment) in inter-molt stage the method described by Siwicki et al. (1994). Briefly, were randomly captured, anesthetized with ice-cold plasma total protein content was measured using a micro- water and hemolymph samples (200 μl) were individu- protein determination method (C-690; Sigma), prior to ally collected from ventral sinus of shrimp using a 1-mL and after precipitating down the Ig molecules, using a syringe. Then, the hemolymph (200 μL) was filled with 12% solution of polyethylene glycol (Sigma). The differ- an equal volume of anticoagulant solution (200 μL) ence in protein content represents the Ig content. GPX ac- (Alsever’s solution, Sigma). The molt stage of the shrimp tivity was assayed using a kit (Biovision, Inc., Milpitas, was determined by an examination of uropoda CA, USA). Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 4 of 7 Challenge test that fed the BF8 diet. The highest survival rate was ob- At the end of the feeding trial, 12 shrimp from each tank tained in BF0.5 group which was significantly different (24 shrimp per treatment) were randomly selected and from other dietary groups. subjected to a bacterial challenge. V. harveyi was used as Shrimp fed BF0.5 and BF4 diets showed significantly the pathogenic agent (provided by the Marine Microbiol- higher NBT activity than shrimp fed the Con diet ogy Laboratory of Jeju National University). The shrimp (Table 4). Significantly higher PO activity was found in were injected intramuscularly with V. harveyi suspension shrimp fed BF6 diet than those fed the Con diet. Signifi- 8 −1 containing 2 × 10 CFU mL and distributed into four- cant increment in GPx activity was observed in BF6 teen 120-L acryl tanks. The pathogenic dose of bacterium groups in comparison to the Con group. Even though had previously been determined in a preliminary test numerically higher values of other examined non- using similar size of shrimp. After injection, the challen- specific immune parameters were observed in shrimp ging shrimp was not fed the diets and the mortality was fed the biofloc supplemented diets, the differences were monitored for 19 days. not significant (P ˃ 0.05). During the challenge test, the first dramatic mortality Statistical analysis was observed on the 13th day after injection and shrimp All dietary treatments were assigned by a completely ran- fed the Con diet showed the lowest disease resistance domized design. Data were subjected to one-way analysis of compared to all other groups (Fig. 1). At the end of the variance (ANOVA) in SPSS version 12.0 (SPSS Inc., Chi- challenge test, significantly higher survival rate was cago, IL, USA). When ANOVA identified differences found in BF4 group compared to the Con groups; how- among groups, the difference in means was made with LSD ever, no significant difference was found among other multiple range tests. Statistical significance was determined biofloc supplemented groups. at P ˂ 0.05. Data are presented as mean ± SD. Percentage data were arcsine transformed before analysis. Discussion Beneficial role of BFT system in penaeid shrimp has well Results been documented (Hari et al., 2006; Xu and Pan 2012). Growth performance and feed utilization of the shrimp Recently, it was reported that use of biofloc as a dietary were significantly affected by dietary supplementation of supplement had enhanced growth rate of L. vannamei the biofloc compared to those of shrimp fed the Con (Ju et al., 2008; Kuhn et al., 2009, 2010). In the present diet (Table 3). Final body weight, weight gain and spe- study, dietary supplementation of biofloc at 4% level sig- cific growth rate of shrimp fed BF4 diet were signifi- nificantly (P < 0.05) enhanced growth, PER and FCR in cantly increased compared to those fed the Con diet. the shrimp. It has been documented that bioflocs are the Dietary inclusion of the biofloc to the Con diet at level rich source of many bioactive compounds, such as carot- of 4.0% resulted in significantly higher protein efficiency enoids, chlorophylls, phytosterols, bromophenols, amino ratio (PER) and lower feed conversion ratio (FCR) com- sugars (Ju et al., 2008) and anti-bacterial compounds pared to the Con diet. Significantly higher feed intake (Crab et al., 2010). Thus, the enhanced growth perform- (FI) was observed in shrimp fed the Con diet than in ance of shrimp fed the biofloc containing diet in the Table 3 Growth performance and feed utilization of L. vannamei (initial BW: 1.01 ± 0.01 g) fed the seven experimental diets for 8 weeks 1 2 3 4 5 6 7 Treatment FBW WG SGR FCR PER FI Survival b b b a b a b Con 6.63 ± 0.55 565 ± 49.6 3.32 ± 0.13 2.25 ± 0.21 1.10 ± 0.11 12.8 ± 0.13 79.6 ± 16.0 ab ab ab ab ab ab a BF0.5 7.12 ± 0.44 610 ± 41.5 3.44 ± 0.10 2.09 ± 0.16 1.15 ± 0.09 12.8 ± 0.02 98.1 ± 3.21 ab ab ab ab ab ab b BF1 7.12 ± 0.82 608 ± 70.2 3.43 ± 0.17 2.10 ± 0.25 1.18 ± 0.15 12.8 ± 0.01 85.2 ± 8.49 ab ab ab ab ab ab b BF2 7.34 ± 0.71 635 ± 67.5 3.50 ± 0.16 2.01 ± 0.22 1.24 ± 0.13 12.7 ± 0.01 88.9 ± 0.00 a a a b a ab b BF4 7.90 ± 0.13 677 ± 8.40 3.60 ± 0.02 1.84 ± 0.04 1.32 ± 0.03 12.7 ± 0.04 85.2 ± 3.21 ab ab ab ab ab ab b BF6 7.27 ± 0.81 627 ± 81.4 3.47 ± 0.19 2.05 ± 0.27 1.20 ± 0.16 12.7 ± 0.04 88.9 ± 5.56 ab ab ab ab ab b b BF8 7.56 ± 0.38 639 ± 39.8 3.51 ± 0.10 1.91 ± 0.15 1.28 ± 0.10 12.5 ± 0.48 90.7 ± 6.42 Values are mean of triplicate groups and presented as mean ± SD. Values in the same column having different letters are significantly different (LSD; P < 0.05) FBW (g) = Final body weight Weight gain (%) = [(final body weight - initial body weight)/initial body weight] x 100 Specific growth rate (%) = 100 x [ln(final body weight) – ln(initial body weight)]/days Feed conversion ratio = dry feed fed/wet weight gain Protein efficiency ratio = fish weight gain (g)/protein Feed intake (g/fish) = dry feed consumed (g)/fish Survival (%) Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 5 of 7 Table 4 Non-specific immune responses of L. vannamei fed the seven experimental diets for 8 weeks 1 2 3 4 5 6 Treatment THC NBT PO SOD Ig GPx b b b Con 0.33 ± 0.01 0.27 ± 0.04 0.16 ± 0.01 238 ± 18.6 31.2 ± 12.7 107 ± 37.7 a b ab BF0.5 1.95 ± 1.30 0.32 ± 0.00 0.18 ± 0.02 240 ± 3.56 44.9 ± 13.6 133 ± 32.4 ab b ab BF1 4.14 ± 0.52 0.28 ± 0.03 0.17 ± 0.02 235 ± 30.8 30.6 ± 4.12 181 ± 25.4 ab b ab BF2 2.26 ± 0.71 0.28 ± 0.02 0.18 ± 0.02 254 ± 15.0 38.1 ± 6.97 157 ± 45.3 a b ab BF4 4.52 ± 3.81 0.32 ± 0.04 0.16 ± 0.03 249 ± 12.0 39.6 ± 6.29 184 ± 76.5 ab a a BF6 3.12 ± 1.46 0.29 ± 0.02 0.24 ± 0.04 250 ± 37.5 43.6 ± 18.3 205 ± 55.3 ab b ab BF8 4.40 ± 4.29 0.31 ± 0.03 0.18 ± 0.04 246 ± 9.13 37.4 ± 2.69 159 ± 40.9 Values are mean of triplicate groups and presented as mean ± SD. Values in the same column having different letters are significantly different (LSD; P < 0.05) 1 3 Total haemocyte count (10 cells/mL) Nitro blue tetrazolium activity Phenoloxidase activity Superoxide dismutase (% inhibition) 5 −1 Total immunoglobulin (mg mL ) 6 −1 Glutathione peroxidase activity (mU mL ) current study might be explained by a bioactive or pro- BF6 and BF8 in the present study were comparable or biotic microbial components, such as Bacillus or Lacto- relatively higher compared to the Con diet, indicating bacillus spp. present in the biofloc. On the other hand, non negative growth effects in shrimp by the dietary bio- biofloc supplementation at 8% level (BF8) did not result floc supplementation up to 8%. in proportionate enhancement in growth rate or FCR Shrimp lack specific or adaptive immune system that re- compared to those of the Con diet. Kuhn et al. (2010) lies entirely on their innate immune mechanisms including supplemented a biofloc in L. vannamei diet and re- both cellular and humoral responses for defense against corded significantly higher growth rate at 10 and 15%, pathogens (Vazquez et al., 2009). NBT activity was in- but no significant difference at 20 and 30% dietary bio- creased in the shrimp fed BF0.5 and BF4, while significantly floc inclusion. The present results also agree with those higher PO activity was observed in BF6 group compared to of Wang (2007) and Anand et al. (2013) who reported those fed the Con diet. It was suggested that shrimp may that increment of dietary probiotic or periphytic algae in releasesomeusefulsubstances into gastrointestinal tract shrimp does not proportionally increase growth per- that could potentially stimulate innate immune response formance of shrimp. Moreover, reduction in growth of (especially phagocytosis) and may release more hemocytes fishes was recorded at high level of microbial supple- into their circulation when they ingested biofloc (Zhao mentation (Kiessling and Askbrandt 1993; Ajiboye et al., et al., 2012). Some beneficial bacteria such as Bacillus sp.in 2012) as microbial products at higher levels tend to re- the ingested biofloc could have facilitate modification of duce feed palatability and digestibility (Kiessling and physiological and immunological status of the host through Askbrandt 1993). However, the growth performance of a colonization in the gastrointestinal tract and have Fig 1 Survival rate of L. vannamei after a challenge against V. harveyi Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 6 of 7 triggered to change the endogenous microbiota (Johnson biofloc as a dietary supplement would be dependent upon et al., 2008; Li et al., 2009). Ju et al. (2008) noted that both the process method of the biofloc and the quantity of or- microbial components (Bacillus sp.) and bioactive com- ganic compounds and beneficial microorganism. pounds (e.g., polysaccharides and carotenoids) in biofloc Abbreviations could exert an immune-stimulating effect and this action BFT: Biofloc technology; FCR: Feed conversion ratio; FI: Feed intake; would be continuous as long as the shrimp consumes bio- GPx: Glutathione peroxidase; Ig: Total immunoglobulin; NBT: Nitro blue tetrazolium; PER: Protein efficiency ratio; PO: Phenoloxidase; SOD: Superoxide floc. However, the modes of action of biofloc on innate im- dismutase; THC: Total hemocyte counts mune mechanism of shrimp are very complicated and still unknown up to date. Further study is required for more de- Acknowledgements This research was supported by the Korea Ministry of Oceans and Fisheries, tailed information and knowledge on this. Research on anti- which provided federal funds to support the “Development of functional feed oxidants has been attracted in aquaculture because fish or additives for shrimp using useful organic matter in biofloc-based rearing water.” shrimp are susceptible to oxidative stress by disease or en- vironmental changes (Liu and Chen, 2004; Castex et al., Funding This study was funded by the Korea Ministry of Oceans and Fisheries, which 2010). Shrimp possess an integrated antioxidant system in- provided federal funds to support the “Development of functional feed additives cluding enzymatic and non-enzymatic antioxidants to for shrimp using useful organic matter in biofloc-based rearing water.” maintain normal oxidant status, especially to cope with nat- Availability of data and materials ural or induced stressors (Castex et al., 2009; Parrilla-Taylor All datasets analyzed during the current study are available from the and Zenteno-Savín, 2011). Generally, the antioxidant cap- corresponding author on reasonable request. ability of an organism under certain condition can reflect Authors’ contributions its health status. In the present study, increased GPx activ- CR and SH conducted the feeding trial, analysis, and drafted the ity was observed in shrimp fed BF6 diet compared to those manuscript. SJ manufactured the experimental diet. KJ designed this fed the Con diet. This result indicates that the antioxidant study, led in analyzing data derived from this study, and revised the manuscript. All authors read and approved the final manuscript. system of the shrimp can be enhanced by unknown com- ponents present in biofloc in some way. Based on its com- Ethics approval and consent to participate position characteristics, the biofloc may play a role in Experimental protocols followed the guidelines of the Animal Care and Use antioxidant activity because of its carotenoids and fat- Committee of Jeju National University. soluble vitamins (Ju et al., 2008) and improve the efficiency Consent for publication of feed utilization by stimulating activities of digestive en- Not applicable. zymes because of high protease and amylase activity in bio- Competing interests floc (Xu and Pan 2012; Xu et al. 2012). Similarly, Xu and The authors declare that they have no competing interests. Pan (2013) reported an increase in total antioxidant cap- acity and SOD activity of L. vannamei reared in a biofloc- Author details Department of Marine Life Sciences, Jeju National University, Jeju 63243, based tanks system. South Korea. Neo Environmental Business Co., Dangjin-si, During the challenge test against V. harveyi, significantly Chungcheongnam-do, South Korea. higher survival rate was found in BF4 group compared to Received: 20 January 2017 Accepted: 6 July 2017 the Con group. Similar observations were found by Ekasari et al. (2014) where following a challenge test by in- jection with infectious myonecrosis virus (IMNV) the sur- References vival of the challenged shrimp from the experimental Ajiboye OO, Yakubu AF, Adams TE. A perspective on the ingestion and nutritional effects of feed additives in farmed fish species. World J Fish & Marine Sci. 2012;4: biofloc groups was significantly higher when compared to 87–101. the challenged shrimp from the control treatment. Anand PSS, Kumar S, Panigrahi A, Ghoshal TK, Dayal JS, Biswas G, et al. Effects of C:N ratio and substrate integration on periphyton biomass, microbial dynamics and growth of Penaeus monodon juveniles. 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Supplemental effects of biofloc powder on growth performance, innate immunity, and disease resistance of Pacific white shrimp Litopenaeus vannamei

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Springer Journals
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Copyright © 2017 by The Author(s)
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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

An 8-week feeding trial was conducted to study the effect of dietary supplementation of a biofloc powder on growth performance and non-specific immune response of Litopenaeus vannamei. Seven experimental diets were prepared with supplementation of graded levels of dried biofloc powder by 0, 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0% (designated as Con, BF0.5, BF1, BF2, BF4, BF6, and BF8, respectively). Triplicate groups of shrimp (1.01 ± 0.01 g) were hand-fed with one of the diets four times a day. At the end of the feeding trial, significantly (P ˂ 0.05) higher growth performance and feed utilization were obtained in BF4 groups compared to those fed the Con diet. The innate immunity of shrimp was improved by the dietary supplementation of biofloc. Dietary inclusion of biofloc at the level of 4.0% significantly increased disease resistance of shrimp against Vibrio harveyi. The results indicate that biofloc might be used as a dietary supplement for growth performance, innate immunity and disease resistance of Pacific white shrimp. Keywords: Biofloc powder, Innate immunity, Growth performance, Disease resistance, Litopenaeus vannamei Background reuse excreted dissolved nitrogen by heterotrophic bacteria Diseases caused by infectious microorganisms are known by controlling carbon and nitrogen ratio in the culture to be one of the major constraints in the shrimp aquacul- water (Avnimelech, 1999). The biofloc in the BFT system is ture industry for the past decades (Ekasari et al., 2014). formed by aggregating many substances such as, other mi- Thus, there is a need to control the disease outbreaks in croorganisms, microalgae, zooplankton, and trapped or- this sector. A concept of a functional feed is an emerging ganic particles or solids from uneaten feeds (De Schryver paradigm in aquaculture industry to develop nutritionally et al., 2008; Crab et al., 2012; Ekasari et al., 2014). It has balanced diets with functions to control the diseases and been demonstrated that the active and condensed microor- culture water system by feed additives (Li and Gatlin ganisms together with suspended organic or inorganic parti- 2004; Lee et al., 2013; Wongsasak et al., 2015). cles tend to from biofloc, which can be consumed The application of biofloc technology (BFT) in shrimp constantly by cultured shrimp as a natural food source aquaculture has gained great attention. BFT offers a prac- (Burford et al., 2004; Wasielesky et al., 2006; Kent et al., tical solution to control culture water quality effectively with 2011). Thus, the biofloc itself provides biomass which is negligible water exchange and improves shrimp growth per- served as nutrients for shrimp especially as a protein source formances in the healthy culture environment (De Schryver or immunostimulants. Previous studies showed that biofloc et al., 2008; Stokstad, 2010; Avnimelech, 2012; Crab et al., had enhanced cellular immune response and antioxidant 2012; Xu and Pan 2013). The mechanism of the BFT is to status of cultured shrimp by its richness in microbes and bioactive compounds (Ju et al., 2008; Xu and Pan 2013). Similar result was found when L. vannamei were fed a for- * Correspondence: kjlee@jejunu.ac.kr Department of Marine Life Sciences, Jeju National University, Jeju 63243, mulated 35% crude protein diet in biofloc-based culture South Korea tanks for a period of 30 days; however, it is not clear if Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 2 of 7 feeding a relatively low protein diet (35%) with the contribu- Table 1 Biofloc chemical composition (dry matter) tion of biofloc could sustain the health status of cultured Parameter Biofloc shrimp in such systems (Xu and Pan 2013). Jang et al. Proximate composition (%) (2011) also reported that the expression of Crude protein 28.7 prophenoloxidase-activating enzyme was significantly in- Crude lipid 2.30 creased in the shrimp reared in a biofloc system. Crude ash 43.0 Pacific white shrimp is one of the most important Amino acid composition (%) shrimp species currently being cultured in many coun- tries. Over the past decade, mass production of L. vanna- Methionine 0.36 mei has been demonstrated in biofloc-based intensive Arginine 1.17 culture systems under high aeration and negligible water Phenylalanine 1.16 exchange (Avnimelech, 2012; Haslun et al., 2012). Re- Leucine 1.81 cently, it was demonstrated that dietary inclusion of bio- Isoleucine 1.02 floc had enhanced growth performance of L. vannamei Lysine 1.11 (Ju et al., 2008; Kuhn et al., 2010; Bauer et al., 2012). There is a lack of information to support the role of biofloc in di- Valine 1.51 ets on growth and immune response in the species. Threonine 1.31 Hence, to investigate the suitability of biofloc as a dietary Histidine 0.41 supplement, we included it in diets at different levels and Trace mineral composition (mg/kg) fed to L. vannamei. The present study aims to evaluate Zinc 521.8 the effect of dietary supplementation of biofloc on growth Aluminum 287.8 performance, non-specific immune response, and suscep- tibility to bacterial infection caused by V. harveyi in L. Manganese 180.7 vannamei juveniles. Iron 146.9 Copper 34.0 Methods Aluminum silicate 3.4 Preparation of biofloc Cadmium 2.7 The biofloc was collected from L. vannamei BFT tanks. A biofloc suspension was prepared in a 300 ton polypropyl- ene tank (45 × 6.5 m) filled with seawater and stocked with South Korea) and pelleted through a pellet machine (SP-50; L. vannamei. The C: N ratio was maintained at 15:1 using Gumgang Engineering, Daegu, Korea). The pelleted diets molasses as a carbon source. Probiotics containing Bacil- were dried at25°Cfor 12 h, andstoredat −24 °C until lus subtilis, Lactobacillus casei and Saccharomyces cerevi- used. Formulation and proximate composition of the ex- siae (Total bacterial account = 1 × 10 CFU/mL) were perimental diets are shown in Table 2. added into the biofloc ponds once a week until enough biofloc sample was collected. Biofloc collection was con- Shrimp and feeding trial tinued for 12 h with enough and strong aeration in the The feeding trial was conducted in indoor shrimp culture BFT tanks. During the aeration, sludges (flocs) were facilities at the Marine Science Institute of the Jeju Na- formed on the water surface. The collected flocs were rap- tional University (Jeju, South Korea). Juvenile L. vannamei idly frozen at −80 °C, and then vacuum freeze-dried. The was obtained from NeoEnBiz shrimp farm (Dangjin, dried flocs were ground into fine powder (<100 μm) and South Korea). Shrimps were fed a commercial diet (35% kept in airtight containers in refrigerator until used in ex- crud protein) twice daily for 10 days to be acclimated to perimental diets. Chemical composition of the biofloc the experimental conditions and facilities. Then, the powder is provided in Table 1. shrimps (initial mean body weight, 1.01 ± 0.01 g) were randomly distributed into twenty one acryl aquaria of Experimental diets and design 96 L capacity at a density of 18 shrimp per aquarium. Each Seven experimental diets were formulated to be isonitro- aquarium was supplied with filtered seawater and aeration −1 genous (400 g kg crude protein) and isocaloric (16 MJ kg to maintain enough dissolved oxygen. Triplicate groups of −1 ). A basal fishmeal based diet was regarded as a control shrimp were hand-fed with one of the test diets four times and supplemented with biofloc at the incremental levels of a day at 08:00, 12.00, 16.00, and 18:00 h for 8 weeks. Daily 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0% (designated as Con, BF0.5, feeding rates were slowly reduced from 10 to 8% of wet BF1, BF2, BF4, BF6, and BF8, respectively) at the expense of body weight during the 8-week feeding period. Water in soybean meal and wheat flour. All dry ingredients were the aquaria was exchanged every 3-day intervals while in- thoroughly mixed in a feed mixer (NVM-16, Gyeonggido, side of the aquaria were cleaned by a sponge to prevent Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 3 of 7 Table 2 Dietary formulation and proximate composition of the (Robertson et al., 1987). The hemolymph-anticoagulant seven experimental diets for L. vannamei (% dry matter) mixture (diluted hemolymph) was placed in five sterile Ingredients Experimental diets eppendorf tubes containing equal volume for the determin- ation of the total hemocyte counts (THC) and respiratory Con BF0.5 BF1 BF2 BF4 BF6 BF8 burst activity. After the abovementioned measurements Brown fishmeal 28.0 28.0 28.0 28.0 28.0 28.0 28.0 with diluted hemolymph, the remained samples was Soybean meal 30.0 30.0 30.0 29.5 28.5 27.5 26.5 centrifuged at 800×g for 20 min using a high-speed refriger- Squid liver meal 3.00 3.00 3.00 3.00 3.00 3.00 3.00 ated microcentrifuge (Micro 17 TR; HanilBioMed Inc., Wheat flour 29.0 28.5 28.0 27.5 26.5 25.5 24.5 Gwangju, Korea) and stored at −70 °C for determination of Starch 5.00 5.00 5.00 5.00 5.00 5.00 5.00 phenoloxidase (PO), superoxide dismutase (SOD) activities, Fish oil 1.00 1.00 1.00 1.00 1.00 1.00 1.00 total immunoglobulin (Ig) level and glutathione peroxidase (GPx) activity. Mineral mix 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Analyses of moisture and ash contents of biofloc Vitamin mix 1.00 1.00 1.00 1.00 1.00 1.00 1.00 powder and diet samples were performed by the Choline chloride 1.00 1.00 1.00 1.00 1.00 1.00 1.00 standard procedures (AOAC, 1995). Crude protein Lecithin 1.00 1.00 1.00 1.00 1.00 1.00 1.00 was measured by using an automatic Kjeltec Analyzer Biofloc 0.00 0.50 1.00 2.00 4.00 6.00 8.00 Unit 2300 (Foss Tecator, Höganäs, Sweden), and Chemical composition (% dry matter) crude lipid was determined using the Soxhlet method with extraction in diethyl ether (Soxhlet Extraction Dry matter 93.2 93.8 94.0 93.2 93.1 93.6 93.5 System C-SH6, Korea). Crude protein 40.8 40.9 40.7 40.6 41.0 41.0 41.0 Crude lipid 6.70 7.50 7.10 7.00 7.60 7.30 7.50 Monitoring of non-specific immune responses Crude Ash 8.00 8.40 8.30 8.60 9.30 10.0 10.8 A drop of the diluted haemolymph was placed in a Energy, MJ/kg diet 16.6 16.6 16.6 16.5 16.4 16.2 16.1 hemocytometer to measure THC using an inverted phase- Mineral premix (g/kg mixture): MgSO .7H O, 80.0; NaH PO .2H O, 370.0; KCl, contrast microscope (Olympus, Model CH30RF200, 4 2 2 4 2 130.0; Ferric citrate, 40.0; ZnSO .7H O, 20.0; Ca-lactate, 356.5; CuCl, 0.2; 4 2 Olympus Optical Co., LTD, Japan). Diluted hemolymph AlCl .6H O, 0.15; Na Se O , 0.01; MnSO .H O, 2.0; CoCl .6H O, 1.0 3 2 2 2 3 4 2 2 2 protein content was measured using a microprotein deter- Vitamin premix (g/kg mixture): L-ascorbic acid, 121.2; DL-tocopheryl acetate, 18.8; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridoxine hydrochloride, 1.8; mination method (C-690; Sigma). Oxidative radical pro- niacin, 36.4; Ca- -pantothenate, 12.7; myo-inositol, 181.8; D-biotin, 0.27; folic duction by hemocytes during respiratory burst was acid, 0.68; p-aminobezoic acid, 18.2; menadione, 1.8; retinyl acetate, 0.73; cholecalficerol, 0.003; cyanocobalamin, 0.003 measured through the nitro blue tetrazolium (NBT) assay described by Dantzler et al. (2001). PO activity was mea- the growth of microflora. A 12:12 h light/dark regime sured spectrophotometrically by recording the formation (08:00–19:00 h, light period) was maintained by timed of dopachrome produced from L-dihydroxyphenylalanine fluorescent lighting. The water temperature was main- (L-DOPA, Sigma) following the procedure of Hernández- tained at 28 ± 1 °C, pH ranged from 7.04 to 8.04, and dis- López et al. (1996). Lysozyme activity was determined fol- −1 solved oxygen was kept above 6.0 mg L and total lowing previously described method (Paglia and Valentine, ammonia nitrogen and nitrite were kept <0.1 and 1967). SOD activity was measured by the percentage reac- −1 0.005 mg L , respectively. Growth of shrimp was mea- tion inhibition rate of enzyme with WST-1 (water-soluble sured with 2-week intervals. Feeding was stopped 16 h tetrazolium dye) substrate and xanthine oxidase using a prior to weighing or hemolymph sampling to minimize SOD Assay Kit (Sigma, 19160) according to the manufac- handling stress on the shrimp. turer’s instructions. Each endpoint assay was monitored by absorbance at 450 nm (the absorbance wavelength for Sample collection and analyses the colored product of WST-1 reaction with superoxide) At the end of the feeding trial, all shrimp in each tank after 20 min of reaction time at 37 °C. The percent inhib- were counted and bulk-weighed for calculation of ition was normalized by mg protein and presented as growth parameters and survival. Five shrimp per tank SOD activity units. Ig level was determined according to (fifteen shrimp per dietary treatment) in inter-molt stage the method described by Siwicki et al. (1994). Briefly, were randomly captured, anesthetized with ice-cold plasma total protein content was measured using a micro- water and hemolymph samples (200 μl) were individu- protein determination method (C-690; Sigma), prior to ally collected from ventral sinus of shrimp using a 1-mL and after precipitating down the Ig molecules, using a syringe. Then, the hemolymph (200 μL) was filled with 12% solution of polyethylene glycol (Sigma). The differ- an equal volume of anticoagulant solution (200 μL) ence in protein content represents the Ig content. GPX ac- (Alsever’s solution, Sigma). The molt stage of the shrimp tivity was assayed using a kit (Biovision, Inc., Milpitas, was determined by an examination of uropoda CA, USA). Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 4 of 7 Challenge test that fed the BF8 diet. The highest survival rate was ob- At the end of the feeding trial, 12 shrimp from each tank tained in BF0.5 group which was significantly different (24 shrimp per treatment) were randomly selected and from other dietary groups. subjected to a bacterial challenge. V. harveyi was used as Shrimp fed BF0.5 and BF4 diets showed significantly the pathogenic agent (provided by the Marine Microbiol- higher NBT activity than shrimp fed the Con diet ogy Laboratory of Jeju National University). The shrimp (Table 4). Significantly higher PO activity was found in were injected intramuscularly with V. harveyi suspension shrimp fed BF6 diet than those fed the Con diet. Signifi- 8 −1 containing 2 × 10 CFU mL and distributed into four- cant increment in GPx activity was observed in BF6 teen 120-L acryl tanks. The pathogenic dose of bacterium groups in comparison to the Con group. Even though had previously been determined in a preliminary test numerically higher values of other examined non- using similar size of shrimp. After injection, the challen- specific immune parameters were observed in shrimp ging shrimp was not fed the diets and the mortality was fed the biofloc supplemented diets, the differences were monitored for 19 days. not significant (P ˃ 0.05). During the challenge test, the first dramatic mortality Statistical analysis was observed on the 13th day after injection and shrimp All dietary treatments were assigned by a completely ran- fed the Con diet showed the lowest disease resistance domized design. Data were subjected to one-way analysis of compared to all other groups (Fig. 1). At the end of the variance (ANOVA) in SPSS version 12.0 (SPSS Inc., Chi- challenge test, significantly higher survival rate was cago, IL, USA). When ANOVA identified differences found in BF4 group compared to the Con groups; how- among groups, the difference in means was made with LSD ever, no significant difference was found among other multiple range tests. Statistical significance was determined biofloc supplemented groups. at P ˂ 0.05. Data are presented as mean ± SD. Percentage data were arcsine transformed before analysis. Discussion Beneficial role of BFT system in penaeid shrimp has well Results been documented (Hari et al., 2006; Xu and Pan 2012). Growth performance and feed utilization of the shrimp Recently, it was reported that use of biofloc as a dietary were significantly affected by dietary supplementation of supplement had enhanced growth rate of L. vannamei the biofloc compared to those of shrimp fed the Con (Ju et al., 2008; Kuhn et al., 2009, 2010). In the present diet (Table 3). Final body weight, weight gain and spe- study, dietary supplementation of biofloc at 4% level sig- cific growth rate of shrimp fed BF4 diet were signifi- nificantly (P < 0.05) enhanced growth, PER and FCR in cantly increased compared to those fed the Con diet. the shrimp. It has been documented that bioflocs are the Dietary inclusion of the biofloc to the Con diet at level rich source of many bioactive compounds, such as carot- of 4.0% resulted in significantly higher protein efficiency enoids, chlorophylls, phytosterols, bromophenols, amino ratio (PER) and lower feed conversion ratio (FCR) com- sugars (Ju et al., 2008) and anti-bacterial compounds pared to the Con diet. Significantly higher feed intake (Crab et al., 2010). Thus, the enhanced growth perform- (FI) was observed in shrimp fed the Con diet than in ance of shrimp fed the biofloc containing diet in the Table 3 Growth performance and feed utilization of L. vannamei (initial BW: 1.01 ± 0.01 g) fed the seven experimental diets for 8 weeks 1 2 3 4 5 6 7 Treatment FBW WG SGR FCR PER FI Survival b b b a b a b Con 6.63 ± 0.55 565 ± 49.6 3.32 ± 0.13 2.25 ± 0.21 1.10 ± 0.11 12.8 ± 0.13 79.6 ± 16.0 ab ab ab ab ab ab a BF0.5 7.12 ± 0.44 610 ± 41.5 3.44 ± 0.10 2.09 ± 0.16 1.15 ± 0.09 12.8 ± 0.02 98.1 ± 3.21 ab ab ab ab ab ab b BF1 7.12 ± 0.82 608 ± 70.2 3.43 ± 0.17 2.10 ± 0.25 1.18 ± 0.15 12.8 ± 0.01 85.2 ± 8.49 ab ab ab ab ab ab b BF2 7.34 ± 0.71 635 ± 67.5 3.50 ± 0.16 2.01 ± 0.22 1.24 ± 0.13 12.7 ± 0.01 88.9 ± 0.00 a a a b a ab b BF4 7.90 ± 0.13 677 ± 8.40 3.60 ± 0.02 1.84 ± 0.04 1.32 ± 0.03 12.7 ± 0.04 85.2 ± 3.21 ab ab ab ab ab ab b BF6 7.27 ± 0.81 627 ± 81.4 3.47 ± 0.19 2.05 ± 0.27 1.20 ± 0.16 12.7 ± 0.04 88.9 ± 5.56 ab ab ab ab ab b b BF8 7.56 ± 0.38 639 ± 39.8 3.51 ± 0.10 1.91 ± 0.15 1.28 ± 0.10 12.5 ± 0.48 90.7 ± 6.42 Values are mean of triplicate groups and presented as mean ± SD. Values in the same column having different letters are significantly different (LSD; P < 0.05) FBW (g) = Final body weight Weight gain (%) = [(final body weight - initial body weight)/initial body weight] x 100 Specific growth rate (%) = 100 x [ln(final body weight) – ln(initial body weight)]/days Feed conversion ratio = dry feed fed/wet weight gain Protein efficiency ratio = fish weight gain (g)/protein Feed intake (g/fish) = dry feed consumed (g)/fish Survival (%) Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 5 of 7 Table 4 Non-specific immune responses of L. vannamei fed the seven experimental diets for 8 weeks 1 2 3 4 5 6 Treatment THC NBT PO SOD Ig GPx b b b Con 0.33 ± 0.01 0.27 ± 0.04 0.16 ± 0.01 238 ± 18.6 31.2 ± 12.7 107 ± 37.7 a b ab BF0.5 1.95 ± 1.30 0.32 ± 0.00 0.18 ± 0.02 240 ± 3.56 44.9 ± 13.6 133 ± 32.4 ab b ab BF1 4.14 ± 0.52 0.28 ± 0.03 0.17 ± 0.02 235 ± 30.8 30.6 ± 4.12 181 ± 25.4 ab b ab BF2 2.26 ± 0.71 0.28 ± 0.02 0.18 ± 0.02 254 ± 15.0 38.1 ± 6.97 157 ± 45.3 a b ab BF4 4.52 ± 3.81 0.32 ± 0.04 0.16 ± 0.03 249 ± 12.0 39.6 ± 6.29 184 ± 76.5 ab a a BF6 3.12 ± 1.46 0.29 ± 0.02 0.24 ± 0.04 250 ± 37.5 43.6 ± 18.3 205 ± 55.3 ab b ab BF8 4.40 ± 4.29 0.31 ± 0.03 0.18 ± 0.04 246 ± 9.13 37.4 ± 2.69 159 ± 40.9 Values are mean of triplicate groups and presented as mean ± SD. Values in the same column having different letters are significantly different (LSD; P < 0.05) 1 3 Total haemocyte count (10 cells/mL) Nitro blue tetrazolium activity Phenoloxidase activity Superoxide dismutase (% inhibition) 5 −1 Total immunoglobulin (mg mL ) 6 −1 Glutathione peroxidase activity (mU mL ) current study might be explained by a bioactive or pro- BF6 and BF8 in the present study were comparable or biotic microbial components, such as Bacillus or Lacto- relatively higher compared to the Con diet, indicating bacillus spp. present in the biofloc. On the other hand, non negative growth effects in shrimp by the dietary bio- biofloc supplementation at 8% level (BF8) did not result floc supplementation up to 8%. in proportionate enhancement in growth rate or FCR Shrimp lack specific or adaptive immune system that re- compared to those of the Con diet. Kuhn et al. (2010) lies entirely on their innate immune mechanisms including supplemented a biofloc in L. vannamei diet and re- both cellular and humoral responses for defense against corded significantly higher growth rate at 10 and 15%, pathogens (Vazquez et al., 2009). NBT activity was in- but no significant difference at 20 and 30% dietary bio- creased in the shrimp fed BF0.5 and BF4, while significantly floc inclusion. The present results also agree with those higher PO activity was observed in BF6 group compared to of Wang (2007) and Anand et al. (2013) who reported those fed the Con diet. It was suggested that shrimp may that increment of dietary probiotic or periphytic algae in releasesomeusefulsubstances into gastrointestinal tract shrimp does not proportionally increase growth per- that could potentially stimulate innate immune response formance of shrimp. Moreover, reduction in growth of (especially phagocytosis) and may release more hemocytes fishes was recorded at high level of microbial supple- into their circulation when they ingested biofloc (Zhao mentation (Kiessling and Askbrandt 1993; Ajiboye et al., et al., 2012). Some beneficial bacteria such as Bacillus sp.in 2012) as microbial products at higher levels tend to re- the ingested biofloc could have facilitate modification of duce feed palatability and digestibility (Kiessling and physiological and immunological status of the host through Askbrandt 1993). However, the growth performance of a colonization in the gastrointestinal tract and have Fig 1 Survival rate of L. vannamei after a challenge against V. harveyi Lee et al. Fisheries and Aquatic Sciences (2017) 20:15 Page 6 of 7 triggered to change the endogenous microbiota (Johnson biofloc as a dietary supplement would be dependent upon et al., 2008; Li et al., 2009). Ju et al. (2008) noted that both the process method of the biofloc and the quantity of or- microbial components (Bacillus sp.) and bioactive com- ganic compounds and beneficial microorganism. pounds (e.g., polysaccharides and carotenoids) in biofloc Abbreviations could exert an immune-stimulating effect and this action BFT: Biofloc technology; FCR: Feed conversion ratio; FI: Feed intake; would be continuous as long as the shrimp consumes bio- GPx: Glutathione peroxidase; Ig: Total immunoglobulin; NBT: Nitro blue tetrazolium; PER: Protein efficiency ratio; PO: Phenoloxidase; SOD: Superoxide floc. However, the modes of action of biofloc on innate im- dismutase; THC: Total hemocyte counts mune mechanism of shrimp are very complicated and still unknown up to date. Further study is required for more de- Acknowledgements This research was supported by the Korea Ministry of Oceans and Fisheries, tailed information and knowledge on this. Research on anti- which provided federal funds to support the “Development of functional feed oxidants has been attracted in aquaculture because fish or additives for shrimp using useful organic matter in biofloc-based rearing water.” shrimp are susceptible to oxidative stress by disease or en- vironmental changes (Liu and Chen, 2004; Castex et al., Funding This study was funded by the Korea Ministry of Oceans and Fisheries, which 2010). Shrimp possess an integrated antioxidant system in- provided federal funds to support the “Development of functional feed additives cluding enzymatic and non-enzymatic antioxidants to for shrimp using useful organic matter in biofloc-based rearing water.” maintain normal oxidant status, especially to cope with nat- Availability of data and materials ural or induced stressors (Castex et al., 2009; Parrilla-Taylor All datasets analyzed during the current study are available from the and Zenteno-Savín, 2011). Generally, the antioxidant cap- corresponding author on reasonable request. ability of an organism under certain condition can reflect Authors’ contributions its health status. In the present study, increased GPx activ- CR and SH conducted the feeding trial, analysis, and drafted the ity was observed in shrimp fed BF6 diet compared to those manuscript. SJ manufactured the experimental diet. KJ designed this fed the Con diet. This result indicates that the antioxidant study, led in analyzing data derived from this study, and revised the manuscript. All authors read and approved the final manuscript. system of the shrimp can be enhanced by unknown com- ponents present in biofloc in some way. Based on its com- Ethics approval and consent to participate position characteristics, the biofloc may play a role in Experimental protocols followed the guidelines of the Animal Care and Use antioxidant activity because of its carotenoids and fat- Committee of Jeju National University. soluble vitamins (Ju et al., 2008) and improve the efficiency Consent for publication of feed utilization by stimulating activities of digestive en- Not applicable. zymes because of high protease and amylase activity in bio- Competing interests floc (Xu and Pan 2012; Xu et al. 2012). Similarly, Xu and The authors declare that they have no competing interests. Pan (2013) reported an increase in total antioxidant cap- acity and SOD activity of L. vannamei reared in a biofloc- Author details Department of Marine Life Sciences, Jeju National University, Jeju 63243, based tanks system. South Korea. Neo Environmental Business Co., Dangjin-si, During the challenge test against V. harveyi, significantly Chungcheongnam-do, South Korea. higher survival rate was found in BF4 group compared to Received: 20 January 2017 Accepted: 6 July 2017 the Con group. Similar observations were found by Ekasari et al. (2014) where following a challenge test by in- jection with infectious myonecrosis virus (IMNV) the sur- References vival of the challenged shrimp from the experimental Ajiboye OO, Yakubu AF, Adams TE. A perspective on the ingestion and nutritional effects of feed additives in farmed fish species. World J Fish & Marine Sci. 2012;4: biofloc groups was significantly higher when compared to 87–101. the challenged shrimp from the control treatment. Anand PSS, Kumar S, Panigrahi A, Ghoshal TK, Dayal JS, Biswas G, et al. Effects of C:N ratio and substrate integration on periphyton biomass, microbial dynamics and growth of Penaeus monodon juveniles. 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