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Effect of feeding strategy of probiotic Enterococcus faecium on growth performance, hematologic, biochemical parameters and non-specific immune response of Nile tilapia
Aquaculture Reports 16 (2020) 100277
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Effect of feeding strategy of probiotic Enterococcus faecium on growth performance, hematologic, biochemical parameters and non-specific immune response of Nile tilapia
T
Leonardo Tachibanaa, Guilherme Silveira Tellia, Danielle de Carla Diasa, Giovani Sampaio Gonçalvesb, Carlos Massatoshi Ishikawaa, Raissa Bertoncello Cavalcantea, Mariene Miyoko Natoria, Said Ben Hameda, Maria José T. Ranzani-Paivaa,* a
Aquaculture Research Center, Scientific Research of Fisheries Institute/APTA/SAA, São Paulo. Av. Francisco Matarazzo, 455 - 05001-900, São Paulo, Brazil Scientific Research of Advanced Technological Research Center for Continental Fishery Agribusiness of Fisheries Institute/APTA/SAA, Abelardo Menezes Avenue, 15092607, São José do Rio Preto, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enterococcus faecium Tilapia Immune system Feeding strategies Challenge Aeromonas hydrophila
The aquaculture industry is in considerable increases. Optimization of husbandary conditions in intensive aquaculture are in continuous interest. Probiotic was proved as promising factors to enhance fish production by remodeling intestinal microbial balance, boosting immune system and reducing antibiotic uses. The efficient strategy of probiotic uses and their protection of fish still making debate. The intake of tilpia fed with basal diet enriched with probiotic (Enterococcus faecium) and its response following to a challenge with pathogenic strain of Aeromonas hydrophila was studied. Four feeding strategies were tested and compared: continuous feeding with only basal diet (CTR), continuous feeding with probiotic-supplemented diet (CON), pulse-administration feeding of 7 days (P7) and 14 days (P14) alternation with probiotic supplemented diet and basal diet. Differences in growth rate, immune system modulation and biochemical parameters were evaluated. P7 strategy showed efficacy in promote fish growth conditions. Body chemical composition and blood biochemical parameters did not show any significative differences between feeding strategies. Burst respiratory analysis showed significant difference in fish fed with P14 strategy. After challenge test, CON strategy showed better fish protection against Aeromonas hydrophila. Enterococcus faecium enhance fish growth, boost imune system if administrated in 14 days period, and efficiently protect fish if it is continuously administrated.
1. Introduction
stress tolerance (Kesarcodi-Watson et al., 2008; Safari et al., 2016; Wang et al., 2008). In addition, probiotics also can be effective in promote the phagocytic activity and lysozyme levels of fish (Harikrishnan et al., 2010). Lactic acid bacteria (LAB) such as Enterococcus faecium is commonly used as probiotic in fish farming and are known to be present in the intestine of healthy fish. According to Wang et al. (2008) the effect of E. faecium in the water at 107 CFU mL−1 concentration (supplemented once every four days) provided higher final weight, daily weight gain, myeloperoxidase activity, respiratory burst and blood phagocytes of the group that receive probiotic in the water than control group. In the other hand, probiotic administration can be a nutritional strategy to improve the immune response and growth performance of fish (Ramos et al., 2017). In fact, probiotics application strategies can be useful in fish intensive production system, in which common
Following the FAO, 2018 tilapia have shown a continuous increase among the major species groups in inland water. In one hand, The Genetic Improvement of Farmed Tilapia (GIFT) project has played an important role in the expansion of Nile tilapia culture (now reported in 87 countries) by helping to avoid the negative impacts of inbreeding or poor genetic management (Gjedrem, 2012). In many countries, antibiotics administration caused concerns about zoonotic bacteria resistances in animal production, causing food borne diseases. Probiotics is one of the few alternatives to substitute these medicaments, promote protective effects against pathogens, and then produce safety foods (Liao and Nyachoti, 2017). In aquaculture, probiotics is widely used to improve growth performance and modulate the immune system in order to inhibit pathogens growth and to increase
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Corresponding author. E-mail address: [email protected] (M.J.T. Ranzani-Paiva).
https://doi.org/10.1016/j.aqrep.2020.100277 Received 5 July 2019; Received in revised form 6 January 2020; Accepted 8 January 2020 Available online 18 February 2020 2352-5134/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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stressors such as handling, high densities of storages, inadequate nutrition cause unbalanced physiological homeostasis, reduced growth, reproductive capacity and suppression of the immune system, allowing the fish susceptible to diseases (Pulkkinen et al., 2010; Van Weerd and Komen, 1998). The dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture. Nevertheless, Ramos et al. (2017) reported that no effect of probiotic feeding was observed after a long period supplementation. The pulse- supplementation probiotic feeding strategies involves alternating short periods of feeding fish with diets containing probiotic and control diets. Effect of probiotic feeding on tilapia could also be related to the probiotic specie used and the pulse-supplementation period. In fact, Apún-Molina et al. (2015) demonstrated that the use of strains considered as potentially probiotic (PPB), did not have any significant differences of hematological dietary supply of PPB, however supplementation every 5 days has an intermediate values of cholesterol in tilapia blood. The protein, glucose, and triglycerides concentration, in relation to the frequency of PPB dietary supply, were not significantly different. Other studies reported that Application of pulse-supplementation probiotic feeding strategies provide the direct benefits of short-term application during the supplemental feeding phase and during the unsupplemented stage where gastric probiotic populations persist for a number of weeks (Balcázar et al., 2007; Kim and Austin, 2006). The pulse feeding strategy may help to avoid over-stimulating the immune response whilst maintaining a level of protection/immuno-stimulation of Nile Tilapia produced in large scale (Merrifield et al., 2010). The present work aimed to evaluate the difference and possible advantages of pulse-administration probiotic feeding strategies for tilapia culture. This goal was evaluated through analysis of growth performance, biochemical parameters. Evolution of immune parameters after bacterial challenge of Nile tilapia fed continuously and pulse feeding schedule with probiotic E. faecium was also performed.
Table 1 Composition of experimental diets (g. 100 g−1 on dry matter basis). Diets
Control
Ingredients Soybean meal 40.5 Corn gluten 5.86 Fish meal 10 Corn 36.45 L-lysine 0.49 DL-methionine 0.4 Threonine 0.15 Bicalcium phosphate 3.8 Vitamin C 0.08 Vitamin and mineral¹ 0.25 Soybean oil 2.0 – Enterococcus faecium (Cylactin®, 1010 CFU g−1) Chemical composition (g 100 g−1) on a dry-matter basis Crude protein (%)* 34.17 Digestible protein (%)* 27.00 −1 3,916 Gross energy (kcal kg )* Digestible energy (kcal kg−1)* 3,042 Ether extract (%)* 2.47 Crude fiber (%)* 5.86 Lysine** 2.25 Methionine** 0.80 Threonine** 1.17 Total calcium (%)** 1.50 Phosphorus available (%)** 0.70 Total ash (%)** 8.85
Probiotic
40.5 5.86 10 36.40 0.49 0.4 0.15 3.8 0.08 0.25 2.0 0.05 34.17 27.00 3,916 3,042 2.47 5.86 2.25 0.80 1.17 1.50 0.70 8.85
*Analyzed; **Calculated
2.3. Sampling and Analyses The fish were fasted 24 hours before biometric measurement and sampling. The animals were anaesthetized with eugenol (50 mg L−1), weighted and lenghted, in order to calculate final weight (FW), weight gain (WG), specific growth rate (SGR), feed consumption (CONS), feed conversion rate (FCR) and final biomass (BIO). Two specimens were randomly collected from each tank (n = 8), anesthetized with clove oil (50 mg L−1), and blood samples were collected. Sampled bled fish were killed by cervical dissection and subjected to the analyses described below.
2. Material and methods 2.1. Experimental design and animals
2.4. Whole-body chemical composition
The experiment was conducted on Fisheries Institute in Pirassununga, Brazil (coordinate 21° 59' 46" South latitude and 47° 25' 33" West longitude). The experimental design was randomized, composed by four treatments and four replications: feeding continiously with only basal diet (CTR), feeding continuously with probiotic-supplemented diet (CON), feeding following pulse-administration of 7 days (P7) and 14 days (P14) cyclically alterning uses of probiotic supplemented diet and basal diet, respectively. During an experimental period of 84 days, 640 tilapias (20,0 ± 0,25 g) were maintained in 16 tanks (capacity of 800 L) (density of 0.05 fish.L−1) and were fed, three times (8:00, 12:00, 16:00hs) a day, with experimental diets and according to the strategies described above. Water quality parameters (temperature, dissolved oxygen and pH) were measured daily. The experiments were conducted according to the ethics committee of the Fisheries Institute protocol 01/2015.
The fish body was dried in an oven at 105 °C until a constant weight was obtained. The crude protein was quatified using the method of Nkjeldahl x6.25 (Miller et al., 2007). Ash residue was obtained after complete combustion at 550 °C. Crude lipid was obtained using the ether extract Soxhlet method (Thiex et al., 2003). 2.5. Probiotic viability Ten fish per aquaria were starved during 24 h to fecal gut depletion before death by super dosage of anesthesic (eugenol at the concentration of 50 mg L−1) and medullar dissection, externally sprayed with alcohol 70 % during 3 min. The intestines were removed under sterile conditions, weighted, macerated inside of test tubes, previously sterilized, and serial dilution until 10-2 fold. A volume of 5 μL were plated on HiCrome E. faecium HiVeg Agar Base (HiMedia Lab, MV1580), in duplicate (Irianto and Austin, 2002). The plates were incubated at 30 °C during 48 h to colony count.
2.2. Experimental diets A basal commercial diet (Table 1) was formulated according to the nutritional requirements of Nile tilapia (Furuya et al., 2004). The probiotic diet was composed by the basal diet with inclusion of commercial lyophilized probiotic E. faecium (CYLACTIN®, DSM, Netherlands) at 1010CFU g−1, diluted in soybean oil (2 % of feed weight) and sprayed on feed. The diets were maintained at 4 °C.
2.6. Hematology and biochemistry analyses Blood samples were collected from the vein of caudal region of anesthetized fish, using a heparin-coated syringe with 25-G needles, 2
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incubated at 37 °C for 1 h to allow adhesion of cells. The supernatant was removed and the wells were washed three times in PBS. Then, 50 μl of 0.2 % NBT (Sigma®) was added and incubated for a further 1 h. The cells were then fixed with 100 % methanol for 2 e 3 min and washed three times with 30 % methanol. The plates were air-dried and 60 μl 2 N potassiumhydroxide and 70 μl dimethyl sulphoxide were added, respectively, to each well. The OD (optical density) was recorded in an ELISA reader at 540 nm.
centrifuged in microtube centrifuge (Hettch Lab Technology, Germany) at 2,000xg for 10 min and plasma samples were obtained and stored at −20 °C until analysis. For serum samples, blood samples were collected by syringes without anticoagulant and allowed to clot for 4 h, centrifuged in microtube centrifuge at 3,000xg for 5 min and then stored at −20 °C. Immediately after blood extraction, the red blood cells were quantified using a Neubauer chamber. The hematocrit percentage was determined through the microhematocrit method (Goldenfarb et al., 1971) and the hemoglobin concentration was accomplished using the cyanmet haemoglobin method (Collier, 1944). The hemoglobin (Hb), hematocrit (Ht), and red blood cell (RBC) values were used to calculate the following haematimetric indexes: the mean corpuscular volume (MCV = (Ht 10)/RBC), meancorpuscular hemoglobin (MCH = (Ht 10)/ Hb), and the mean corpuscular hemoglobin concentration (MCHC = (Hb100)/Ht). The serum cortisol and glucose levels were determined using ELISA (DBC kit; Diagnostics Biochem. Canada, Inc., Ontario, Canada) (Sathish et al., 2012) and a glucose kit (Labtest, Minas Gerais, Brazil) (Borges et al., 2008), respectively. For the fish chalanged against A. hydrophila, in addition to the haematological analyzes described above, the counts of the differential and total leukocyte and thrombocyte was performed according to Hrubec and Smith (1998).
2.10. Challenge against A. hydrophila Experimental infection was carried out, in triplicate, for all the treatments cited above. In this challenge experiment, a control group was added, the fish of this group were injected (intraperitoneal injection) with 0.1 ml of PBS (0, 7 %). Fifty specimens of tilapia (30.00 g average bodyweight) were bred in tanks of 40 L capacity (10 fish/tank). After a feeding period of 21 days, the fish were experimentally infected via intraperitoneal injection with the pathogenic bacteria A. hydrophila isolated and identified from disesed tilapia. infection volume was 0.1 mL of bacteria at a concentration of 108 CFU mL−1 (LD50). The fish were observed for 14 days for mortality detection. The relative level of protection (RLP) was calculated according Newman and Mainrich (1982) with the following formula: RLP: 1-(percent of mortality in treated group/percent of mortality in control group) x 100.
2.7. Phagocytic activity The phagocytic activity was determined following the in vivo method described by Dias et al. (2016) with slight modifications of Telli et al. (2014). The fish randomly sampled were anesthetized, and 3.0 mL of a Saccharomyces cerevisiae Baker's yeast, Type II, Sigma, USA yeast solution at concentration of 8000 cells μL−1 was injected into the celomic cavity. After four hours of incubation (time required for the migration of phagocytic cells to the celomic cavity), the animals were killed by a high dosage of anesthetic and spinal cord dissection; the coelomic cavity of each fish was washed with 3.0 mL of PBS. The liquid was collected using a Pasteur pipette and centrifuged at 251.5 x g for 5 min, and the supernatant was discarded. Aliquots of the sediments, containing the yeast phagocyted by the leucocytes, were examined between a slide and coverslip under a contrast phase microscope (400x) with a green filter. One hundred phagocytes were counted to determine the phagocytic capacity (PC), PC: (number of phagocytes that phagocyted yeast/number of phagocytes observed), as well as the phagocytic index (PI), PI: (number of yeasts ingested/number of phagocytes observed).
2.11. Statistical analyzes The results were analyzed by one-way ANOVA using the SAS 9.1software. When significant differences were observed, the group means were compared with Duncan's test at a significance level of 5 %. 3. Results 3.1. Growth, body composition and survival During the experiment, the four feeding strategies did not cause any mortality of animals. However, the 7 days pulse feeding strategy (P7) demonstred efficacy in promote bether fish growth conditions, showed by the higher final weight, weight gain and final biomass (P < 0.01) (Table 2). The same tendency were not verified in whole body chemical composition. Effectively, the comparaison of the dried fish body, the crude protein and the ash residue did not showed any significative differences between all the treatments. The mean of values was 72.33
2.8. Lysozyme
Table 2 Final weight (FW), weight gain (WG), specific growth rate (SGR), feed consumption (CONS), feed conversion rate (FCR) and total final biomass (BIO) of Nile tilapia fed following pulse-administration with probiotic (E. faecium) supplemented in the diet during 84 days.
The lysozyme activity was determined following the method used by Kim and Austin (2006), in 100 μL serum samples diluted in two fold serial dilutions in a 96-well microtitre flat-bottomed plate (400 μL). Each well was mixed with 100 μL of a 0.4 mg mL−1Micrococcus lysodeikticus (Sigma) suspension in PBS (0.04 M). The microtitre plates were incubated at 22 °C, and the O.D. values after 0.0, 5.0, and 10.0 min of incubation were measured at 570 nm using PBS (Phosphatebuffered saline) as the blank solution. PBS was used as the negative control instead of the serum blood. A unit of lysozyme activity was defined as the amount of serum that caused a decrease in the absorbance of 0.001 min−1.
CTR FW (g) WG (g) SGR CONS (g) FCR BIO (g)
2.9. Respiratory burst activity AB
CON B
104.33 ± 2.05 84.51B ± 2.55 1.98 ± 0.05 98.87A ± 3.67 1.17 ± 0.05 4,173.27 B ± 82.20
P7 B
103.96 ± 4.62 83.87B ± 4.72 1.96 ± 0.06 96.45A ± 4.38 1.15 ± 0.04 4,158.38 B ± 184.98
P14 A
109.00 ± 0.70 88.96A ± 0.49 2.02 ± 0.02 98.75A ± 2.60 1.11 ± 0.03 4,359.90 A ± 28.03
103.65 B ± 1.79 83.59B ± 1.74 1.95 ± 0.02 91.94B ± 3.39 1.10 ± 0.04 4,146.42 B ± 88.85
Different letters in the column are significantly different by Duncan's test (P < 0.05). *CTR: control diet; CON: continuous supply of feed with probiotic; P7: pulse administration with probiotic feed every seven days; P14: pulse administration with probiotic feed every 14 days.
The phagocytic respiratory burst activity was done by nitroblue tetrazolium (NBT) assay following the method used by Secombes (1996) and modified by Stasiak and Bauman (1996). Blood samples (50 μl) were placed into the wells of flat-bottomed microtitre plates and 3
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starting from 48 h after inoculation with the pathogenic bacteria. There was no mortality in fish injected with PBS (0.7 %). There were no significant differences in hematological parameters (haematocrit, hemoglobin, erythrocytes counts, leucocytes total number, monocytes, neutrophyls, limphocyts and trombocyts total numbers) between the groups infected with A. hydrophila (Table 4). Significant differences were observed between the infected groups and the control group (injected with PBS), and these groups (CTR, P7 and P14) showed higher leukocyte and lymphocyte counts. The level of relative protection against A. hydrophila of groups fed diets with probiotics (CON, P7 and P14) showed higher percentage compared to control group (CTR). Treatment with continuously supply of probiotic supplementation showed the highest level of protection against A. hydrophila among all the tested groups.
% ± 0.96, 9.09 % ± 1.09, 14.28 % ± 0.66 and 3.86 % ± 0.30 respectively for moisture, ether extract, crude protein and ash. 3.2. Probiotic viability It was possible to recover the probiotic bacteria from the intestine of fish fed the probiotic-supplemented diet in any fed strategy CON, P7 and P14. The probiotic (E. faecium) was absent in the control group. The values of probiotic (log 10 CFUg−1) of E. faecium) recovered were as follow: CTR: 0, CON: 3.49, P7: 2.94, P14: 3.18. 3.3. Blood biochemical parameters Fish serum analysis did not show any significative difference in biochemical parameters between control and probiotic diet feeding even if supplied in pulse scheme. The mean values and standard deviation were 3.49 ± 0.12 g dL−1 to total protein, 0.68 ± 0,05 g dL−1 to albumin, 2.81 ± 0,11 g dL−1 to globulin, 0.24 ± 0,02 to albumin/ globulin and 41.05 ± 4,89 units mL−1 to lysozyme activity.
4. Discussion The importance of probiotic is incresing in europe with the continous restriction of chemotherapeutic treatment of diseases in animals raised in high density. In fish, the dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture (Ramos et al., 2017). Probiotics have also effects on growth rate and enhance immune system function (Hai, 2015). In this study, we noted that the wieght gain was obtained in fish group fed in Pulse administration with probiotic every 7 days, however, no significative difference was registered in the other feed strategies (Table 2). Merrifield et al. (2010) reported also that the feeding strategy of probiotic delivery to fish may provoke different responses to growth. The intermittent supply of probiotic bacteria can stimulate the immune system in order to always maintain the animal's defenses against possible infestations. In intestinal tracts lactic acid bacteria such as E. faecium, produces bacteriocins (Sonsa-Ard et al., 2015) and enzymes that facilitate the digestion of food, reduce the intestinal pH and consequently increase the absorption of minerals (Sun et al., 2011). The important caracteristics of these lactic acid bacteria to adapt to the gastro-intestinal tract allow them to dominate the intestinal flora (Gupta et al., 2019) and inhibit pathogens either by competition for nutrients or by secretion of inhibitory substrates (Allameh et al., 2017). One of the prerequisites for a bacterium to be considered as probiotic is to resist stomach acidity and digestive enzymes (Hamon et al., 2013). In this work, the presence of E. faecium in the fish intestine was verified by spreading in specific medium (Hicrome-Himedia) in all the treatments that received the probiotic. In the control, E. faecium was not detected, avoiding possible interferences in the results of the experiment (Table 3). In this study, we did not found any significative difference in serum analisis (albumin, globulin, lysosym, total protein) between all the treatments. This could be explicated by a constant regulation of the total protein in fish. The equilibre of these parameters in fish could be breaked by other stress conditions such as temperature. In fact, Mohapatra et al. (2014) showed that Serum albumin to globulin ratio (A/G ratio) was significantly (P < 0.01) reduced in fish fed with the probiotic incorporated diet and increased with elevated water temperature. In the experiment conducted with supplentation of probable probiotic bacteria (PPB), Apún-Molina et al. (2015) reported that highest concentration of cholesterol in tilapia blood occurred in tilapia that did not receive any PPB or were treated every 10 days as compared with tilapia without PPB (P < 0.05), with intermediate values for tilapia fed PPB every 5 days. The protein, glucose, and triglycerides concentration, in relation to the frequency of PPB dietary supply, were not significantly different. The phagocytic activity differ also with species of tilapia in fact, Cai et al. (2004) noted that phagocytic activity of nile tilapia and blue tilapia fed with probiotic was respectively (61 %) and (39 %). The
3.4. Respiratory burst and phagocytic activity Higher levels (P < 0.05) of respiratory burst activity were observed in fish that received feed pulse-administration of 14 days (P14), being statistically significant in comparison with the other groups (Fig. 1). No differences were observed in the phagocytic activity between the treatments. 3.5. Hematological parameters The Table 3 displays the data regarding the hematological parameters in the serum and the plasma after a after 84 days of experimental feeding for all the experimental trials. The hematological parameters HT, HB, ER, CHCM, and MVC in the serum, as well as the concentration of glucose and cortisol in the plasma did not show any significative difference between all the experimental trials during the experimental period. 3.6. Challenge with Aeromonas hydrophila The evolution of hematological parameters and relative protection level (RPL) of all the trials challenged with A. hydrophila and the control group (injected with PBS) were displayed in the Table 4. The onset of clinical signs such as isolation of fish, petechiae, ascites and hemorrhage were observed 24 h after challenge, followed by death between
Fig. 1. Respiratory burst activity of Nile tilapia blood cells, feeding following pulse-administration with probiotic (Enterococcus faecium) supplemented in the diet during 84 days. OD (optical density). ab Different letter differ by Duncan’s test (p < 0.05). CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days. 4
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Table 3 Hematological parameters of glucose, cortisol (CORT), hematocrit (HT), hemoglobin (HB), erythrocyte count (ER) and calculated mean corpuscular hemoglobin (CHCM), mean corpuscular volume (MVC), of Nile tilapia with different schedule of feeding during 84 days. The data are expressed as the means ± S.E.M (n = 8).
GLUCOSE (g dL-1) CORT ng mL-1 HT (%) HB (g dL-1) ER (106 mm-3) MCHC (%) MVC (%)
CTR
CON
P7
P14
49.88 ± 9.34 6.62 ± 1.26 35.31 ± 2.63 6.14 ± 0.64 1.21 ± 0.06 17.49 ± 2.36 297.16 ± 36.69
55.38 ± 11.34 7.09 ± 2.56 35.56 ± 2.11 6.34 ± 0.96 1.22 ± 0.02 17.85 ± 2.58 288.58 ± 12.60
53.63 ± 17.98 8.33 ± 2.25 36.06 ± 3.34 6.13 ± 0.72 1.20 ± 0.07 17.17 ± 3.05 301.09 ± 29.92
49.50 ± 10.14 9.24 ± 4.01 33.81 ± 1.85 5.86 ± 0.76 1.35 ± 0.17 17.29 ± 1.78 247.72 ± 20.41
*CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days.
may exist (Killie and Jorgensen, 1994) but are poorly understood (Bricknell and Dalmo, 2005). It was reported that the short term ciclyc delivery of the probiotic may be a viable alternative for immunostimulation of the fish (Merrifield et al., 2010), in our work the cyclic delivery was gives significant results only after 14 days period. This may suggest that the cyclic period could depend on fish specie and probiotic specie. Besides, this suggest that an over-stimulation of the immune system can be prevented by the constant presence of the probiotic in the diet. Our results confirm the conclusion of Merrifield et al. (2010) that argues that probably the use of probiotic continuously over a long period in fish feeding should not be relevant. As well, the implementation of this kind of continuous feeding strategy in commercial fish farming may be difficult due to the feed logistic distribution and control complications. However, a 14 day pulse administration of probiotic could stimulate periodically the immune system of fish. These results could be interesting for fish farmer since they, in one hand, reduce the cost of fish supplement feeding, and in the other hand stimulate periodically the immune system to defend fish from eventual disease risk. After challenge test with A. hydrophila, we registrated a significative decreases in the number of the lymphocytes compared to the fish injected only with PBS. The effectiveness of probiotics to protect fish from infection still in debate. Pirarat et al. (2006) has suggested that the probiotic Lactobacillus rhamnosus GG protect against Edwardsiella tarda infection by enhancing the alternative complement system thereby increasing phagocytic cell aggregation and phagocytic activity. Shelby et al. (2006) found that feeding commercial probiotics for 94 days did not prevent streptococcal disease (Welker and Lim, 2011).
phagocytic activity of three species of tilapia and their hybrid ranged between 74.3 % and 88.1 % (Casas Solis et al., 2007). Khalkhali and Mojgani (2017) revealed that E. faecium enhances mucosal immunity by increasing numbers of intraepithelial T lymphocytes, production of IgA, stimulates macrophages and dendritic cells to produce nitric oxid and digest microbes. Panigrahi et al. (2007) showed that, in rainbow trout fed with probiotics, expression levels of interleukin 1 alpha (IL-1α), Tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta (TGF-β) were increased significantly in the spleen and the kidney. Bricknell and Dalmo (2005) reported that continuous feeding of an immunostimulant can either up-regulates the immune system to heightened levels and this is maintained until the immunostimulant is withdrawn, or cause adverse effects such as tolerance or immunosuppression. Conversely, pulse administration may oscillate the immune response from a resting level to an enhanced response then back to resting again. The same author revealed that most immunostimulation strategies involve pulse feeding the immunostimulant for a short period, usually 4–6 weeks, to up-regulate the immune response. In our study we used different strategies. In fact, we used a continuous feeding (CON), seven days pulse-administration and 14 days pulse-administration. In the trial P7 and CON of our study, we did not registered any significative difference in the immune parameters (phagocytic activity and respiratory burst activity). This could be explicated by an immuno tolerance of fish. Tolerance has been reported in some fish species (Nakamura et al., 1998; Waagbo et al., 1994; Zapata et al., 1997) and antigen competition has been observed in Atlantic salmon suggesting that the mechanisms for the induction of tolerance
Table 4 Hematological parameters and relative protection level (RPL) of Nile tilapia submitted to challenge infection with A. hydrophila after probiotic, E. faecium feeding during 21 days. Hematological parameters were measured from surviving fish 14 days post challenge.
Hematocrit (%) Hemoglobin (g.dL
−1
)
Erythrocytes (104 cel mm−3) Leucocytes (cel mm−3) Monocytes (cel mm−3) Neutrophils (cel mm−3) Lymphocytes (cel mm−3) Trombocytes (cel mm−3) Relative protection level (%)
CTR
CON
P7
P14
PBS
29.13 ± 4.60 4.43 ± 0.73 106.75 ± 22.56 1,065.10b ± 535.30 40.66 ± 26.12 421.46 ± 349.46 601.00b ± 262.00 11,194.00 ± 8,412.00 0.00
33.20 ± 7.72 5.19 ± 0.69 110.70 ± 25.63 1,528.40b ± 1,070.30 60.19 ± 20.52 585.95 ± 537.31 882.00b ± 546.00 11,164.00 ± 4,915.00 83.33
32.13 ± 3.04 4.79 ± 0.43 126.13 ± 12.46 2,075.10b ± 1,358.50 63.32 ± 44.51 949.01 ± 699.03 1,062.00b ± 658.00 16,579.00 ± 8,068.00 62.96
30.70 ± 3.81 4.39 ± 0.91 109.00 ± 16.93 1,272.20b ± 1,586.00 26.49 ± 39.28 578.49 ± 370.47 664.00b ± 1,457.00 8,245.00 ± 5,830.00 66.60
33.10 ± 6.04 5.24 ± 0.59 119.60 ± 17.05 3,393.00ª ± 961.40 137.90 ± 148.38 239.76 ± 136.10 3,007.00ª ± 828.00 16,245.00 ± 4,489.00 100.00
CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days. 5
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The enduring protection of probiotics, seems to be strongly depending on the probiotic specie, the concentration administrated and the feeding duration. Aly et al. (2008a) found that supplementing Bacillus pumilus at 1012/g diet increased protection of Nile tilapia against A. hydrophila after 1 and 2 but not 8 months of feeding. The lower dietary concentration tested (106/g) did not provide any protection. In another study, Aly et al. (2008b) also found that dietary supplementation of L. acidophilus, B. subtilis, or a mixture of the two generally provided greater protection against A. hydrophila, P. fluorescens, and S. iniae after 2 months of feeding compared to 1 month (Welker and Lim, 2011).
Cai, W.Q., Li, S.F., Ma, J.Y., 2004. Diseases resistance of Nile tilapia (Oreochromis niloticus), blue tilapia (Oreochromis aureus) and their hybrid (female Nile tilapia x male blue tilapia) to Aeromonas sobria. Aquaculture 229, 79–87. https://doi.org/10.1016/ S0044-8486(03)00357-0. Casas Solis, J., Santerre, A., Girón Pérez, M.I., Reynoso Orozco, R., Zaitseva, G., 2007. A comparative sudy of phagocytic activity and lymphoproliferative response in five varieties of tilapia Oreochromis spp. J. Fish Biol. 71, 1541–1545. https://doi.org/10. 1111/j.1095-8649.2007.01601.x. Collier, H.B., 1944. The standardization of blood haemoglobin determinations. Can. Med. Assoc. J. 50, 550–552. Dias, M., Brochetta, C., Marchetti, A., Bodinier, R., Brückert, F., Cosson, P., 2016. Role of SpdA in cell spreading and Phagocytosis in dictyostelium. PLoS One 11 (8), e0160376. https://doi.org/10.1371/journal.pone.0160376. FAO, 2018. The State of World Fisheries and Aquaculture 2018 - Meeting the Sustainable Development Goals. Rome. Licence: CC BY-NC-SA 3.0 IGO. . Furuya, W.M., Pezzato, L.E., Barros, M.M., Pezzato, A.C., Furuya, V.R.B., Miranda, E.C., 2004. Use of ideal protein concept for precision formulationof amino acid levels in fish-meal-free diets for juvenile Nile tilapia (Oreochromis niloticus L.). Aquac. Res. 35, 1110–1116. https://doi.org/10.1111/j.1365-2109.2004.01133.x. Gjedrem, T., 2012. Genetic improvement for the development of efficient global aquaculture: a personal opinion review. Aquaculture 344–349, 12–22. https://doi.org/10. 1016/j.aquaculture.2012.03.003. Goldenfarb, P.B., Bowyer, F.P., Hall, E., Brosius, E., 1971. Reproducibility in the hematology laboratory: the microhematocrit determinations. Am. J. Clin. Pathol. 56, 35–39. Gupta, S., Feckaninová, A., Lokesh, J., Košcová, J., Sørensen, M., Fernandes, J., Kiron, V., 2019. 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Liao, S.F., Nyachoti, M., 2017. Using probiotics to improve swine gut health and nutrient utilization. Anim. Nutr. (Zhongguo xu mu shou yi xue hui). 3 (4), 331–343. https:// doi.org/10.1016/j.aninu.2017.06.007. Merrifield, D.L., Bradley, G., Baker, R., Davies, S., 2010. Probiotic applications for rainbow trout (Oncorhynchus mykiss Walbaum) II. Effects on growth performance, feed utilization, intestinal microbiota and related health criteria postantibiotic treatment. Aquacult. Nutr. 16, 496–503. https://doi.org/10.1111/j.1365-2095.2009. 00688.x. Miller, E.L., Bimbo, A.P., Barlow, S.M., Sheridan, B., 2007. Repeatability and reproducibility of determination of the nitrogen content of fishmeal by the combustion (Dumas) method and comparison with the kjeldahl method: interlaboratory study. J. AOAC Int. 90 (1), 1–15. Mohapatra, S., Chakraborty, T., Prusty, A.K., PaniPrasad, K., Mohanta, K.N., 2014. Beneficial effects of dietary probiotics mixture on hemato-immunology and cell apoptosis ofLabeo rohitaFingerlings reared at higher water temperatures. PLoS One 9 (6), e100929. https://doi.org/10.1371/journal.pone.0100929. Nakamura, O., Suzuki, Y., Aida, K., 1998. Humoral immune response against orally administered human gamma globulin in the carp. Fish. Sci. 64, 558–562. Newman, S.G., Mainrich, J.J., 1982. Direct immersion vaccination of juvenile rainbow trout (Salmo gairdneri) and juvenile coho salmon (Oncorhynchus kisutch) with a (Yarsinina Yuckerli) bacterin. J. Fish Dis. 5, 339–341. Panigrahi, A., Kiron, V., Satoh, S., Hirono, I., Kobayashi, T., Sugita, H., Puangkaew, J., Aoki, T., 2007. Immune modulation and expression of cytokine genes in rainbow trout Oncorhynchus mykiss upon probiotic feeding. Dev. Comp. Immunol. 31 (4), 372–382. https://doi.org/10.1016/j.dci.2006.07.004. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Vet. Immunol. Immunopathol. 113, 339–347. https://doi.org/10.1016/j.vetimm.2006.06.003. Pulkkinen, K., Suomalainen, L.R., Read, A.F., Ebert, D., Rintamäki, P., Valtonen, E.T., 2010. Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland. Proc. Biol. Sci. 277 (1681), 593–600. https://doi.org/ 10.1098/rspb.2009.1659.
5. Conclusion With this study we proved the benefic effect of E. faecium as a probiotic to be used as a complement for feeding tilapia. Impact of the different feeding strategies was not detected in all the studied parameters but we confirmed that E. faecium enhances fish growth, boost imune system if administrated in 7 days period, and efficiently protect fish if it is continuously administrated. This motivates further investigation that could be undertaken using other single or multiple probiotics to be used in intensive aquaculture industry. Funding This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), projects numbers: 2013/09731-8 and 2016/19816-9. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The authors thank to Claudio Cirineu Ciola, Jair Donizetti Mazzaferro and Hélio Sanches Mariscal, Eliana Oshiro for thecnical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2020.100277. References Allameh, S.K., Noaman, V., Nahavandi, R., 2017. Effects of probiotic bacteria on fish performance. Adv. Tech. Clin. Microbiol. 1, 2. Aly, S.M., Ahmed, Y.A., Ghareeb, A.A., Mohamed, M.F., 2008a. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immunol. 25, 128–136. https://doi.org/10.1016/j.fsi.2008.03.013. Aly, S.M., Mohamed, M.F., John, G., 2008b. Effect of probiotics on the survival, growth and challenge infection in Tilapia nilotica (Oreochromis niloticus). Aquac. Res. 39, 647–656. https://doi.org/10.1111/j.1365-2109.2008.01932.x. Apún-Molina, J.P., Santamaría-Miranda, A., Luna-González, A., Ibarra-Gámez, J.C., Medina-Alcantar, V., Racotta, I., 2015. Growth and metabolic responses of whiteleg shrimp Litopenaeus vannameiandNile tilapia Oreochromis niloticusin polyculture fed with potentialprobiotic microorganisms on different schedules. Lat. Am. J. Aquat. Res. 43, 435–445 DOI: 10.3856. Balcázar, J.L., de Blas, I., Ruiz-Zarzuela, I., Vendrell, D., Calvo, A.C., Marquez, I., Girones, O., Muzquiz, J.L., 2007. Changes in intestinal microbiota and humoral immune response following probiotic administration in brown trout (Salmo trutta). Br. J. Nutr. 97, 522–527. https://doi.org/10.1017/S0007114507432986. Borges, L.P., Brandão, R., Godoi, B., Nogueira, C.W., Zeni, G., 2008. Oral administration of diphenyl diselenide protects against cadmium-induced liver damage in rats. Chem.-Biol. Interact. 171, 15–25. https://doi.org/10.1016/j.cbi.2007.09.005. Bricknell, I., Dalmo, R.A., 2005. The use of immunostimulants in fish larval aquaculture. Fish Shellfish Immunol. 19, 457–472. https://doi.org/10.1016/j.fsi.2005.03.008.
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Sun, Y.Z., Yang, H.L., Ma, R.L., Song, K., Li, J.S., 2011. Effect of Lactococcus lactis and Enterococcus faecium on growth performance, digestive enzymes and immune response of grouper Epinephelus coioides. Aquac. Nut. 18 (3), 281–289. https://doi.org/ 10.1111/j.1365-2095.2011.00894.x. Telli, G.S., Ranzani Paiva, M.J.T., de Carla Dias, D., Sussel, F.R., Ishikawa, C.M., Tachibana, L., 2014. Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities. Fish Shellfish Immunol. 39 (2), 305–311. https://doi.org/10.1016/j.fsi. 2014.05.025. Thiex, N.J., Anderson, S., Gildemeister, B., 2003. Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (Randall/Soxtec/Submersion method): collaborative study. J. AOAC Int. 86 (5), 888–898. Van Weerd, J.H., Komen, J., 1998. The effects of chronic stress on growth in fish: a critical appraisal. Comp. Biochem. Physiol. A. 120, 107–112. Waagbo, R., Glette, J., Sandnes, K., Hemre, G.I., 1994. Influence of dietary carbohydrate on blood chemistry, immunity and disease resistance in Atlantic salmon, Salmo salar L. J. Fish Dis. 17, 245–258. Wang, Y.B., Tian, Z.Q., Yao, J.T., Li, W.F., 2008. Effect of probiotics, Enteroccus faecium on tilapia (Oreochromis niloticus) growth performance and immune response. Aquaculture. 277, 203–207. https://doi.org/10.1016/j.aquaculture.2008.03.007. Welker, T.L., Lim, C., 2011. Use of probiotics in diets of Tilapia. J. Aquac. Res. Dev. https://doi.org/10.4172/2155-9546.S1-014. S1:014. Zapata, A.G., Torroba, M., Varas, A., Jimenez, E., 1997. Immunity in fish larvae. Dev. Biol. Stand. 90, 23–32.
Ramos, M.A., Batista, S., Pires, M.A., Silva, A.P., Pereira, L.F., Saavedra, M.J., Ozório, R.O.A., Rema, P., 2017. Dietary probiotic supplementation improves growth and the intestinal morphology of Nile tilapia. Animal 11 (8), 1259–1269. https://doi.org/10. 1017/S1751731116002792. Safari, R., Adel, M., Lazado, C.C., Caipang, C.M., Dadar, M., 2016. Host-derived probiotics Enterococcus casseliflavus improves resistance against Streptococcus iniae infection in rainbow trout (Oncorhynchus mykiss) via immunomodulation. Fish Shellfish Immunol. 52, 198–205. https://doi.org/10.1016/j.fsi.2016.03.020. Sathish Babu, M., Bobby, Z., Habeebullah, S., 2012. Increased inflammatory response and imbalance in blood and urinary oxidant–antioxidant status in South Indian women with gestational hypertension and preeclampsia. Clin. Biochem. 45, 835–838. https://doi.org/10.1016/j.clinbiochem.2012.04.018. Secombes, C.J., 1996. The nonspecific immune system: cellular defenses. In: Iwama, G., Nakanishi, T. (Eds.), The Fish Immune System. Organism, Pathogen and Environment. Academic Press, San Diego, pp. 63–95. Shelby, R.A., Lim, C.E., Aksoy, M., Klesius, P.H., 2006. Effect of probiotic diet supplements on disease resistance and immune response of young Nile tilapia, Oreochromis niloticus. J. App. Aquac. 18, 23–34. https://doi.org/10.1300/J028v18n02_02. Sonsa-Ard, N., Rodtong, S., Chikindas, M.L., Yongsawatdigul, J., 2015. Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe. Food Control 54, 308–316. https://doi.org/10.1016/j. foodcont.2015.02.010. Stasiak, A.S., Bauman, C.P., 1996. Neutrophil activity as a potent indicator for concomitant analysis. Fish Shellfish Immunol. 6 (537), 539.
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Effect of feeding strategy of probiotic Enterococcus faecium on growth performance, hematologic, biochemical parameters and non-specific immune response of Nile tilapia
T
Leonardo Tachibanaa, Guilherme Silveira Tellia, Danielle de Carla Diasa, Giovani Sampaio Gonçalvesb, Carlos Massatoshi Ishikawaa, Raissa Bertoncello Cavalcantea, Mariene Miyoko Natoria, Said Ben Hameda, Maria José T. Ranzani-Paivaa,* a
Aquaculture Research Center, Scientific Research of Fisheries Institute/APTA/SAA, São Paulo. Av. Francisco Matarazzo, 455 - 05001-900, São Paulo, Brazil Scientific Research of Advanced Technological Research Center for Continental Fishery Agribusiness of Fisheries Institute/APTA/SAA, Abelardo Menezes Avenue, 15092607, São José do Rio Preto, SP, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Enterococcus faecium Tilapia Immune system Feeding strategies Challenge Aeromonas hydrophila
The aquaculture industry is in considerable increases. Optimization of husbandary conditions in intensive aquaculture are in continuous interest. Probiotic was proved as promising factors to enhance fish production by remodeling intestinal microbial balance, boosting immune system and reducing antibiotic uses. The efficient strategy of probiotic uses and their protection of fish still making debate. The intake of tilpia fed with basal diet enriched with probiotic (Enterococcus faecium) and its response following to a challenge with pathogenic strain of Aeromonas hydrophila was studied. Four feeding strategies were tested and compared: continuous feeding with only basal diet (CTR), continuous feeding with probiotic-supplemented diet (CON), pulse-administration feeding of 7 days (P7) and 14 days (P14) alternation with probiotic supplemented diet and basal diet. Differences in growth rate, immune system modulation and biochemical parameters were evaluated. P7 strategy showed efficacy in promote fish growth conditions. Body chemical composition and blood biochemical parameters did not show any significative differences between feeding strategies. Burst respiratory analysis showed significant difference in fish fed with P14 strategy. After challenge test, CON strategy showed better fish protection against Aeromonas hydrophila. Enterococcus faecium enhance fish growth, boost imune system if administrated in 14 days period, and efficiently protect fish if it is continuously administrated.
1. Introduction
stress tolerance (Kesarcodi-Watson et al., 2008; Safari et al., 2016; Wang et al., 2008). In addition, probiotics also can be effective in promote the phagocytic activity and lysozyme levels of fish (Harikrishnan et al., 2010). Lactic acid bacteria (LAB) such as Enterococcus faecium is commonly used as probiotic in fish farming and are known to be present in the intestine of healthy fish. According to Wang et al. (2008) the effect of E. faecium in the water at 107 CFU mL−1 concentration (supplemented once every four days) provided higher final weight, daily weight gain, myeloperoxidase activity, respiratory burst and blood phagocytes of the group that receive probiotic in the water than control group. In the other hand, probiotic administration can be a nutritional strategy to improve the immune response and growth performance of fish (Ramos et al., 2017). In fact, probiotics application strategies can be useful in fish intensive production system, in which common
Following the FAO, 2018 tilapia have shown a continuous increase among the major species groups in inland water. In one hand, The Genetic Improvement of Farmed Tilapia (GIFT) project has played an important role in the expansion of Nile tilapia culture (now reported in 87 countries) by helping to avoid the negative impacts of inbreeding or poor genetic management (Gjedrem, 2012). In many countries, antibiotics administration caused concerns about zoonotic bacteria resistances in animal production, causing food borne diseases. Probiotics is one of the few alternatives to substitute these medicaments, promote protective effects against pathogens, and then produce safety foods (Liao and Nyachoti, 2017). In aquaculture, probiotics is widely used to improve growth performance and modulate the immune system in order to inhibit pathogens growth and to increase
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Corresponding author. E-mail address: [email protected] (M.J.T. Ranzani-Paiva).
https://doi.org/10.1016/j.aqrep.2020.100277 Received 5 July 2019; Received in revised form 6 January 2020; Accepted 8 January 2020 Available online 18 February 2020 2352-5134/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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stressors such as handling, high densities of storages, inadequate nutrition cause unbalanced physiological homeostasis, reduced growth, reproductive capacity and suppression of the immune system, allowing the fish susceptible to diseases (Pulkkinen et al., 2010; Van Weerd and Komen, 1998). The dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture. Nevertheless, Ramos et al. (2017) reported that no effect of probiotic feeding was observed after a long period supplementation. The pulse- supplementation probiotic feeding strategies involves alternating short periods of feeding fish with diets containing probiotic and control diets. Effect of probiotic feeding on tilapia could also be related to the probiotic specie used and the pulse-supplementation period. In fact, Apún-Molina et al. (2015) demonstrated that the use of strains considered as potentially probiotic (PPB), did not have any significant differences of hematological dietary supply of PPB, however supplementation every 5 days has an intermediate values of cholesterol in tilapia blood. The protein, glucose, and triglycerides concentration, in relation to the frequency of PPB dietary supply, were not significantly different. Other studies reported that Application of pulse-supplementation probiotic feeding strategies provide the direct benefits of short-term application during the supplemental feeding phase and during the unsupplemented stage where gastric probiotic populations persist for a number of weeks (Balcázar et al., 2007; Kim and Austin, 2006). The pulse feeding strategy may help to avoid over-stimulating the immune response whilst maintaining a level of protection/immuno-stimulation of Nile Tilapia produced in large scale (Merrifield et al., 2010). The present work aimed to evaluate the difference and possible advantages of pulse-administration probiotic feeding strategies for tilapia culture. This goal was evaluated through analysis of growth performance, biochemical parameters. Evolution of immune parameters after bacterial challenge of Nile tilapia fed continuously and pulse feeding schedule with probiotic E. faecium was also performed.
Table 1 Composition of experimental diets (g. 100 g−1 on dry matter basis). Diets
Control
Ingredients Soybean meal 40.5 Corn gluten 5.86 Fish meal 10 Corn 36.45 L-lysine 0.49 DL-methionine 0.4 Threonine 0.15 Bicalcium phosphate 3.8 Vitamin C 0.08 Vitamin and mineral¹ 0.25 Soybean oil 2.0 – Enterococcus faecium (Cylactin®, 1010 CFU g−1) Chemical composition (g 100 g−1) on a dry-matter basis Crude protein (%)* 34.17 Digestible protein (%)* 27.00 −1 3,916 Gross energy (kcal kg )* Digestible energy (kcal kg−1)* 3,042 Ether extract (%)* 2.47 Crude fiber (%)* 5.86 Lysine** 2.25 Methionine** 0.80 Threonine** 1.17 Total calcium (%)** 1.50 Phosphorus available (%)** 0.70 Total ash (%)** 8.85
Probiotic
40.5 5.86 10 36.40 0.49 0.4 0.15 3.8 0.08 0.25 2.0 0.05 34.17 27.00 3,916 3,042 2.47 5.86 2.25 0.80 1.17 1.50 0.70 8.85
*Analyzed; **Calculated
2.3. Sampling and Analyses The fish were fasted 24 hours before biometric measurement and sampling. The animals were anaesthetized with eugenol (50 mg L−1), weighted and lenghted, in order to calculate final weight (FW), weight gain (WG), specific growth rate (SGR), feed consumption (CONS), feed conversion rate (FCR) and final biomass (BIO). Two specimens were randomly collected from each tank (n = 8), anesthetized with clove oil (50 mg L−1), and blood samples were collected. Sampled bled fish were killed by cervical dissection and subjected to the analyses described below.
2. Material and methods 2.1. Experimental design and animals
2.4. Whole-body chemical composition
The experiment was conducted on Fisheries Institute in Pirassununga, Brazil (coordinate 21° 59' 46" South latitude and 47° 25' 33" West longitude). The experimental design was randomized, composed by four treatments and four replications: feeding continiously with only basal diet (CTR), feeding continuously with probiotic-supplemented diet (CON), feeding following pulse-administration of 7 days (P7) and 14 days (P14) cyclically alterning uses of probiotic supplemented diet and basal diet, respectively. During an experimental period of 84 days, 640 tilapias (20,0 ± 0,25 g) were maintained in 16 tanks (capacity of 800 L) (density of 0.05 fish.L−1) and were fed, three times (8:00, 12:00, 16:00hs) a day, with experimental diets and according to the strategies described above. Water quality parameters (temperature, dissolved oxygen and pH) were measured daily. The experiments were conducted according to the ethics committee of the Fisheries Institute protocol 01/2015.
The fish body was dried in an oven at 105 °C until a constant weight was obtained. The crude protein was quatified using the method of Nkjeldahl x6.25 (Miller et al., 2007). Ash residue was obtained after complete combustion at 550 °C. Crude lipid was obtained using the ether extract Soxhlet method (Thiex et al., 2003). 2.5. Probiotic viability Ten fish per aquaria were starved during 24 h to fecal gut depletion before death by super dosage of anesthesic (eugenol at the concentration of 50 mg L−1) and medullar dissection, externally sprayed with alcohol 70 % during 3 min. The intestines were removed under sterile conditions, weighted, macerated inside of test tubes, previously sterilized, and serial dilution until 10-2 fold. A volume of 5 μL were plated on HiCrome E. faecium HiVeg Agar Base (HiMedia Lab, MV1580), in duplicate (Irianto and Austin, 2002). The plates were incubated at 30 °C during 48 h to colony count.
2.2. Experimental diets A basal commercial diet (Table 1) was formulated according to the nutritional requirements of Nile tilapia (Furuya et al., 2004). The probiotic diet was composed by the basal diet with inclusion of commercial lyophilized probiotic E. faecium (CYLACTIN®, DSM, Netherlands) at 1010CFU g−1, diluted in soybean oil (2 % of feed weight) and sprayed on feed. The diets were maintained at 4 °C.
2.6. Hematology and biochemistry analyses Blood samples were collected from the vein of caudal region of anesthetized fish, using a heparin-coated syringe with 25-G needles, 2
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incubated at 37 °C for 1 h to allow adhesion of cells. The supernatant was removed and the wells were washed three times in PBS. Then, 50 μl of 0.2 % NBT (Sigma®) was added and incubated for a further 1 h. The cells were then fixed with 100 % methanol for 2 e 3 min and washed three times with 30 % methanol. The plates were air-dried and 60 μl 2 N potassiumhydroxide and 70 μl dimethyl sulphoxide were added, respectively, to each well. The OD (optical density) was recorded in an ELISA reader at 540 nm.
centrifuged in microtube centrifuge (Hettch Lab Technology, Germany) at 2,000xg for 10 min and plasma samples were obtained and stored at −20 °C until analysis. For serum samples, blood samples were collected by syringes without anticoagulant and allowed to clot for 4 h, centrifuged in microtube centrifuge at 3,000xg for 5 min and then stored at −20 °C. Immediately after blood extraction, the red blood cells were quantified using a Neubauer chamber. The hematocrit percentage was determined through the microhematocrit method (Goldenfarb et al., 1971) and the hemoglobin concentration was accomplished using the cyanmet haemoglobin method (Collier, 1944). The hemoglobin (Hb), hematocrit (Ht), and red blood cell (RBC) values were used to calculate the following haematimetric indexes: the mean corpuscular volume (MCV = (Ht 10)/RBC), meancorpuscular hemoglobin (MCH = (Ht 10)/ Hb), and the mean corpuscular hemoglobin concentration (MCHC = (Hb100)/Ht). The serum cortisol and glucose levels were determined using ELISA (DBC kit; Diagnostics Biochem. Canada, Inc., Ontario, Canada) (Sathish et al., 2012) and a glucose kit (Labtest, Minas Gerais, Brazil) (Borges et al., 2008), respectively. For the fish chalanged against A. hydrophila, in addition to the haematological analyzes described above, the counts of the differential and total leukocyte and thrombocyte was performed according to Hrubec and Smith (1998).
2.10. Challenge against A. hydrophila Experimental infection was carried out, in triplicate, for all the treatments cited above. In this challenge experiment, a control group was added, the fish of this group were injected (intraperitoneal injection) with 0.1 ml of PBS (0, 7 %). Fifty specimens of tilapia (30.00 g average bodyweight) were bred in tanks of 40 L capacity (10 fish/tank). After a feeding period of 21 days, the fish were experimentally infected via intraperitoneal injection with the pathogenic bacteria A. hydrophila isolated and identified from disesed tilapia. infection volume was 0.1 mL of bacteria at a concentration of 108 CFU mL−1 (LD50). The fish were observed for 14 days for mortality detection. The relative level of protection (RLP) was calculated according Newman and Mainrich (1982) with the following formula: RLP: 1-(percent of mortality in treated group/percent of mortality in control group) x 100.
2.7. Phagocytic activity The phagocytic activity was determined following the in vivo method described by Dias et al. (2016) with slight modifications of Telli et al. (2014). The fish randomly sampled were anesthetized, and 3.0 mL of a Saccharomyces cerevisiae Baker's yeast, Type II, Sigma, USA yeast solution at concentration of 8000 cells μL−1 was injected into the celomic cavity. After four hours of incubation (time required for the migration of phagocytic cells to the celomic cavity), the animals were killed by a high dosage of anesthetic and spinal cord dissection; the coelomic cavity of each fish was washed with 3.0 mL of PBS. The liquid was collected using a Pasteur pipette and centrifuged at 251.5 x g for 5 min, and the supernatant was discarded. Aliquots of the sediments, containing the yeast phagocyted by the leucocytes, were examined between a slide and coverslip under a contrast phase microscope (400x) with a green filter. One hundred phagocytes were counted to determine the phagocytic capacity (PC), PC: (number of phagocytes that phagocyted yeast/number of phagocytes observed), as well as the phagocytic index (PI), PI: (number of yeasts ingested/number of phagocytes observed).
2.11. Statistical analyzes The results were analyzed by one-way ANOVA using the SAS 9.1software. When significant differences were observed, the group means were compared with Duncan's test at a significance level of 5 %. 3. Results 3.1. Growth, body composition and survival During the experiment, the four feeding strategies did not cause any mortality of animals. However, the 7 days pulse feeding strategy (P7) demonstred efficacy in promote bether fish growth conditions, showed by the higher final weight, weight gain and final biomass (P < 0.01) (Table 2). The same tendency were not verified in whole body chemical composition. Effectively, the comparaison of the dried fish body, the crude protein and the ash residue did not showed any significative differences between all the treatments. The mean of values was 72.33
2.8. Lysozyme
Table 2 Final weight (FW), weight gain (WG), specific growth rate (SGR), feed consumption (CONS), feed conversion rate (FCR) and total final biomass (BIO) of Nile tilapia fed following pulse-administration with probiotic (E. faecium) supplemented in the diet during 84 days.
The lysozyme activity was determined following the method used by Kim and Austin (2006), in 100 μL serum samples diluted in two fold serial dilutions in a 96-well microtitre flat-bottomed plate (400 μL). Each well was mixed with 100 μL of a 0.4 mg mL−1Micrococcus lysodeikticus (Sigma) suspension in PBS (0.04 M). The microtitre plates were incubated at 22 °C, and the O.D. values after 0.0, 5.0, and 10.0 min of incubation were measured at 570 nm using PBS (Phosphatebuffered saline) as the blank solution. PBS was used as the negative control instead of the serum blood. A unit of lysozyme activity was defined as the amount of serum that caused a decrease in the absorbance of 0.001 min−1.
CTR FW (g) WG (g) SGR CONS (g) FCR BIO (g)
2.9. Respiratory burst activity AB
CON B
104.33 ± 2.05 84.51B ± 2.55 1.98 ± 0.05 98.87A ± 3.67 1.17 ± 0.05 4,173.27 B ± 82.20
P7 B
103.96 ± 4.62 83.87B ± 4.72 1.96 ± 0.06 96.45A ± 4.38 1.15 ± 0.04 4,158.38 B ± 184.98
P14 A
109.00 ± 0.70 88.96A ± 0.49 2.02 ± 0.02 98.75A ± 2.60 1.11 ± 0.03 4,359.90 A ± 28.03
103.65 B ± 1.79 83.59B ± 1.74 1.95 ± 0.02 91.94B ± 3.39 1.10 ± 0.04 4,146.42 B ± 88.85
Different letters in the column are significantly different by Duncan's test (P < 0.05). *CTR: control diet; CON: continuous supply of feed with probiotic; P7: pulse administration with probiotic feed every seven days; P14: pulse administration with probiotic feed every 14 days.
The phagocytic respiratory burst activity was done by nitroblue tetrazolium (NBT) assay following the method used by Secombes (1996) and modified by Stasiak and Bauman (1996). Blood samples (50 μl) were placed into the wells of flat-bottomed microtitre plates and 3
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starting from 48 h after inoculation with the pathogenic bacteria. There was no mortality in fish injected with PBS (0.7 %). There were no significant differences in hematological parameters (haematocrit, hemoglobin, erythrocytes counts, leucocytes total number, monocytes, neutrophyls, limphocyts and trombocyts total numbers) between the groups infected with A. hydrophila (Table 4). Significant differences were observed between the infected groups and the control group (injected with PBS), and these groups (CTR, P7 and P14) showed higher leukocyte and lymphocyte counts. The level of relative protection against A. hydrophila of groups fed diets with probiotics (CON, P7 and P14) showed higher percentage compared to control group (CTR). Treatment with continuously supply of probiotic supplementation showed the highest level of protection against A. hydrophila among all the tested groups.
% ± 0.96, 9.09 % ± 1.09, 14.28 % ± 0.66 and 3.86 % ± 0.30 respectively for moisture, ether extract, crude protein and ash. 3.2. Probiotic viability It was possible to recover the probiotic bacteria from the intestine of fish fed the probiotic-supplemented diet in any fed strategy CON, P7 and P14. The probiotic (E. faecium) was absent in the control group. The values of probiotic (log 10 CFUg−1) of E. faecium) recovered were as follow: CTR: 0, CON: 3.49, P7: 2.94, P14: 3.18. 3.3. Blood biochemical parameters Fish serum analysis did not show any significative difference in biochemical parameters between control and probiotic diet feeding even if supplied in pulse scheme. The mean values and standard deviation were 3.49 ± 0.12 g dL−1 to total protein, 0.68 ± 0,05 g dL−1 to albumin, 2.81 ± 0,11 g dL−1 to globulin, 0.24 ± 0,02 to albumin/ globulin and 41.05 ± 4,89 units mL−1 to lysozyme activity.
4. Discussion The importance of probiotic is incresing in europe with the continous restriction of chemotherapeutic treatment of diseases in animals raised in high density. In fish, the dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture (Ramos et al., 2017). Probiotics have also effects on growth rate and enhance immune system function (Hai, 2015). In this study, we noted that the wieght gain was obtained in fish group fed in Pulse administration with probiotic every 7 days, however, no significative difference was registered in the other feed strategies (Table 2). Merrifield et al. (2010) reported also that the feeding strategy of probiotic delivery to fish may provoke different responses to growth. The intermittent supply of probiotic bacteria can stimulate the immune system in order to always maintain the animal's defenses against possible infestations. In intestinal tracts lactic acid bacteria such as E. faecium, produces bacteriocins (Sonsa-Ard et al., 2015) and enzymes that facilitate the digestion of food, reduce the intestinal pH and consequently increase the absorption of minerals (Sun et al., 2011). The important caracteristics of these lactic acid bacteria to adapt to the gastro-intestinal tract allow them to dominate the intestinal flora (Gupta et al., 2019) and inhibit pathogens either by competition for nutrients or by secretion of inhibitory substrates (Allameh et al., 2017). One of the prerequisites for a bacterium to be considered as probiotic is to resist stomach acidity and digestive enzymes (Hamon et al., 2013). In this work, the presence of E. faecium in the fish intestine was verified by spreading in specific medium (Hicrome-Himedia) in all the treatments that received the probiotic. In the control, E. faecium was not detected, avoiding possible interferences in the results of the experiment (Table 3). In this study, we did not found any significative difference in serum analisis (albumin, globulin, lysosym, total protein) between all the treatments. This could be explicated by a constant regulation of the total protein in fish. The equilibre of these parameters in fish could be breaked by other stress conditions such as temperature. In fact, Mohapatra et al. (2014) showed that Serum albumin to globulin ratio (A/G ratio) was significantly (P < 0.01) reduced in fish fed with the probiotic incorporated diet and increased with elevated water temperature. In the experiment conducted with supplentation of probable probiotic bacteria (PPB), Apún-Molina et al. (2015) reported that highest concentration of cholesterol in tilapia blood occurred in tilapia that did not receive any PPB or were treated every 10 days as compared with tilapia without PPB (P < 0.05), with intermediate values for tilapia fed PPB every 5 days. The protein, glucose, and triglycerides concentration, in relation to the frequency of PPB dietary supply, were not significantly different. The phagocytic activity differ also with species of tilapia in fact, Cai et al. (2004) noted that phagocytic activity of nile tilapia and blue tilapia fed with probiotic was respectively (61 %) and (39 %). The
3.4. Respiratory burst and phagocytic activity Higher levels (P < 0.05) of respiratory burst activity were observed in fish that received feed pulse-administration of 14 days (P14), being statistically significant in comparison with the other groups (Fig. 1). No differences were observed in the phagocytic activity between the treatments. 3.5. Hematological parameters The Table 3 displays the data regarding the hematological parameters in the serum and the plasma after a after 84 days of experimental feeding for all the experimental trials. The hematological parameters HT, HB, ER, CHCM, and MVC in the serum, as well as the concentration of glucose and cortisol in the plasma did not show any significative difference between all the experimental trials during the experimental period. 3.6. Challenge with Aeromonas hydrophila The evolution of hematological parameters and relative protection level (RPL) of all the trials challenged with A. hydrophila and the control group (injected with PBS) were displayed in the Table 4. The onset of clinical signs such as isolation of fish, petechiae, ascites and hemorrhage were observed 24 h after challenge, followed by death between
Fig. 1. Respiratory burst activity of Nile tilapia blood cells, feeding following pulse-administration with probiotic (Enterococcus faecium) supplemented in the diet during 84 days. OD (optical density). ab Different letter differ by Duncan’s test (p < 0.05). CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days. 4
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Table 3 Hematological parameters of glucose, cortisol (CORT), hematocrit (HT), hemoglobin (HB), erythrocyte count (ER) and calculated mean corpuscular hemoglobin (CHCM), mean corpuscular volume (MVC), of Nile tilapia with different schedule of feeding during 84 days. The data are expressed as the means ± S.E.M (n = 8).
GLUCOSE (g dL-1) CORT ng mL-1 HT (%) HB (g dL-1) ER (106 mm-3) MCHC (%) MVC (%)
CTR
CON
P7
P14
49.88 ± 9.34 6.62 ± 1.26 35.31 ± 2.63 6.14 ± 0.64 1.21 ± 0.06 17.49 ± 2.36 297.16 ± 36.69
55.38 ± 11.34 7.09 ± 2.56 35.56 ± 2.11 6.34 ± 0.96 1.22 ± 0.02 17.85 ± 2.58 288.58 ± 12.60
53.63 ± 17.98 8.33 ± 2.25 36.06 ± 3.34 6.13 ± 0.72 1.20 ± 0.07 17.17 ± 3.05 301.09 ± 29.92
49.50 ± 10.14 9.24 ± 4.01 33.81 ± 1.85 5.86 ± 0.76 1.35 ± 0.17 17.29 ± 1.78 247.72 ± 20.41
*CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days.
may exist (Killie and Jorgensen, 1994) but are poorly understood (Bricknell and Dalmo, 2005). It was reported that the short term ciclyc delivery of the probiotic may be a viable alternative for immunostimulation of the fish (Merrifield et al., 2010), in our work the cyclic delivery was gives significant results only after 14 days period. This may suggest that the cyclic period could depend on fish specie and probiotic specie. Besides, this suggest that an over-stimulation of the immune system can be prevented by the constant presence of the probiotic in the diet. Our results confirm the conclusion of Merrifield et al. (2010) that argues that probably the use of probiotic continuously over a long period in fish feeding should not be relevant. As well, the implementation of this kind of continuous feeding strategy in commercial fish farming may be difficult due to the feed logistic distribution and control complications. However, a 14 day pulse administration of probiotic could stimulate periodically the immune system of fish. These results could be interesting for fish farmer since they, in one hand, reduce the cost of fish supplement feeding, and in the other hand stimulate periodically the immune system to defend fish from eventual disease risk. After challenge test with A. hydrophila, we registrated a significative decreases in the number of the lymphocytes compared to the fish injected only with PBS. The effectiveness of probiotics to protect fish from infection still in debate. Pirarat et al. (2006) has suggested that the probiotic Lactobacillus rhamnosus GG protect against Edwardsiella tarda infection by enhancing the alternative complement system thereby increasing phagocytic cell aggregation and phagocytic activity. Shelby et al. (2006) found that feeding commercial probiotics for 94 days did not prevent streptococcal disease (Welker and Lim, 2011).
phagocytic activity of three species of tilapia and their hybrid ranged between 74.3 % and 88.1 % (Casas Solis et al., 2007). Khalkhali and Mojgani (2017) revealed that E. faecium enhances mucosal immunity by increasing numbers of intraepithelial T lymphocytes, production of IgA, stimulates macrophages and dendritic cells to produce nitric oxid and digest microbes. Panigrahi et al. (2007) showed that, in rainbow trout fed with probiotics, expression levels of interleukin 1 alpha (IL-1α), Tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta (TGF-β) were increased significantly in the spleen and the kidney. Bricknell and Dalmo (2005) reported that continuous feeding of an immunostimulant can either up-regulates the immune system to heightened levels and this is maintained until the immunostimulant is withdrawn, or cause adverse effects such as tolerance or immunosuppression. Conversely, pulse administration may oscillate the immune response from a resting level to an enhanced response then back to resting again. The same author revealed that most immunostimulation strategies involve pulse feeding the immunostimulant for a short period, usually 4–6 weeks, to up-regulate the immune response. In our study we used different strategies. In fact, we used a continuous feeding (CON), seven days pulse-administration and 14 days pulse-administration. In the trial P7 and CON of our study, we did not registered any significative difference in the immune parameters (phagocytic activity and respiratory burst activity). This could be explicated by an immuno tolerance of fish. Tolerance has been reported in some fish species (Nakamura et al., 1998; Waagbo et al., 1994; Zapata et al., 1997) and antigen competition has been observed in Atlantic salmon suggesting that the mechanisms for the induction of tolerance
Table 4 Hematological parameters and relative protection level (RPL) of Nile tilapia submitted to challenge infection with A. hydrophila after probiotic, E. faecium feeding during 21 days. Hematological parameters were measured from surviving fish 14 days post challenge.
Hematocrit (%) Hemoglobin (g.dL
−1
)
Erythrocytes (104 cel mm−3) Leucocytes (cel mm−3) Monocytes (cel mm−3) Neutrophils (cel mm−3) Lymphocytes (cel mm−3) Trombocytes (cel mm−3) Relative protection level (%)
CTR
CON
P7
P14
PBS
29.13 ± 4.60 4.43 ± 0.73 106.75 ± 22.56 1,065.10b ± 535.30 40.66 ± 26.12 421.46 ± 349.46 601.00b ± 262.00 11,194.00 ± 8,412.00 0.00
33.20 ± 7.72 5.19 ± 0.69 110.70 ± 25.63 1,528.40b ± 1,070.30 60.19 ± 20.52 585.95 ± 537.31 882.00b ± 546.00 11,164.00 ± 4,915.00 83.33
32.13 ± 3.04 4.79 ± 0.43 126.13 ± 12.46 2,075.10b ± 1,358.50 63.32 ± 44.51 949.01 ± 699.03 1,062.00b ± 658.00 16,579.00 ± 8,068.00 62.96
30.70 ± 3.81 4.39 ± 0.91 109.00 ± 16.93 1,272.20b ± 1,586.00 26.49 ± 39.28 578.49 ± 370.47 664.00b ± 1,457.00 8,245.00 ± 5,830.00 66.60
33.10 ± 6.04 5.24 ± 0.59 119.60 ± 17.05 3,393.00ª ± 961.40 137.90 ± 148.38 239.76 ± 136.10 3,007.00ª ± 828.00 16,245.00 ± 4,489.00 100.00
CTR: feed without probiotic; CON: continuous supply of feed with probiotic; P7: Pulse administration with probiotic feed every seven days; P14: Pulse administration with probiotic feed every 14 days. 5
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The enduring protection of probiotics, seems to be strongly depending on the probiotic specie, the concentration administrated and the feeding duration. Aly et al. (2008a) found that supplementing Bacillus pumilus at 1012/g diet increased protection of Nile tilapia against A. hydrophila after 1 and 2 but not 8 months of feeding. The lower dietary concentration tested (106/g) did not provide any protection. In another study, Aly et al. (2008b) also found that dietary supplementation of L. acidophilus, B. subtilis, or a mixture of the two generally provided greater protection against A. hydrophila, P. fluorescens, and S. iniae after 2 months of feeding compared to 1 month (Welker and Lim, 2011).
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5. Conclusion With this study we proved the benefic effect of E. faecium as a probiotic to be used as a complement for feeding tilapia. Impact of the different feeding strategies was not detected in all the studied parameters but we confirmed that E. faecium enhances fish growth, boost imune system if administrated in 7 days period, and efficiently protect fish if it is continuously administrated. This motivates further investigation that could be undertaken using other single or multiple probiotics to be used in intensive aquaculture industry. Funding This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), projects numbers: 2013/09731-8 and 2016/19816-9. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement The authors thank to Claudio Cirineu Ciola, Jair Donizetti Mazzaferro and Hélio Sanches Mariscal, Eliana Oshiro for thecnical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aqrep.2020.100277. References Allameh, S.K., Noaman, V., Nahavandi, R., 2017. Effects of probiotic bacteria on fish performance. Adv. Tech. Clin. Microbiol. 1, 2. Aly, S.M., Ahmed, Y.A., Ghareeb, A.A., Mohamed, M.F., 2008a. Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immunol. 25, 128–136. https://doi.org/10.1016/j.fsi.2008.03.013. Aly, S.M., Mohamed, M.F., John, G., 2008b. Effect of probiotics on the survival, growth and challenge infection in Tilapia nilotica (Oreochromis niloticus). Aquac. Res. 39, 647–656. https://doi.org/10.1111/j.1365-2109.2008.01932.x. Apún-Molina, J.P., Santamaría-Miranda, A., Luna-González, A., Ibarra-Gámez, J.C., Medina-Alcantar, V., Racotta, I., 2015. Growth and metabolic responses of whiteleg shrimp Litopenaeus vannameiandNile tilapia Oreochromis niloticusin polyculture fed with potentialprobiotic microorganisms on different schedules. Lat. Am. J. Aquat. Res. 43, 435–445 DOI: 10.3856. Balcázar, J.L., de Blas, I., Ruiz-Zarzuela, I., Vendrell, D., Calvo, A.C., Marquez, I., Girones, O., Muzquiz, J.L., 2007. Changes in intestinal microbiota and humoral immune response following probiotic administration in brown trout (Salmo trutta). Br. J. Nutr. 97, 522–527. https://doi.org/10.1017/S0007114507432986. Borges, L.P., Brandão, R., Godoi, B., Nogueira, C.W., Zeni, G., 2008. Oral administration of diphenyl diselenide protects against cadmium-induced liver damage in rats. Chem.-Biol. Interact. 171, 15–25. https://doi.org/10.1016/j.cbi.2007.09.005. Bricknell, I., Dalmo, R.A., 2005. The use of immunostimulants in fish larval aquaculture. Fish Shellfish Immunol. 19, 457–472. https://doi.org/10.1016/j.fsi.2005.03.008.
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