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Effects of dietary prebiotic GroBiotic®‐A on growth performance, plasma thyroid hormones and mucosal immunity of great sturgeon, Huso huso (Linnaeus, 1758)

Effects of dietary prebiotic GroBiotic®‐A on growth performance, plasma thyroid hormones and... Received: 27 October 2015    Accepted: 27 May 2016 DOI: 10.1111/jai.13153 ORIGINAL AR TICLE Eects o ff f dietary prebiotic GroBiotic - A on growth performance, plasma thyroid hormones and mucosal immunity of great sturgeon, Huso huso (Linnaeus, 1758) 1 2 3 4 M. Adel  | S. Nayak  | C. C. Lazado  | S. Yeganeh Department of Aquatic Animal Health and Summary Diseases, Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research The present study was conducted to evaluate the effects of Grobiotic - A, a commer- Education and Extension Organization cial prebiotics, when administered in feed on the growth performance, plasma thyroid (AREEO), Tehran, Iran hormones and mucosal immunity of great sturgeon (Huso huso). The commercial prebi- Department of Biotechnology, North Orissa University, Baripada, Odisha, India otic mixture was supplemented in the diets at four different levels (i.e. 0.0% as control, Section for Aquaculture, National Institute 0.5%, 1% and 2%, in three replicates, 20 fish per replicate) and fed to the fish for an of Aquatic Resources, Technical University of Denmark, North Sea Science Park, Hirtshals, 8- week period wherein 240 fish were cultured in 1,800- L fiberglass tanks that formed Denmark part of a flow- through system. Water temperature was maintained at 20.4 ± 1.5°C. Department of Fisheries, Sari Agricultural Significant changes in growth performance parameters were observed, but only in Sciences and Natural Resources University, Sari, Iran those groups fed with 1% and 2% prebiotics. Specifically, marked improvements rela- tive to the control group were observed in percentage weight gain, body weight gain, Correspondence feed conversion ratio and specific growth rate in prebiotic- fed fish. The levels of Milad Adel, Department of Aquatic Animal Health and Diseases, Iranian Fisheries plasma thyroid hormones, specifically thyroxine and thyroid stimulating hormones Science Research Institute (IFSRI), were significantly elevated in the group receiving 2% prebiotics. Activities of lysozyme Agricultural Research Education and Extension Organization (AREEO), Tehran, and alkaline phosphatase in skin mucus were significantly enhanced in prebiotics- fed Iran. groups, particularly at an inclusion level of 1% and higher (2% group compared to the Emails: miladadel85@yahoo.com; miladadel65@gmail.com control). Inhibitory activity of the skin mucus against pathogens, particularly Strepto- coccus iniae and Yersinia ruckeri, was significantly improved following prebiotic feed- ing. Taken together, dietary inclusion of GroBiotic - A promoted growth, modulated thyroid hormones, and enhanced mucosal immunity of H. huso. This prebiotic mixture has the potential for use in improving the growth performance and health status of farmed great sturgeon. 1  |  INTR ODUCTION conditions, and their high value for meat and caviar (Jalali, Ahmadifar, Sudagar, & Azari Takami, 2009). In the 1990s, the great sturgeon (Huso huso) was already listed in the Under culture conditions, sturgeons are easily infected by several CITES (Convention on International Trade in Endangered Species) opportunistic pathogenic bacteria (e.g. Streptococcus sp., Aeromonas as an endangered species (Nazari, Sohrabnejad, & Ghomi, 2009). To sp., Yersinia sp., and Vibrio sp.), in addition to stressful conditions such address the declining wild stocks, the potential of the great sturgeon as poor water quality and high stocking density, all of which continue for culture under farmed conditions has been actively explored in the to be perennial challenges in many intensive aquaculture farms (Yang last two decades. Some of their features for potential domestication & Li, 2009). The increasing resistance to antibiotics, medication, and are a fast growth rate, high adaptability to controlled environmental other side eects ff from drugs associated with the overdependence of This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. J. Appl. Ichthyol. 2016; 32: 825–831 wileyonlinelibrary.com/journal/jai © 2016 The Authors Journal of Applied Ichthyology 825     Published by Blackwell Verlag GmbH Adel et Al . 826       the aquaculture industry on synthetic antimicrobials to combat diseas- daily in each tank and maintained as follows: average dissolved oxygen es necessitates the search for alternative strategies to improve health content (5.7 ± 0.4 mg/L), pH (7.76 ± 0.4), temperature (20.4 ± 1.5°C), and minimize dependence on these drugs (Lazado & Caipang, 2014; salinity (2.4 ± 0.11 g/L) and electrical conductivity (5826.4 ± 159.3 MM/ Nayak, 2010). In recent years, significant aen tt tion has been paid cm). In addition, a daily 14L:10D photoperiod (light intensisty 180 lux towards dietary supplements such as probiotics, prebiotics and synbi- during the feeding experiment) was provided to the cultured fish, which otics in order to improve growth performance as well as the health and were acclimated to these conditions for approximately 2 weeks before welfare status of farmed aquatic animals (Caipang & Lazado, 2015; the start of the feeding experiment. During this period, the fish were fed Cerezuela, Meseguer, & Esteban, 2011; Yousefian et al., 2012). three times daily at a ration of 3%–4% body weight. Prebiotics are non- digestible food ingredients that selectively stimulate the growth and/or activate the metabolism of certain health- 2.2 | Bacterial pathogens promoting bacteria in the gut, thereby improving the host intestinal balance (Fooks, Fuller, & Gibson, 1999; Ghaedi, Keyvanshokooh, Bacterial pathogens including Streptococcus iniae (ATCC29178), Mohammadi Azarm, & Akhlaghi, 2015). Some of the prebiotic mix - Yersinia ruckeri (KC291153), Listeria monocytogenes (ATCC1143) and tures widely used in aquaculture at experimental and farming levels Escherichia coli (PTCC 1037), were obtained from the Persian Type include inulin, oligofructose, mannanoligosaccharide (MOS), galactool- Culture Collection. ™ ® igosaccharide (GOS), β- 1, 3 glucan, ImmunoWall and Grobiotic - A (Burr, Hume, Ricke, Nisbet, & Gatlin, 2010; Caipang & Lazado, 2015; 2.3 | Preparation of experimental diets Cerezuela, Cuesta, Meseguer, & Esteban, 2008). Previous studies on the use of some of these dietary supplements revealed growth perfor- The prebiotic used in the study is commercially known as GroBiotic - A mance and increased immunity of great sturgeon (Ahmadifar, Akrami, (International Ingredient Corporation, Fenton, Missouri, USA) consist - Ghelichi, & Mohammadi Zarejabad, 2011; Akrami, Hajimoradloo, ing of partially autolyzed brewer’s yeast, dairy ingredient components Matinfar, & Abedian Kinari, 2009; Akrami et al., 2013; Hoseinifar et al., 2010; Ta’ati, Soltani, Bahmani, & Zamini, 2011), hence affirming their TABLE 1  Dietary formulation and proximate composition of basal potential in improving farming conditions. diet used in the study Grobiotic - A is a commercial prebiotic mixture composed of par - Ingredient Composition (%) tially autolyzed brewer’s yeast, dairy ingredient components, and dried Kilka fishmeal 58.0 fermentation products (Li & Gatlin, 2004). The beneficial properties of this probiotic mixture include growth promotion, efficient nutrient Wheat flour 19.0 utilization, immunomodulation and conferment of disease resistance, Fish oil 5.2 among others (Hoseinifar et al., 2010; Ta’ati et al., 2011). Dietary sup - Soybean oil 5.8 plementation of Grobiotic - A has been evaluated in several aqua- a Vitamin premix 3.0 culture species such as rainbow trout Oncorhynchus mykiss (Sealey Mineral premix 2.5 et al., 2007), hybrid striped bass Morone chrysops × M. saxatilis (Li & Cellulose 2.5 Gatlin, 2004), goldfish Carassius auratus (Raggi & Gatlin, 2012), red Binder 2.0 drum Sciaenops ocellatus (Buentello, Neill, & Gatlin, 2010), Caspian Salt 1.0 kutum Rutilus frissi (Yousefian et al., 2012), and juvenile Nile tila- Anti- fungi 0.4 pia Oreochromis niloticus (Vechklang et al., 2012). To the best of our Antioxidant 0.25 knowledge, this prebiotic mixture has not been tested on farmed Huso Proximate composition (% dry matter) huso. This study was therefore designed to evaluate the eects ff of Dry matter 98.84 dietary inclusion of Grobiotic - A on the growth performance, thyroid Crude protein 40.32 and cutaneous innate immunity of the great sturgeon, to determine if Crude lipid 18.86 it would be a good supplement for use in aquaculture. Ash 9.6 Fiber 2.8 Moisture 8.1 2  |  MATERIALS AND METHODS NFE 18.8 P/E ratio (mg protein/kJ) 19.68 Gross energy (kJ/g diet) 21.84 2.1 | Fish Premix detailed by Jalali et al. (2009). The experiment was carried out at the Sturgeon Culture Center (Samandak, Amet binder (MehrTaban- e- Yazd, Iran). ToxiBan antifungal (Vet- A- Mix, Shenandoah, IA). Sari), Mazandaran Province, northern Iran. Apparently healthy fish with Butylatedhydroxytoluene (BHT; Merck, Germany). an average body weight of 40.8 ± 6 g (mean ± SD, n = 240) were ran- Nitrogen- free extracts (NFE) = dry matter − (crude protein + crude domly selected from the holding tanks. The fish were distributed among lipid + ash + fibre). the 12 1,800-L fiberglass tanks (2.0 × 2.0 × 0.5 m), with a stocking den- Gross energy (kJ/g diet) calculated according to 23.6 kJ/g forprotein, sity of 20 fish per tank. Water parameters (mean ± SD) were monitored 39.5 kJ/g for lipid and 17.0 kJ/g for NFE. Adel et Al .       827 and dried fermentation products. Four experimental diets were pre- Feed conversion ratio (FCR)= Feed intake (g)∕Weight gain (g), pared for the study: the basal diet (Table 1) as the control, plus three prebiotic test diet groups supplemented at levels of 0.5%, 1% and Survival rate% = (Final number of fish∕initial number of fish)×100. 2%. All diets were prepared at the same time and stored in sterile plastic bags at 4°C until used. GroBiotic - A was replaced with cel- 2.7 | Quantification of thyroid hormones lulose in the basal diet (control diet, Table 1). Proximate composition At the end of the experiment, the levels of plasma thyroid hor- of the basal diet was according to the standard methods described mones were analyzed by commercially available ELISA kits (Delaware by the Association of Official Analytical Chemists (AOAC, 2005): Biotech Inc., Heidelberg, Germany). The hormones quantified based moisture was determined by oven- drying the samples at 105°C on the manufacturer’s standardized protocols were: thyroxine (Behr, Germany); crude lipid was determined by chloroform methanol (T4; Delaware Biotech Inc., Heidelberg, Germany), tri-iodo thyronine extraction (2:1, v/v); crude protein was determined (Kjeldahl proce - (T3) and thyroid stimulating hormone (TSH; DiaMetra Co., Milano, Italy). dure: N × 6.25) using an automatic Kjeldahl system; ash was measured by incineration in a muffle furnace at 500°C for 6 hr. 2.8 | Cutaneous innate immune responses 2.4 | Feeding Total protein concentration of skin mucus was measured colorimet - rically at 540 nm using bovine serum albumin as a standard (Lowry, The feeding trial lasted for 8 weeks. The basal and supplemented Rosebrough, Farr, & Randall, 1951). feeds were manually administered at three designated times of day, Alkaline phosphatase activity was quantified using a Pars Azmoon with a feeding ration of 3% body weight per day. Water parameters kit (Tehran Company, Iran) according to the manufacturer’s instruc- as enumerated above were monitored daily to ensure that they were tions, and absorbance was read at 405 nm with a spectrophotometer within the range of the biological requirements of the fish throughout (Sanchooli, Hajimoradloo, & Ghorbani, 2012). the feeding experiment. Lysozyme activity was determined as earlier described by Ellis (1990), with minor modifications. Briefly, 25 μl mucus was added to 2.5 | Sampling strategies 1 ml of a suspension of Micrococcus lysodeikticus (0.2 mg/ml in a 0.05 mol/L sodium phosphate buffer [pH 6.2]) and absorbance was No feed was given 24 hr prior to weighing and sampling the fish. Fish measured at 450 nm aer ft 0.5 and 3 min. were anesthetized with clove oil (100 mg/L) and thereafter circa 5 ml At the end of the experiment, antimicrobial activities of the skin of blood was collected from the caudal vein with heparinized syringes. mucus (from nine fish per replicate tank) against known pathogens The collection tubes with blood samples were immediately placed in were determined by disc diffusion assay (Bauer, Kirby, Sherris, & Turck, ice. Plasma was separated by centrifugation at 3,000 × g for 15 min, 1966). From overnight cultures in tryptic soy broth of each bacterial divided into aliquots and stored at −80°C until used (Adel, Abedian pathogen, 0.1 ml (containing 1.5 × 10 CFU/ml bacteria) was individ- Amiri, Zorriehzahra, Nematolahi, & Esteban, 2015). ually cultured on Mueller Hinton agar plates. Sterile filter paper discs At the end of the experiment mucus samples were collected from containing 10 μl mucus samples were placed above the seeded plate nine fish per replicate tank following previously described protocol agar and then incubated at 25°C for 48 hr (Turker, Birinci Yildirım, & (Balasubramanian et al., 2013). Briefly, mucus from an individual fish Pehlivan Karakaş, 2009). Aer ft the incubation period, the zone of inhi- was scraped in an anterior to posterior direction of the dorsal body bition was measured. All plates were performed in triplicate. surface using a sterile spatula. The collected mucus was then thor- Minimum inhibitory concentration (MIC) of skin mucus against oughly mixed with an equal volume of sterile Tris- buffered saline bacterial pathogens was determined by serial dilution method as (TBS, 50 mmol/L Tris HCl, pH 8.0, 150 mmol/L NaCl) and centrifuged described by Wei, Xavier, and Marimuthu (2010). Different concen- at 30,000 g at 4°C for 15 min. The supernatant was then collected, trations of mucus extract (25, 50, 75, 100, 125, 150, 200, 250 and filtered and kept at −80°C until further analysis. 300 μl) were added into a tube containing 1.5 × 10 CFU/ml of a spe- cific bacterial pathogen and incubated at 37°C for 24 hr. The MIC is 2.6 | Evaluation of growth performance defined as the lowest concentration of the mucus extract at which the pathogen does not demonstrate visible growth (Turker et al., 2009). The following calculations were undertaken at the end of feeding trial, based on the measurements described above: Weight gain =W (g)−W (g); 2.9 | Statistical analysis 2 1 The data were subjected to statistical analysis using the SPSS soft- Protein efficiency ratio (PER)= WG (g)∕protein intake (g); ware version no. 18 (SPSS Inc., Chicago, IL, USA). Aer ft satisfying the Specific growth rate (SGR%)= 100 (lnW −lnW )∕T. 2 1 assumptions of normality and equal variance, the data were analyzed where W is the initial weight, W is the final weight and T is the num- by one- way analysis of variance (ANOVA) followed by Duncan’s multi - 1 2 ber of days in the feeding period. ple range tests. Significance was determined at α = .05. Adel et Al . 828       3  |  RESULT S 3.3 | Eects ff on skin mucus innate immune responses The 0.5%- GA and 1%- GA groups showed no significant changes 3.1 | Eects on gr ff owth performance in their skin mucus protein levels after 8- week exposure (Fig. 1). The growth performance of juvenile beluga fed diets supplemented However, the group that received 2% prebiotics in the diet exhibited with different levels of dietary GBA are presented in Table 2. All stud - a significant elevation of approx. 20% in skin mucus protein. ied parameters were affected by prebiotic feeding, but only in groups Prebiotic feeding resulted in significantly elevated levels of skin with 1% and 2% in- feed prebiotic supplements. Distinctively clear mucus lysozyme, especially in the 1%- GA and 2%- GA groups (Fig. 2). effects were noted with body weight gain where the 1%- GA group Mean lysozyme activity in the 1%- GA group was 43.6 (±0.23) IU/mg, increased by 30% relative to control, while an almost 50% increment which was 34% higher compared to the control. On the other hand, was observed in the 2%- GA group. FCR and SGR also improved sig- lysozyme activity in the 2%- GA group was 50.08 ± 0.36 (mean ± SD, nificantly in groups receiving 1% and 2% prebiotics in their diets. PER n = 10) IU/mg, equal to an almost 55% increase relative to the control and %SR were significantly improved but only in the group receiving group. 2% prebiotics. The activity of skin mucus alkaline phosphatase was influenced in almost the same manner with that observed in lysozyme activity (Fig. 3). Significant changes were observed only in groups receiving an 3.2 | Eects on plasma th ff yroid hormones inclusion level of 1% and higher (Fig. 3). Skin mucus from 1%- GA and The effects of dietary inclusion of GroBiotic - A on three thyroid hor- mones of H. huso are detailed in Table 3, whereby significant increases 2.0 in thyroid hormones were observed only in the 2%- GA group. The b ab remaining inclusion levels did not show any significant alterations in 1.5 the level of thyroid hormones. The level T4 hormone level increased significantly—by almost 80% in the 2%- GA group. On the other hand, 1.0 the plasma TSH level was elevated by a significant 8% in the same 0.5 prebiotic- fed group. 0.0 TABLE 2  Effects of dietary inclusion of GroBiotic - A on growth performance parameters of H. huso. Values are mean ± SD of 10 individual fish per replicate tank Treatment Parameters Control 0.5%- GA 1%- GA 2%- GA FIGURE 1  Protein levels in skin mucus of Huso huso fed with a a b b Weight gain 57.96 ± 5.6 56.5 ± 4.6 94.79 ± 6.32 98.68 ± 7.14 GroBiotic - A for 8 weeks. Values presented here are mean ± SE of 10 (%) individual fish per replicate tank. Different letter notations indicate a a b c BW gain (g) 23.64 ± 1.29 23.06 ± 1.23 30.22 ± 1.86 34.75 ± 1.16 significant differences at p < .05 a a a b PER 0.82 ± 0.05 0.83 ± 0.06 0.88 ± 0.08 0.96 ± 0.1 a a b c FCR 2.09 ± 0.2 2.16 ± 0.4 1.81 ± 0.3 1.72 ± 0.2 a a b b SGR (%/day) 1.84 ± 0.16 1.76 ± 0.12 2.04 ± 0.24 2.22 ± 0.32 c a a ab b SR% 88 ± 1.6 93 ± 2.2 96 ± 1.1 98.2 ± 1.3 BW gain, Body weight gain; PER, Protein efficiency ratio; FCR, fed conver- 40 sion ratio; SGR, specific growth rate; %SR, percentage survival rate. Values in a row with different superscripts show significant differences (p < .05; n = 10). TABLE 3  Plasma thyroid hormones of H. huso fed GroBiotic - A for 8 weeks. Values are mean ± SD of 10 fish per replicate tank 0 Thyroid hormones Control 0.5%- GA 1%- GA 2%- GA a a ab b T4 (μg/dl) 0.87 ± 0.09 1.08 ± 0.13 1.26 ± 0.18 1.58 ± 0.22 Treatment a a a a T3 (μg/dl) 0.64 ± 0.02 0.74 ± 0.06 0.68 ± 0.09 0.61 ± 0.04 FIGURE 2  Lysozyme activity (IU/mg) in skin mucus of Huso a a a b TSH (μg/dl) 0.58 ± 0.04 0.52 ± 0.08 0.50 ± 0.07 0.63 ± 0.05 huso fed with GroBiotic - A for 8 weeks. Values presented here are Values in a row with different superscripts show significant differences mean ± SE of 10 individual fish per replicate tank. Different letter (p < .05; n = 10). notations indicate significant differences at p < .05 Control G-A % 0.5 G-A %1 G- A %2 Control G-A % 0.5 G-A % 1 G-A % 2 Lysozyme (IU/mg) Protein levels (mg/ml) Adel et Al .       829 of the prebiotics in the diet significantly impacted growth perfor- mance, plasma thyroid hormones and mucosal immunity of Huso huso. The application of Grobiotic - A in other fish species resulted in increased weight gain, improved feed efficiency, and beer tt nutrient digestibility as well as enhanced disease resistance (Buentello et al., 2010; Li & Gatlin, 2004, 2005; Vechklang et al., 2012). Th present study corroborated those earlier findings on the beneficial eects ff of growth performance. In particular, 2% Grobiotic - A in the diet signifi- cantly influenced the growth performance parameters of the fish, spe- cifically in weight gain. However, in a study with hybrid striped bass, Li and Gatlin (2004) did not observe such an increase in weight gain. Treatment This apparent difference could be due to the biological peculiarities between fish species and/or the duration of prebiotic feeding. FIGURE 3  Alkaline phosphatase (IU/L) activity in skin mucus of Hormones play pivotal roles in the regulation of growth and Huso huso fed GroBiotic - A for 8 weeks. Values presented here are nutrient intake in fish, and are sensitive to changes in feed intake mean ± SE of 10 individual fish per replicate tank. Different letter (MacKenzie, Van Putte, & Leiner, 1998). Thyroid hormones such as thy- notations indicate significant differences at p < .05 roxine and tri- iodothyronine, which are the principal thyroid hormones 2%- GA groups displayed an elevated alkaline phosphatase activity of secreted from the hypothalamic- pituitary- thyroid axis, are involved in at least 40% relative to the control group. many physiological processes during growth, development, behav- Marked enhancement in the antibacterial activity of H. huso ior, and stress in fish (Peter, 2011; Power et al., 2001). In the present skin mucus against different bacterial pathogens was observed in study, dietary supplementation of this prebiotic at 2% level was found groups receiving 1% and 2% in- feed prebiotics (Table 4). In these two to have an enhancing eect ff on thyroid hormones. Metabolic functions prebiotic- fed groups, for instance, inhibitory activity against S. iniae of thyroid hormones have been documented in a number of studies was promoted by at least 44% while inhibition of Y. ruckeri improved and which display a strong correlation with growth- promoting activ- by no less than 33%. MIC of skin mucus from control fish performed ities (Garg, 2007; Leatherland, 1994). It is plausible that the indirect at highest concentration against S. iniae and at lowest concentration influence of prebiotics on thyroid hormones is related to the growth- with Y. ruckeri. The MICs of skin mucus from fish groups receiving 1% promoting features of this feed additive. However, the mechanism and 2% prebiotics were generally lower compared with the 0.1%- GA behind such a benefit has yet to be unraveled in future studies. group. The MIC of skin mucus against S. iniae and Y. ruckeri for 1%- GA While the role of prebiotics on the growth, nutrition and physio- and 2%- GA groups was identical. logical responses of fish has been widely demonstrated in a number of studies, the involvement of prebiotics in stimulating fish immunity has been documented less frequently (Buentello et al., 2010; Cerezuela 4  |  DISCUSSION et al., 2008; Sealey et al., 2007). In the present study, we explored the eects ff of dietary prebiotics in mucosal immunity of H. huso. The present study demonstrated the benefits from Grobiotic - A, Lysozyme is an important component of the innate immunity in fish a commercial prebiotics, which consist of partially autolyzed brewer’s and which catalytically hydrolyzes the bond between N- acetyl muram- yeast, dairy ingredient components, and dried fermentation products ic acid and N- acetyl glucosamine in the cell wall of bacteria (Shailesh (Li & Gatlin, 2004, 2005; Zheng, Wang, Gatlin, & Ye, 2011). Inclusion & Sahoo, 2008). On the other hand, alkaline phosphatase has been TABLE 4  Inhibitory activity of skin mucus of H. huso fed GroBiotic - A for 8 weeks. Values are mean ± SD of nine fish per replicate tank Control 0.5%- GA 1%- GA 2%- GA Inhibition Inhibition Inhibition zone size zone size zone size Inhibition zone Pathogens (mm) MIC (μl/ml) (mm) MIC (μl/ml) (mm) MIC (μl/ml) size (mm) MIC (μl/ml) a a b b Streptococcus 5.4 ± 0.4 250 5.8 ± 0.2 250 8.5 ± 0.4 200 7.8 ± 0.2 200 iniae a a b c Yersinia ruckeri 8.1 ± 0.32 75 7.9 ± 0.6 75 10.8 ± 0.5 50 12.3 ± 0.6 50 a a b b Escherichia coli 5.8 ± 0.4 100 6.2 ± 0.2 75 10.8 ± 0.3 >50 9.8 ± 0.5 75 a a b b Listeria 6.3 ± 0.3 125 5.9 ± 0.2 >100 11.2 ± 1.2 100 10.6 ± 0.9 75 monocytogenes Values in a row with different superscripts show significant differences (p < .05; n = 9). Control G-A % 0.5 G-A % 1 G-A % 2 Alkaline phosphatase activity (IU/L) Adel et Al . 830       Balasubramanian, S., Gunasekaran, G., Baby Rani, P., Arul Prakash, A., demonstrated as a potential stress indicator in the epidermal mucus of Prakash, M., & Senthil Raja, J. A. P. (2013). A study on the antifungal fish and has a protective role during the first stages of wound healing properties of skin mucus from selected fresh water fishes. Golden (Iger & Abraham, 1994). Our results demonstrated that prebiotic feed- Research Thoughts, 2, 23–29. ing enhances these cutaneous immune components. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Turck, M. (1966). Antibiotic The fish mucus is known to contain a number of anti- microbial susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45, 493–496. substances, mainly attributed to the presence of lysozyme, agglu- Buentello, J. A., Neill, W. H., & Gatlin, D. M. III (2010). Eects ff of dietary tinins, thermolabile complement factors, or immunoglobulin (Hellio, prebiotics on the growth, feed efficiency and non- specific immunity of Pons, Beaupoil, Bourgougnon, & Le Gal, 2002). In the present study, juvenile red drum Sciaenops ocellatus fed soybean- based diets. Aqua- prebiotic- fed groups showed enhanced inhibitory activities with least culture Research, 41, 411–418. Burr, G., Hume, M., Ricke, S., Nisbet, D., & Gatlin, D. M. III (2010). In vitro MIC values against all tested pathogens under in vitro conditions. This and in vivo evaluation of the prebiotics GroBiotic - A, Inulin, Mannano- enhancing eect ff will therefore be helpful in preventing the adherence ligosaccharide, and Galactooligosaccharide on the digestive microbiota and multiplication of pathogens in fish, specifically in the mucus, which and performance of hybrid striped bass (Morone chrysops×Morone sax- has an intimate contact with the immediate environment. atilis). Microbial Ecology, 59, 187–198. Caipang, C. M. A., & Lazado, C. C. (2015). Nutritional impacts on fish In conclusion, the results of the present study revealed that pre- ® mucosa: Immunostimulants, pre- and probiotics. In B. Beck & H. Peat- biotic Grobiotic - A could be a potential dietary additive for farmed man (Eds.), Mucosal health in aquaculture (pp. 4–22). London, UK: Aca- great sturgeon. In particular, its dietary inclusion appears to improve demic Press, Inc. ISBN 9780124171862. the growth performance and health status of fish. The changes Cerezuela, R., Cuesta, A., Meseguer, J., & Esteban, M. A. (2008). Eects ff of insulin on gilthead seabream (Sparus aurata L.) innate immune parame- identified following prebiotic feeding accounted for the response ters. Fish & Shellfish Immunology, 24, 663–668. only in the short- term, experimental set- up. Future studies will Cerezuela, R., Meseguer, J., & Esteban, M. A. (2011). Current knowledge be directed toward looking into the eects ff of administering pre- in synbiotic use for fish aquaculture: A review. Journal of Aquaculture biotics during the entire production cycle and/or at the long- term Research & Development, 2, 1–7. Ellis, A. E. (1990). Lysozoyme assays. In J. S. Stolen, T. C. Fletcher, D. P. Ander- eects ff of short- term prebiotic feeding. The findings discussed here son, B. S. Robertson & W. R. Van Muisvinkel (Eds.), Techniques in fish add to the growing evidence on the prospects of using prebiotics immunology, 3rd edn (pp. 101–103). Fair Haven, NJ: SOS Publications. as health- promoting feed additives in aquaculture, which in turn is Fooks, L. J., Fuller, R., & Gibson, G. R. (1999). Prebiotics, probiotics and currently fostering more sustainable eco- and consumer- friendly human gut microbiology. International Dairy Journal, 9, 53–61. dietary supplements. Garg, S. K. (2007). 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Aquaculture Research, 39, 223–239. 42, 549–557. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Ichthyology Wiley

Effects of dietary prebiotic GroBiotic®‐A on growth performance, plasma thyroid hormones and mucosal immunity of great sturgeon, Huso huso (Linnaeus, 1758)

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10.1111/jai.13153
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Received: 27 October 2015    Accepted: 27 May 2016 DOI: 10.1111/jai.13153 ORIGINAL AR TICLE Eects o ff f dietary prebiotic GroBiotic - A on growth performance, plasma thyroid hormones and mucosal immunity of great sturgeon, Huso huso (Linnaeus, 1758) 1 2 3 4 M. Adel  | S. Nayak  | C. C. Lazado  | S. Yeganeh Department of Aquatic Animal Health and Summary Diseases, Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research The present study was conducted to evaluate the effects of Grobiotic - A, a commer- Education and Extension Organization cial prebiotics, when administered in feed on the growth performance, plasma thyroid (AREEO), Tehran, Iran hormones and mucosal immunity of great sturgeon (Huso huso). The commercial prebi- Department of Biotechnology, North Orissa University, Baripada, Odisha, India otic mixture was supplemented in the diets at four different levels (i.e. 0.0% as control, Section for Aquaculture, National Institute 0.5%, 1% and 2%, in three replicates, 20 fish per replicate) and fed to the fish for an of Aquatic Resources, Technical University of Denmark, North Sea Science Park, Hirtshals, 8- week period wherein 240 fish were cultured in 1,800- L fiberglass tanks that formed Denmark part of a flow- through system. Water temperature was maintained at 20.4 ± 1.5°C. Department of Fisheries, Sari Agricultural Significant changes in growth performance parameters were observed, but only in Sciences and Natural Resources University, Sari, Iran those groups fed with 1% and 2% prebiotics. Specifically, marked improvements rela- tive to the control group were observed in percentage weight gain, body weight gain, Correspondence feed conversion ratio and specific growth rate in prebiotic- fed fish. The levels of Milad Adel, Department of Aquatic Animal Health and Diseases, Iranian Fisheries plasma thyroid hormones, specifically thyroxine and thyroid stimulating hormones Science Research Institute (IFSRI), were significantly elevated in the group receiving 2% prebiotics. Activities of lysozyme Agricultural Research Education and Extension Organization (AREEO), Tehran, and alkaline phosphatase in skin mucus were significantly enhanced in prebiotics- fed Iran. groups, particularly at an inclusion level of 1% and higher (2% group compared to the Emails: miladadel85@yahoo.com; miladadel65@gmail.com control). Inhibitory activity of the skin mucus against pathogens, particularly Strepto- coccus iniae and Yersinia ruckeri, was significantly improved following prebiotic feed- ing. Taken together, dietary inclusion of GroBiotic - A promoted growth, modulated thyroid hormones, and enhanced mucosal immunity of H. huso. This prebiotic mixture has the potential for use in improving the growth performance and health status of farmed great sturgeon. 1  |  INTR ODUCTION conditions, and their high value for meat and caviar (Jalali, Ahmadifar, Sudagar, & Azari Takami, 2009). In the 1990s, the great sturgeon (Huso huso) was already listed in the Under culture conditions, sturgeons are easily infected by several CITES (Convention on International Trade in Endangered Species) opportunistic pathogenic bacteria (e.g. Streptococcus sp., Aeromonas as an endangered species (Nazari, Sohrabnejad, & Ghomi, 2009). To sp., Yersinia sp., and Vibrio sp.), in addition to stressful conditions such address the declining wild stocks, the potential of the great sturgeon as poor water quality and high stocking density, all of which continue for culture under farmed conditions has been actively explored in the to be perennial challenges in many intensive aquaculture farms (Yang last two decades. Some of their features for potential domestication & Li, 2009). The increasing resistance to antibiotics, medication, and are a fast growth rate, high adaptability to controlled environmental other side eects ff from drugs associated with the overdependence of This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. J. Appl. Ichthyol. 2016; 32: 825–831 wileyonlinelibrary.com/journal/jai © 2016 The Authors Journal of Applied Ichthyology 825     Published by Blackwell Verlag GmbH Adel et Al . 826       the aquaculture industry on synthetic antimicrobials to combat diseas- daily in each tank and maintained as follows: average dissolved oxygen es necessitates the search for alternative strategies to improve health content (5.7 ± 0.4 mg/L), pH (7.76 ± 0.4), temperature (20.4 ± 1.5°C), and minimize dependence on these drugs (Lazado & Caipang, 2014; salinity (2.4 ± 0.11 g/L) and electrical conductivity (5826.4 ± 159.3 MM/ Nayak, 2010). In recent years, significant aen tt tion has been paid cm). In addition, a daily 14L:10D photoperiod (light intensisty 180 lux towards dietary supplements such as probiotics, prebiotics and synbi- during the feeding experiment) was provided to the cultured fish, which otics in order to improve growth performance as well as the health and were acclimated to these conditions for approximately 2 weeks before welfare status of farmed aquatic animals (Caipang & Lazado, 2015; the start of the feeding experiment. During this period, the fish were fed Cerezuela, Meseguer, & Esteban, 2011; Yousefian et al., 2012). three times daily at a ration of 3%–4% body weight. Prebiotics are non- digestible food ingredients that selectively stimulate the growth and/or activate the metabolism of certain health- 2.2 | Bacterial pathogens promoting bacteria in the gut, thereby improving the host intestinal balance (Fooks, Fuller, & Gibson, 1999; Ghaedi, Keyvanshokooh, Bacterial pathogens including Streptococcus iniae (ATCC29178), Mohammadi Azarm, & Akhlaghi, 2015). Some of the prebiotic mix - Yersinia ruckeri (KC291153), Listeria monocytogenes (ATCC1143) and tures widely used in aquaculture at experimental and farming levels Escherichia coli (PTCC 1037), were obtained from the Persian Type include inulin, oligofructose, mannanoligosaccharide (MOS), galactool- Culture Collection. ™ ® igosaccharide (GOS), β- 1, 3 glucan, ImmunoWall and Grobiotic - A (Burr, Hume, Ricke, Nisbet, & Gatlin, 2010; Caipang & Lazado, 2015; 2.3 | Preparation of experimental diets Cerezuela, Cuesta, Meseguer, & Esteban, 2008). Previous studies on the use of some of these dietary supplements revealed growth perfor- The prebiotic used in the study is commercially known as GroBiotic - A mance and increased immunity of great sturgeon (Ahmadifar, Akrami, (International Ingredient Corporation, Fenton, Missouri, USA) consist - Ghelichi, & Mohammadi Zarejabad, 2011; Akrami, Hajimoradloo, ing of partially autolyzed brewer’s yeast, dairy ingredient components Matinfar, & Abedian Kinari, 2009; Akrami et al., 2013; Hoseinifar et al., 2010; Ta’ati, Soltani, Bahmani, & Zamini, 2011), hence affirming their TABLE 1  Dietary formulation and proximate composition of basal potential in improving farming conditions. diet used in the study Grobiotic - A is a commercial prebiotic mixture composed of par - Ingredient Composition (%) tially autolyzed brewer’s yeast, dairy ingredient components, and dried Kilka fishmeal 58.0 fermentation products (Li & Gatlin, 2004). The beneficial properties of this probiotic mixture include growth promotion, efficient nutrient Wheat flour 19.0 utilization, immunomodulation and conferment of disease resistance, Fish oil 5.2 among others (Hoseinifar et al., 2010; Ta’ati et al., 2011). Dietary sup - Soybean oil 5.8 plementation of Grobiotic - A has been evaluated in several aqua- a Vitamin premix 3.0 culture species such as rainbow trout Oncorhynchus mykiss (Sealey Mineral premix 2.5 et al., 2007), hybrid striped bass Morone chrysops × M. saxatilis (Li & Cellulose 2.5 Gatlin, 2004), goldfish Carassius auratus (Raggi & Gatlin, 2012), red Binder 2.0 drum Sciaenops ocellatus (Buentello, Neill, & Gatlin, 2010), Caspian Salt 1.0 kutum Rutilus frissi (Yousefian et al., 2012), and juvenile Nile tila- Anti- fungi 0.4 pia Oreochromis niloticus (Vechklang et al., 2012). To the best of our Antioxidant 0.25 knowledge, this prebiotic mixture has not been tested on farmed Huso Proximate composition (% dry matter) huso. This study was therefore designed to evaluate the eects ff of Dry matter 98.84 dietary inclusion of Grobiotic - A on the growth performance, thyroid Crude protein 40.32 and cutaneous innate immunity of the great sturgeon, to determine if Crude lipid 18.86 it would be a good supplement for use in aquaculture. Ash 9.6 Fiber 2.8 Moisture 8.1 2  |  MATERIALS AND METHODS NFE 18.8 P/E ratio (mg protein/kJ) 19.68 Gross energy (kJ/g diet) 21.84 2.1 | Fish Premix detailed by Jalali et al. (2009). The experiment was carried out at the Sturgeon Culture Center (Samandak, Amet binder (MehrTaban- e- Yazd, Iran). ToxiBan antifungal (Vet- A- Mix, Shenandoah, IA). Sari), Mazandaran Province, northern Iran. Apparently healthy fish with Butylatedhydroxytoluene (BHT; Merck, Germany). an average body weight of 40.8 ± 6 g (mean ± SD, n = 240) were ran- Nitrogen- free extracts (NFE) = dry matter − (crude protein + crude domly selected from the holding tanks. The fish were distributed among lipid + ash + fibre). the 12 1,800-L fiberglass tanks (2.0 × 2.0 × 0.5 m), with a stocking den- Gross energy (kJ/g diet) calculated according to 23.6 kJ/g forprotein, sity of 20 fish per tank. Water parameters (mean ± SD) were monitored 39.5 kJ/g for lipid and 17.0 kJ/g for NFE. Adel et Al .       827 and dried fermentation products. Four experimental diets were pre- Feed conversion ratio (FCR)= Feed intake (g)∕Weight gain (g), pared for the study: the basal diet (Table 1) as the control, plus three prebiotic test diet groups supplemented at levels of 0.5%, 1% and Survival rate% = (Final number of fish∕initial number of fish)×100. 2%. All diets were prepared at the same time and stored in sterile plastic bags at 4°C until used. GroBiotic - A was replaced with cel- 2.7 | Quantification of thyroid hormones lulose in the basal diet (control diet, Table 1). Proximate composition At the end of the experiment, the levels of plasma thyroid hor- of the basal diet was according to the standard methods described mones were analyzed by commercially available ELISA kits (Delaware by the Association of Official Analytical Chemists (AOAC, 2005): Biotech Inc., Heidelberg, Germany). The hormones quantified based moisture was determined by oven- drying the samples at 105°C on the manufacturer’s standardized protocols were: thyroxine (Behr, Germany); crude lipid was determined by chloroform methanol (T4; Delaware Biotech Inc., Heidelberg, Germany), tri-iodo thyronine extraction (2:1, v/v); crude protein was determined (Kjeldahl proce - (T3) and thyroid stimulating hormone (TSH; DiaMetra Co., Milano, Italy). dure: N × 6.25) using an automatic Kjeldahl system; ash was measured by incineration in a muffle furnace at 500°C for 6 hr. 2.8 | Cutaneous innate immune responses 2.4 | Feeding Total protein concentration of skin mucus was measured colorimet - rically at 540 nm using bovine serum albumin as a standard (Lowry, The feeding trial lasted for 8 weeks. The basal and supplemented Rosebrough, Farr, & Randall, 1951). feeds were manually administered at three designated times of day, Alkaline phosphatase activity was quantified using a Pars Azmoon with a feeding ration of 3% body weight per day. Water parameters kit (Tehran Company, Iran) according to the manufacturer’s instruc- as enumerated above were monitored daily to ensure that they were tions, and absorbance was read at 405 nm with a spectrophotometer within the range of the biological requirements of the fish throughout (Sanchooli, Hajimoradloo, & Ghorbani, 2012). the feeding experiment. Lysozyme activity was determined as earlier described by Ellis (1990), with minor modifications. Briefly, 25 μl mucus was added to 2.5 | Sampling strategies 1 ml of a suspension of Micrococcus lysodeikticus (0.2 mg/ml in a 0.05 mol/L sodium phosphate buffer [pH 6.2]) and absorbance was No feed was given 24 hr prior to weighing and sampling the fish. Fish measured at 450 nm aer ft 0.5 and 3 min. were anesthetized with clove oil (100 mg/L) and thereafter circa 5 ml At the end of the experiment, antimicrobial activities of the skin of blood was collected from the caudal vein with heparinized syringes. mucus (from nine fish per replicate tank) against known pathogens The collection tubes with blood samples were immediately placed in were determined by disc diffusion assay (Bauer, Kirby, Sherris, & Turck, ice. Plasma was separated by centrifugation at 3,000 × g for 15 min, 1966). From overnight cultures in tryptic soy broth of each bacterial divided into aliquots and stored at −80°C until used (Adel, Abedian pathogen, 0.1 ml (containing 1.5 × 10 CFU/ml bacteria) was individ- Amiri, Zorriehzahra, Nematolahi, & Esteban, 2015). ually cultured on Mueller Hinton agar plates. Sterile filter paper discs At the end of the experiment mucus samples were collected from containing 10 μl mucus samples were placed above the seeded plate nine fish per replicate tank following previously described protocol agar and then incubated at 25°C for 48 hr (Turker, Birinci Yildirım, & (Balasubramanian et al., 2013). Briefly, mucus from an individual fish Pehlivan Karakaş, 2009). Aer ft the incubation period, the zone of inhi- was scraped in an anterior to posterior direction of the dorsal body bition was measured. All plates were performed in triplicate. surface using a sterile spatula. The collected mucus was then thor- Minimum inhibitory concentration (MIC) of skin mucus against oughly mixed with an equal volume of sterile Tris- buffered saline bacterial pathogens was determined by serial dilution method as (TBS, 50 mmol/L Tris HCl, pH 8.0, 150 mmol/L NaCl) and centrifuged described by Wei, Xavier, and Marimuthu (2010). Different concen- at 30,000 g at 4°C for 15 min. The supernatant was then collected, trations of mucus extract (25, 50, 75, 100, 125, 150, 200, 250 and filtered and kept at −80°C until further analysis. 300 μl) were added into a tube containing 1.5 × 10 CFU/ml of a spe- cific bacterial pathogen and incubated at 37°C for 24 hr. The MIC is 2.6 | Evaluation of growth performance defined as the lowest concentration of the mucus extract at which the pathogen does not demonstrate visible growth (Turker et al., 2009). The following calculations were undertaken at the end of feeding trial, based on the measurements described above: Weight gain =W (g)−W (g); 2.9 | Statistical analysis 2 1 The data were subjected to statistical analysis using the SPSS soft- Protein efficiency ratio (PER)= WG (g)∕protein intake (g); ware version no. 18 (SPSS Inc., Chicago, IL, USA). Aer ft satisfying the Specific growth rate (SGR%)= 100 (lnW −lnW )∕T. 2 1 assumptions of normality and equal variance, the data were analyzed where W is the initial weight, W is the final weight and T is the num- by one- way analysis of variance (ANOVA) followed by Duncan’s multi - 1 2 ber of days in the feeding period. ple range tests. Significance was determined at α = .05. Adel et Al . 828       3  |  RESULT S 3.3 | Eects ff on skin mucus innate immune responses The 0.5%- GA and 1%- GA groups showed no significant changes 3.1 | Eects on gr ff owth performance in their skin mucus protein levels after 8- week exposure (Fig. 1). The growth performance of juvenile beluga fed diets supplemented However, the group that received 2% prebiotics in the diet exhibited with different levels of dietary GBA are presented in Table 2. All stud - a significant elevation of approx. 20% in skin mucus protein. ied parameters were affected by prebiotic feeding, but only in groups Prebiotic feeding resulted in significantly elevated levels of skin with 1% and 2% in- feed prebiotic supplements. Distinctively clear mucus lysozyme, especially in the 1%- GA and 2%- GA groups (Fig. 2). effects were noted with body weight gain where the 1%- GA group Mean lysozyme activity in the 1%- GA group was 43.6 (±0.23) IU/mg, increased by 30% relative to control, while an almost 50% increment which was 34% higher compared to the control. On the other hand, was observed in the 2%- GA group. FCR and SGR also improved sig- lysozyme activity in the 2%- GA group was 50.08 ± 0.36 (mean ± SD, nificantly in groups receiving 1% and 2% prebiotics in their diets. PER n = 10) IU/mg, equal to an almost 55% increase relative to the control and %SR were significantly improved but only in the group receiving group. 2% prebiotics. The activity of skin mucus alkaline phosphatase was influenced in almost the same manner with that observed in lysozyme activity (Fig. 3). Significant changes were observed only in groups receiving an 3.2 | Eects on plasma th ff yroid hormones inclusion level of 1% and higher (Fig. 3). Skin mucus from 1%- GA and The effects of dietary inclusion of GroBiotic - A on three thyroid hor- mones of H. huso are detailed in Table 3, whereby significant increases 2.0 in thyroid hormones were observed only in the 2%- GA group. The b ab remaining inclusion levels did not show any significant alterations in 1.5 the level of thyroid hormones. The level T4 hormone level increased significantly—by almost 80% in the 2%- GA group. On the other hand, 1.0 the plasma TSH level was elevated by a significant 8% in the same 0.5 prebiotic- fed group. 0.0 TABLE 2  Effects of dietary inclusion of GroBiotic - A on growth performance parameters of H. huso. Values are mean ± SD of 10 individual fish per replicate tank Treatment Parameters Control 0.5%- GA 1%- GA 2%- GA FIGURE 1  Protein levels in skin mucus of Huso huso fed with a a b b Weight gain 57.96 ± 5.6 56.5 ± 4.6 94.79 ± 6.32 98.68 ± 7.14 GroBiotic - A for 8 weeks. Values presented here are mean ± SE of 10 (%) individual fish per replicate tank. Different letter notations indicate a a b c BW gain (g) 23.64 ± 1.29 23.06 ± 1.23 30.22 ± 1.86 34.75 ± 1.16 significant differences at p < .05 a a a b PER 0.82 ± 0.05 0.83 ± 0.06 0.88 ± 0.08 0.96 ± 0.1 a a b c FCR 2.09 ± 0.2 2.16 ± 0.4 1.81 ± 0.3 1.72 ± 0.2 a a b b SGR (%/day) 1.84 ± 0.16 1.76 ± 0.12 2.04 ± 0.24 2.22 ± 0.32 c a a ab b SR% 88 ± 1.6 93 ± 2.2 96 ± 1.1 98.2 ± 1.3 BW gain, Body weight gain; PER, Protein efficiency ratio; FCR, fed conver- 40 sion ratio; SGR, specific growth rate; %SR, percentage survival rate. Values in a row with different superscripts show significant differences (p < .05; n = 10). TABLE 3  Plasma thyroid hormones of H. huso fed GroBiotic - A for 8 weeks. Values are mean ± SD of 10 fish per replicate tank 0 Thyroid hormones Control 0.5%- GA 1%- GA 2%- GA a a ab b T4 (μg/dl) 0.87 ± 0.09 1.08 ± 0.13 1.26 ± 0.18 1.58 ± 0.22 Treatment a a a a T3 (μg/dl) 0.64 ± 0.02 0.74 ± 0.06 0.68 ± 0.09 0.61 ± 0.04 FIGURE 2  Lysozyme activity (IU/mg) in skin mucus of Huso a a a b TSH (μg/dl) 0.58 ± 0.04 0.52 ± 0.08 0.50 ± 0.07 0.63 ± 0.05 huso fed with GroBiotic - A for 8 weeks. Values presented here are Values in a row with different superscripts show significant differences mean ± SE of 10 individual fish per replicate tank. Different letter (p < .05; n = 10). notations indicate significant differences at p < .05 Control G-A % 0.5 G-A %1 G- A %2 Control G-A % 0.5 G-A % 1 G-A % 2 Lysozyme (IU/mg) Protein levels (mg/ml) Adel et Al .       829 of the prebiotics in the diet significantly impacted growth perfor- mance, plasma thyroid hormones and mucosal immunity of Huso huso. The application of Grobiotic - A in other fish species resulted in increased weight gain, improved feed efficiency, and beer tt nutrient digestibility as well as enhanced disease resistance (Buentello et al., 2010; Li & Gatlin, 2004, 2005; Vechklang et al., 2012). Th present study corroborated those earlier findings on the beneficial eects ff of growth performance. In particular, 2% Grobiotic - A in the diet signifi- cantly influenced the growth performance parameters of the fish, spe- cifically in weight gain. However, in a study with hybrid striped bass, Li and Gatlin (2004) did not observe such an increase in weight gain. Treatment This apparent difference could be due to the biological peculiarities between fish species and/or the duration of prebiotic feeding. FIGURE 3  Alkaline phosphatase (IU/L) activity in skin mucus of Hormones play pivotal roles in the regulation of growth and Huso huso fed GroBiotic - A for 8 weeks. Values presented here are nutrient intake in fish, and are sensitive to changes in feed intake mean ± SE of 10 individual fish per replicate tank. Different letter (MacKenzie, Van Putte, & Leiner, 1998). Thyroid hormones such as thy- notations indicate significant differences at p < .05 roxine and tri- iodothyronine, which are the principal thyroid hormones 2%- GA groups displayed an elevated alkaline phosphatase activity of secreted from the hypothalamic- pituitary- thyroid axis, are involved in at least 40% relative to the control group. many physiological processes during growth, development, behav- Marked enhancement in the antibacterial activity of H. huso ior, and stress in fish (Peter, 2011; Power et al., 2001). In the present skin mucus against different bacterial pathogens was observed in study, dietary supplementation of this prebiotic at 2% level was found groups receiving 1% and 2% in- feed prebiotics (Table 4). In these two to have an enhancing eect ff on thyroid hormones. Metabolic functions prebiotic- fed groups, for instance, inhibitory activity against S. iniae of thyroid hormones have been documented in a number of studies was promoted by at least 44% while inhibition of Y. ruckeri improved and which display a strong correlation with growth- promoting activ- by no less than 33%. MIC of skin mucus from control fish performed ities (Garg, 2007; Leatherland, 1994). It is plausible that the indirect at highest concentration against S. iniae and at lowest concentration influence of prebiotics on thyroid hormones is related to the growth- with Y. ruckeri. The MICs of skin mucus from fish groups receiving 1% promoting features of this feed additive. However, the mechanism and 2% prebiotics were generally lower compared with the 0.1%- GA behind such a benefit has yet to be unraveled in future studies. group. The MIC of skin mucus against S. iniae and Y. ruckeri for 1%- GA While the role of prebiotics on the growth, nutrition and physio- and 2%- GA groups was identical. logical responses of fish has been widely demonstrated in a number of studies, the involvement of prebiotics in stimulating fish immunity has been documented less frequently (Buentello et al., 2010; Cerezuela 4  |  DISCUSSION et al., 2008; Sealey et al., 2007). In the present study, we explored the eects ff of dietary prebiotics in mucosal immunity of H. huso. The present study demonstrated the benefits from Grobiotic - A, Lysozyme is an important component of the innate immunity in fish a commercial prebiotics, which consist of partially autolyzed brewer’s and which catalytically hydrolyzes the bond between N- acetyl muram- yeast, dairy ingredient components, and dried fermentation products ic acid and N- acetyl glucosamine in the cell wall of bacteria (Shailesh (Li & Gatlin, 2004, 2005; Zheng, Wang, Gatlin, & Ye, 2011). Inclusion & Sahoo, 2008). On the other hand, alkaline phosphatase has been TABLE 4  Inhibitory activity of skin mucus of H. huso fed GroBiotic - A for 8 weeks. Values are mean ± SD of nine fish per replicate tank Control 0.5%- GA 1%- GA 2%- GA Inhibition Inhibition Inhibition zone size zone size zone size Inhibition zone Pathogens (mm) MIC (μl/ml) (mm) MIC (μl/ml) (mm) MIC (μl/ml) size (mm) MIC (μl/ml) a a b b Streptococcus 5.4 ± 0.4 250 5.8 ± 0.2 250 8.5 ± 0.4 200 7.8 ± 0.2 200 iniae a a b c Yersinia ruckeri 8.1 ± 0.32 75 7.9 ± 0.6 75 10.8 ± 0.5 50 12.3 ± 0.6 50 a a b b Escherichia coli 5.8 ± 0.4 100 6.2 ± 0.2 75 10.8 ± 0.3 >50 9.8 ± 0.5 75 a a b b Listeria 6.3 ± 0.3 125 5.9 ± 0.2 >100 11.2 ± 1.2 100 10.6 ± 0.9 75 monocytogenes Values in a row with different superscripts show significant differences (p < .05; n = 9). Control G-A % 0.5 G-A % 1 G-A % 2 Alkaline phosphatase activity (IU/L) Adel et Al . 830       Balasubramanian, S., Gunasekaran, G., Baby Rani, P., Arul Prakash, A., demonstrated as a potential stress indicator in the epidermal mucus of Prakash, M., & Senthil Raja, J. A. P. (2013). A study on the antifungal fish and has a protective role during the first stages of wound healing properties of skin mucus from selected fresh water fishes. Golden (Iger & Abraham, 1994). Our results demonstrated that prebiotic feed- Research Thoughts, 2, 23–29. ing enhances these cutaneous immune components. Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Turck, M. (1966). Antibiotic The fish mucus is known to contain a number of anti- microbial susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45, 493–496. substances, mainly attributed to the presence of lysozyme, agglu- Buentello, J. A., Neill, W. H., & Gatlin, D. M. III (2010). Eects ff of dietary tinins, thermolabile complement factors, or immunoglobulin (Hellio, prebiotics on the growth, feed efficiency and non- specific immunity of Pons, Beaupoil, Bourgougnon, & Le Gal, 2002). In the present study, juvenile red drum Sciaenops ocellatus fed soybean- based diets. Aqua- prebiotic- fed groups showed enhanced inhibitory activities with least culture Research, 41, 411–418. Burr, G., Hume, M., Ricke, S., Nisbet, D., & Gatlin, D. M. III (2010). In vitro MIC values against all tested pathogens under in vitro conditions. This and in vivo evaluation of the prebiotics GroBiotic - A, Inulin, Mannano- enhancing eect ff will therefore be helpful in preventing the adherence ligosaccharide, and Galactooligosaccharide on the digestive microbiota and multiplication of pathogens in fish, specifically in the mucus, which and performance of hybrid striped bass (Morone chrysops×Morone sax- has an intimate contact with the immediate environment. atilis). Microbial Ecology, 59, 187–198. Caipang, C. M. A., & Lazado, C. C. (2015). Nutritional impacts on fish In conclusion, the results of the present study revealed that pre- ® mucosa: Immunostimulants, pre- and probiotics. In B. Beck & H. Peat- biotic Grobiotic - A could be a potential dietary additive for farmed man (Eds.), Mucosal health in aquaculture (pp. 4–22). London, UK: Aca- great sturgeon. In particular, its dietary inclusion appears to improve demic Press, Inc. ISBN 9780124171862. the growth performance and health status of fish. The changes Cerezuela, R., Cuesta, A., Meseguer, J., & Esteban, M. A. (2008). Eects ff of insulin on gilthead seabream (Sparus aurata L.) innate immune parame- identified following prebiotic feeding accounted for the response ters. Fish & Shellfish Immunology, 24, 663–668. only in the short- term, experimental set- up. Future studies will Cerezuela, R., Meseguer, J., & Esteban, M. A. (2011). 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Journal of Applied Ichthyology – Wiley

Published: Oct 1, 2016

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