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Frequency in the supply of Lactobacillus influence its probiotic effect for yellow tail lambari

Frequência no fornecimento de Lactobacillus influencia seu efeito probiótico para o ambari-do-rabo-amarelo

ABSTRACT:

The aim of this research was to evaluate the changes in intestinal tract microbiota, hematological parameters and zootechnical indexes of yellowtail lambaris (Astyanax bimaculatus) fed with different probiotic supply frequencies (Lactobacillus spp.). Lambaris (1,200) were distributed in twelve boxes, divided in three treatments, 100% supply; 25% and 0% (control) of feeds supplemented with probiotic. After 87 days, fish fed with supplemented diet, regardless of frequency (100% and 25%), showed an increase in the total lactic acid bacteria count of 7.99±0.15 log CFU mL-1 and 7.04±1, 29 log UFC mL-¹ respectively, in the intestinal tract and decrease of Pseudomonas spp. in both 100% and 25% (4.09±0.84log UFC mL-1 and 4.00±1.12log UFC mL-1, respectively) in relation to the control treatment (6.74±1.04log UFC mL-1). Fish fed with 100% of diets supplemented with probiotics had a higher leukocyte count (47.7±6.7x103μL-1), lymphocytes (36.0±2.6x103μL1), monocytes (12.0±4.2x103μl-1), as well as higher mean final weight (10.60±0.89g) and weekly growth rate (0.80±0.08g.week-1) than the other treatments (25% of probiotic offered and control). Probiotic (Lactobacillus spp.) offered in 25% of feeds (or more) alters the yellowtail lambari microbiota (A. bimaculatus); however, must be offer in 100% of feeds to improve the hematology profile and productive performance.

Key words:
food handling; lactic acid bacteria; Astyanax; hematology; microbiota

RESUMO:

O objetivo deste trabalho foi avaliar as alterações na microbiota do trato intestinal, parâmetros hematológicos e índices zootécnicos de lambaris-do-rabo-amarelo (Astyanax bimaculatus) alimentados com diferentes frequências de oferta do probiótico (Lactobacillus spp.). Os lambaris (1.200) foram distribuídos em doze caixas, divididos em três tratamentos, oferta de 100%, 25% e 0% (controle) de alimentações suplementadas com probiótico. Após 87 dias, os peixes alimentados com dieta suplementada, independente da frequência (100% e 25%), apresentaram aumento na contagem total de bactérias ácido láticas 7,99±0,15log UFC mL e -7,04±1,29log UFC mL, respectivamente, no trato intestinal e redução de Pseudomonas spp. tanto em 100% quanto em 25% (4,09 ± 0,84 log UFC mL e 4,00±1,12 log UFC mL, respectivamente) em relação ao tratamento controle (6,74±1,04log UFC mL). Os peixes alimentados com 100% das dietas suplementadas com probiótico apresentaram maior contagem de leucócitos (47,7±6,7x103µL-1), linfócitos (36,0±2,6x103µL-1), monócitos (12,0±4,2x103µL-1), assim como maior peso médio final (10,60±0,89g) e taxa de crescimento semanal (0,80±0,08g.semana-1) que os demais tratamentos (25% de oferta de probiótico e controle). O probiótico (Lactobacillus spp.) oferecido em 25% de alimentos (ou mais) altera a microbiota de lambari de rabo amarelo (A. bimaculatus), no entanto, deve ser oferecido em 100% de alimentos para melhorar o perfil hematologia e desempenho produtivo.

Palavras-chave:
manejo alimentar; bactérias ácido láticas; Astyanax; hematologia; microbiota

INTRODUCTION:

In conventional fish culture animals are exposed directly to several microorganisms present in the environment, both beneficial and pathogenic. Probiotics reduce the need for investments with antimicrobials and parasiticides; consequently, reducing the use of chemotherapeutics and their environmental impacts caused by fish culture (HAI, 2015HAI, N.V. Research findings from the use of probiotics in tilapia aquaculture: a review. Fish & Shellfish Immunology, v.45, n.2, p.592-597, 2015. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815002454 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.fsi.2015.05.026.
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), its use offers a very promising alternative to the use of antibiotics in production systems of aquaculture (VASEEHARAN & RAMASAMY, 2003VASEEHARAN, B. A. R. P.; RAMASAMY, P. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon. Letters in Applied Microbiology, v. 36, n. 2, p. 83-87, 2003. Available from: <Available from: https://onlinelibrary.wiley.com/doi/full/10.1046/j.1472-765X.2003.01255.x >. Accessed: May 11, 2018. doi: 10.1046/j.1472-765X.2003.01255.x.
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).

A probiotic is defined as a living microbial adjuvant that has a beneficial effect on the host by modifying the microbial community associated with the host or environment (VERSCHUERE et al., 2000VERSCHUERE, L. et al. Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Reviews, v.64, n.4, p.655-671, 2000. Available from: <Available from: http://mmbr.asm.org/content/64/4/655.full.pdf+html >. Accessed: Jan. 23, 2018. doi: 10.1128/MMBR.64.4.655-671.2000.
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) as vibrio reduction on water (VASEEHARAN et al, 2004VASEEHARAN, B. et al. Effect of probiotics, antibiotic sensitivity, pathogenicity, and plasmid profiles of Listonella anguillarum-like bacteria isolated from Penaeus monodon culture systems. Aquaculture, v. 241, n. 1-4, p. 77-91, 2004. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0044848604005289 >. Accessed: May 11, 2018. doi: 10.1046/j.1472-765X.2003.01255.x.
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). Among probiotic microorganisms, lactic acid bacteria (LAB) have shown great efficacy to fish with results in the improvement of zootechnical indexes, weight gain, specific growth rate, feed conversion ratio (JATOBÁ et al., 2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
; STANDEN et al., 2016STANDEN, B.T. et al. Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus. Fish & Shellfish Immunology, v.49, p.427-435, 2016. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815302564 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2015.11.037.
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), as a histological changes of the gastrointestinal tract, inducing an increase in villus height and width as well as epithelial cell thickness (MELLO et al., 2013MELLO, H. et al. Efeitos benéficos de probióticos no intestino de juvenis de Tilápia-do-Nilo. Pesquisa Veterinária Brasileira, p.724-730, 2013. Available from: <Available from: https://repositorio.unesp.br/bitstream/handle/11449/76301/2s2.084881530200.pdf?sequence=1&isAllowed=y >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-736X2013000600006.
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), besides acting as promoters of the immune system (JATOBÁ et al., 2008;JATOBÁ, A. et al. Lactic-acid bacteria isolated from the intestinal tract of Nile tilapia utilized as probiotic. Pesquisa Agropecuária Brasileira, v.43, n.9, p.1201-1207, 2008. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0100-204X2008000900015&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-204X2008000900015.
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TELLI et al., 2014TELLI, G.S. et al. Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities. Fish & Shellfish Immunology, v.39, n.2, p.305-311, 2014. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464814001740 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2014.05.025.
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).

Several factors may alter the efficiency of probiotics, such as method of administration, mode of incorporation, inoculum/feed ratio, quantity supplied by fish (%/kg), as well as frequency of supply (HAI, 2015HAI, N.V. Research findings from the use of probiotics in tilapia aquaculture: a review. Fish & Shellfish Immunology, v.45, n.2, p.592-597, 2015. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815002454 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.fsi.2015.05.026.
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). However, few studies have evaluated different methods of application, mainly for Brazilian native species. Among them, the yellowtail lambari (Astyanax bimaculatus) has been highlighted due its desirable characteristics to fish culture such as: omnivorous food habit, short life cycle, and can be used as a main, secondary or forage species, as well as can be reared in the different culture systems (PORTO-FORESTI et al., 2005PORTO-FORESTI, F. et al. Biologia e criação do lambari-do-rabo-amarelo (Astyanax altiparanae). Espécies nativas para piscicultura no Brasil, v.2, p.101-116, 2005.).

The objective of this research was to evaluate the intestinal tract microbiota, hematological parameters and growth performance of yellow tail lambaris (A. bimaculatus), fed with different frequencies in the supply of probiotic (Lactobacillus spp.).

MATERIALS AND METHODS:

The study was carried out in the Laboratório de Aquicultura (LAQ), Instituto Federal Catarinense (IFC), campus Araquari, (Protocol number 0219/2017 approved by animal ethics committee).

The Lactobacillus strain CPQBA 1168-15 DRM-01 with proven probiotic effect in vitro and in vivo (JATOBÁ et al., 2017JATOBÁ, A. et al. Selection of autochtone probiotic for Astyanax bimaculatus. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.69, n.6, p.1645-1652, 2017. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0102-09352017000601645&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/1678-4162-9158.
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) and 1200 yellow tail lambari (A. bimaculatus), average weight 1.0±0.2g, were used.

Fish were distributed in 12 experimental units under outdoor conditions, 100 fish per box, with a capacity of 800L, equipped with biological filters and recirculation system. Boxes were divided into three treatments (Table 1), with four replicates. According to the protocols established by JATOBÁ et al. (2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
), 10% of inoculum was included in the probiotic diets with Lactobacillus strain and MRS medium (Lactobacillus MRS Broth, HiMedia Laboratories Pvt., India), and only sterile MRS medium (Lactobacillus MRS Broth, HiMedia Laboratories Pvt., India) in the control diet. Probiotic diets were only used when concentrations ≥1.0x107 CFU.g-1 were observed.

Table 1
Experimental treatments, probiotic supply frequency during yellowtail lambari (A. bimaculatus) culture.

Fish were fed three times a day (09:00, 13:00 and 16:00) with a commercial diet (GUABI®, 1.7mm, 36% crude protein and 6.5% ethereal extract, manufacturer’s warranty levels), with 4% of their biomass, and biometrics were performed weekly for food management. Dissolved oxygen and temperature were measured twice a day, while pH and ammonia were measured weekly.

After 87 days of rearing and a 24h period of starvation, eight animals per experimental unit were anesthetized with Eugenol (50mg L-1) and sacrificed by cerebral concussion. Three were used for the microbiological assays of digestive tract, and the others were used for hematological analysis.

For microbiological assays of the digestive tract, the guts of three fish from each tank were removed and pooled to microbiological and histological tests. Pooled fish guts were homogenized and serially diluted 1:10 in 0.65% of NaCl sterile saline. Samples from each dilution were cultured in TSA (Triptone Soy Agar, HiMedia Laboratories Pvt. Ltd., India), TCBS (Agar Thiosulfate Citrate Bile Sucrose, HiMedia Laboratories Pvt. Ltd., India), Cetrimide (Cetrimide Agar, HiMedia Laboratories Pvt. Ltd., India), and then incubated for 24h at 30°C, as well as MRS (Lactobacillus MRS Agar, HiMedia Laboratories Pvt. Ltd., India), followed by incubation for 48h at 35°C, for viable cultivable heterotrophic bacterial counts, including Vibrio spp., Pseudomonas spp. and lactic acid bacteria (LAB), respectively.

For hematological analysis, approximately 1.0mL of blood was drawn from the caudal vein of each fish for the preparation of blood smears, in duplicate, and the following hematological analyses: determination of hematocrit by standard microhematocrit method; glucose with digital readers (G-TECH free®, Accumed-Glicomed, Brazil); total hemocyte count by Neubauer hemocytometer. Blood smear slides were stained with Giemsa and May Grünwald stain (ROSENFELD, 1947ROSENFELD, G. Corante pancrônico para hematologia e citologia clínica. Nova combinação dos componentes do May-Grünwald e do Giemsa num só corante de emprego rápido. Memórias do Instituto Butantan, v.20, p.329-334, 1947.) for total and differential leukocyte count.

Survival (SR), final weight (FW), weekly weight gain (WWG), consumption, specific growth rate (SGR), apparent feed efficiency (SGR) and yield were all determined at the end of the experiment, according to the formulas below:

SR%=Nº of live individualsNº of individuals introduces*100

WWGg.week-1=Final weight-Initial weightnº of week

SGR %day-1=loglogFinal weight-loglogInitial weightnº of days*100

AFE=(Final weight-Initial weight)/consumption

Yield kgm-3=Final Biomass-Initial Biomasspond area

All data were first subjected to Bartlett’s analysis, to verify the homogeneity of variance, one-way ANOVA and significant differences among treatments were analyzed using the Student-Newman-Keuls (SNK) test of means separation. Microbiological data were log (x+1) transformed. All tests were conducted at a 5% level of significance.

RESULTS AND DISCUSSION:

Diets supplemented, regardless of the probiotic supply frequency, had a higher LAB and lower Pseudomonas spp. in the intestinal tract in relation to the control (Table 2), while in counting of total heterotrophic bacteria and Vibrionaceas there was no difference between treatments.

Table 2
Means±standard deviation of bacterial counts (log UFC mL-) of intestinal tract of yellowtail lambari (A. bimaculatus) supplemented with probiotic (Lactobacillus spp.), offered on different frequencies.

Results corroborate with JATOBÁ & MOURIÑO (2015JATOBÁ, A.; MOURIÑO, J.L.P. Efeito do Lactobacillus plantarum no trato intestinal de alevinos de Oreochromis niloticus. Ciência Animal Brasileira, v.16, n.1, p.45-53, 2015. Available from: <Available from: http://189.126.110.61/cab/article/view/25259 >. Accessed: Jan. 19, 2018. doi: 10.1590/1089-68916i127789.
http://189.126.110.61/cab/article/view/2...
) who observed the increase of LAB in the intestinal tract of Nile tilapia fed with probiotic diet (L. plantarum) during 35 days. However, they were divergent with STANDEN et al. (2016STANDEN, B.T. et al. Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus. Fish & Shellfish Immunology, v.49, p.427-435, 2016. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815302564 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2015.11.037.
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) who recorded a higher amount of Bacillus in the intestinal tract of Nile tilapia (O. nilotcus) only in the treatment that received the highest concentration (3g of probiotic per kg) in the diet, already for total heterotrophic bacteria both works did not observe changes in the microbiota.

Some researches (JATOBÁ et al., 2008JATOBÁ, A. et al. Lactic-acid bacteria isolated from the intestinal tract of Nile tilapia utilized as probiotic. Pesquisa Agropecuária Brasileira, v.43, n.9, p.1201-1207, 2008. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0100-204X2008000900015&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-204X2008000900015.
http://www.scielo.br/scielo.php?pid=S010...
; 2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
; JATOBÁ & MOURIÑO, 2015JATOBÁ, A.; MOURIÑO, J.L.P. Efeito do Lactobacillus plantarum no trato intestinal de alevinos de Oreochromis niloticus. Ciência Animal Brasileira, v.16, n.1, p.45-53, 2015. Available from: <Available from: http://189.126.110.61/cab/article/view/25259 >. Accessed: Jan. 19, 2018. doi: 10.1590/1089-68916i127789.
http://189.126.110.61/cab/article/view/2...
) have observed a reduction in the counts of total heterotrophic bacteria in the intestinal tract of fingerlings and juveniles of Nile tilapia fed with probiotic (L. plantarum), differently from that observed in this study, independent of the frequency in the probiotic supply the total heterotrophic bacteria counts was not changed. The same fact were observed by YAMASHITA et al. (2017YAMASHITA, M.M. et al. Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis. Aquaculture Nutrition, v. 23, n. 6, p. 1235-1243, 2017. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1111/anu.12498/full >. Accessed: Jan. 23, 2018. doi: 10.1111/anu.12498.
http://onlinelibrary.wiley.com/doi/10.11...
) and STANDEN et al. (2016STANDEN, B.T. et al. Dietary administration of a commercial mixed-species probiotic improves growth performance and modulates the intestinal immunity of tilapia, Oreochromis niloticus. Fish & Shellfish Immunology, v.49, p.427-435, 2016. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815302564 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2015.11.037.
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), in which the supply of the probiotic did not interfere in the counting of total heterotrophic bacteria. In all these studies the changes or not of the amount of total heterotrophic bacteria in the intestinal tract of the fish did not interfere in the LAB probiotic effect.

Fish fed with probiotic, regardless of frequency, showed lower count of Pseudomonas spp. corroborating with the results of DEL’DUCA et al. (2013DEL’DUCA, A. et al. Evaluation of the presence and efficiency of potential probiotic bacteria in the gut of tilapia (Oreochromis niloticus) using the fluorescent in situ hybridization technique. Aquaculture, v.388, p.115-121, 2013. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0044848613000343 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.aquaculture.2013.01.019.
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), that observed a reduction of Pseudomonas sp. and Aeromonas sp. in the intestinal microbiota of Nile tilapia fed with Bacillus sp. and Enterococcus sp. together or separately for 30 days.

The three main mechanisms of action of LAB are: competitive exclusion of pathogenic bacteria; production of inhibitory compounds (VERSCHUERE et al., 2000VERSCHUERE, L. et al. Probiotic bacteria as biological control agents in aquaculture. Microbiology and Molecular Biology Reviews, v.64, n.4, p.655-671, 2000. Available from: <Available from: http://mmbr.asm.org/content/64/4/655.full.pdf+html >. Accessed: Jan. 23, 2018. doi: 10.1128/MMBR.64.4.655-671.2000.
http://mmbr.asm.org/content/64/4/655.ful...
; LAZADO & CAIPANG, 2014LAZADO, C.C.; CAIPANG, C.M.A. Mucosal immunity and probiotics in fish. Fish & Shellfish Immunology, v.39, n.1, p.78-89, 2014. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464814001314 >. Accessed: Jan. 19, 2018. doi: 10.1016/j.fsi.2014.04.015.
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) and improvement of the immune response against pathogenic microorganisms (JATOBÁ et al., 2008JATOBÁ, A. et al. Lactic-acid bacteria isolated from the intestinal tract of Nile tilapia utilized as probiotic. Pesquisa Agropecuária Brasileira, v.43, n.9, p.1201-1207, 2008. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0100-204X2008000900015&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-204X2008000900015.
http://www.scielo.br/scielo.php?pid=S010...
; YAMASHITA et al., 2017YAMASHITA, M.M. et al. Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis. Aquaculture Nutrition, v. 23, n. 6, p. 1235-1243, 2017. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1111/anu.12498/full >. Accessed: Jan. 23, 2018. doi: 10.1111/anu.12498.
http://onlinelibrary.wiley.com/doi/10.11...
) which consequently promotes general improvement of animal health (HAI, 2015HAI, N.V. Research findings from the use of probiotics in tilapia aquaculture: a review. Fish & Shellfish Immunology, v.45, n.2, p.592-597, 2015. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464815002454 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.fsi.2015.05.026.
http://www.sciencedirect.com/science/art...
).

Hybrid tilapia (O. niloticus X O. aureus) supplemented with Lactobacillus brevis were protected against the challenge with Aeromonas hydrophila (LIU et al., 2013LIU, W. et al. Comparison of adhesive gut bacteria composition, immunity, and disease resistance in juvenile hybrid tilapia fed two different Lactobacillus strains. Fish & Shellfish Immunology, v.35, n.1, p.54-62, 2013. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464813004993 >. Accessed: Jan. 19, 2018. doi: 10.1016/j.fsi.2013.04.010.
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). Nile tilapia supplemented with Bacillus subtilis and B. cereus obtained a better percentage of relative survival and also improving intestinal morphology (MELLO et al., 2013MELLO, H. et al. Efeitos benéficos de probióticos no intestino de juvenis de Tilápia-do-Nilo. Pesquisa Veterinária Brasileira, p.724-730, 2013. Available from: <Available from: https://repositorio.unesp.br/bitstream/handle/11449/76301/2s2.084881530200.pdf?sequence=1&isAllowed=y >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-736X2013000600006.
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), the same was observed for yellow tail lambari fed with probiotic Lactobacillus spp. (same strain used in this study) that survival rate was 16.2% higher after experimental challenge against Aeromonas hydrophila compared to the unsupplemented group (JATOBÁ et al., 2017JATOBÁ, A. et al. Selection of autochtone probiotic for Astyanax bimaculatus. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.69, n.6, p.1645-1652, 2017. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0102-09352017000601645&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/1678-4162-9158.
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). Due to the changes presented in the intestinal microbiota in this study it is suggested higher resistance of the animals fed with probiotic against bacterial infections.

A higher number of total leukocytes, lymphocytes and circulating monocytes were observed in lambari fed at 100% frequency in relation to the other treatments, while glucose, hematocrit and other blood parameters did not differ among treatments (Table 3).

Table 3
Hematological variables of yellowtail lambari (A. bimaculatus) supplemented with probiotic (Lactobacillus spp.), offered on different frequencies.

Changes in hematocrit and glucose levels may be associated with animal stress (GONÇALVES et al., 2011GONÇALVES, A.T. et al. Effects of a probiotic bacterial Lactobacillus rhamnosus dietary supplement on the crowding stress response of juvenile Nile tilapia Oreochromis niloticus. Fisheries Science, v.77, n.4, p.633-642, 2011. Available from: <https://link.springer.com/article/10.1007/s12562-011-0367-2>. Accessed: Jan. 18, 2018. doi: 10.1007/s12562-011-0367-2.
https://doi.org/https://link.springer.co...
; DE PAIVA et al., 2013DE PAIVA, M.J.T.R. et al. Métodos para análise hematológica em peixes. Editora da Universidade Estadual de Maringá-EDUEM, 2013.), the equality in these parameters showed that probiotic, regardless of frequency, did not alter fish homeostasis, suggesting that all the animals were at the same level of welfare.

The increase in leukocyte cells indicates an immunostimulation of the bloodstream cells of fish (DE PAIVA et al., 2013DE PAIVA, M.J.T.R. et al. Métodos para análise hematológica em peixes. Editora da Universidade Estadual de Maringá-EDUEM, 2013.), demonstrating that only the constant supply (100%) of the probiotic causes a greater recruitment of monocytes and lymphocytes in yellow tail lambari. Besides this, increasing humoral and cellular immune defenses is other way to shown probiotic benefices, as shown by GOBI et al. (2016GOBI, Narayanan et al. GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus. Fish & Shellfish Immunology, v. 52, p. 230-238, 2016. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S1050464816300857 >. Accessed: May 11, 2018. doi: 10.1016/j.fsi.2016.03.006.
https://www.sciencedirect.com/science/ar...
) whom demonstrated an increase in serum immunological enzymes and antioxidant enzyme levels of Pangasius hypophthalmus fed with Bacillus licheniformis Dahb1. This same strain also demonstrated probiotic effects for tilapia enhancing immune parameters and activities of antioxidant enzymes in the serum and mucus (GOBI et al., 2016GOBI, Narayanan et al. GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus. Fish & Shellfish Immunology, v. 52, p. 230-238, 2016. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S1050464816300857 >. Accessed: May 11, 2018. doi: 10.1016/j.fsi.2016.03.006.
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).

The change/improvement in leukogram is one of the most well-known beneficial effects of the probiotic use for fish (ALY et al., 2008ALY, S.M. et al. 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 Immunology, v.25, n.1, p.128-136, 2008. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464808000831 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.fsi.2008.03.013.
http://www.sciencedirect.com/science/art...
; JATOBÁ et al., 2008JATOBÁ, A. et al. Lactic-acid bacteria isolated from the intestinal tract of Nile tilapia utilized as probiotic. Pesquisa Agropecuária Brasileira, v.43, n.9, p.1201-1207, 2008. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0100-204X2008000900015&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-204X2008000900015.
http://www.scielo.br/scielo.php?pid=S010...
; WANG et al., 2008WANG, Y.B. et al. Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune response. Aquaculture, v.277, n.3, p.203-207, 2008. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S0044848608001488 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.aquaculture.2008.03.007.
http://www.sciencedirect.com/science/art...
; JATOBÁ et al., 2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
; STANDEN et al., 2013STANDEN, B.T. et al. Probiotic Pediococcus acidilactici modulates both localised intestinal-and peripheral-immunity in tilapia (Oreochromis niloticus). Fish & Shellfish Immunology, v.35, n.4, p.1097-1104, 2013. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464813006566 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2013.07.018.
http://www.sciencedirect.com/science/art...
; EISSA & ABOU-ELGHEIT, 2014EISSA, N.; ABOU-ELGHEIT, E. Dietary supplementation impacts of potential non-pathogenic isolates on growth performance, hematological parameters and disease resistance in Nile Tilapia (Oreochromis Niloticus). Journal of Veterinary Advances, v.4, n.10, p.712-719, 2014. Available from: <Available from: https://s3.amazonaws.com/academia.edu.documents/35339762/741413257869.pdf?AWSAccessKeyId=AKIAIWOWYYGZ2Y53UL3A&Expires=1516368051&Signature=QAQwAypMw74zQMPbYKkrNqPNG4w%3D&response-content-disposition=inline%3B%20filename%3DDietary_Supplementation_Impacts_of_Poten.pdf >. Accessed: Jan. 18, 2018. doi: 10.5455/jva.20141025045451.
https://s3.amazonaws.com/academia.edu.do...
; YAMASHITA et al., 2017YAMASHITA, M.M. et al. Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis. Aquaculture Nutrition, v. 23, n. 6, p. 1235-1243, 2017. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1111/anu.12498/full >. Accessed: Jan. 23, 2018. doi: 10.1111/anu.12498.
http://onlinelibrary.wiley.com/doi/10.11...
). The increase in circulating leukocytes corroborates IWASHITA et al. (2015IWASHITA, M.K.P. et al. Dietary supplementation with Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae enhance immunity and disease resistance against Aeromonas hydrophila and Streptococcus iniae infection in juvenile tilapia Oreochromis niloticus. Fish & Shellfish Immunology, v.43, n.1, p.60-66, 2015. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464814004793 >. Accessed: Jan. 18, 2018. doi: 10.1016/j.fsi.2014.12.008.
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) which had higher levels of white blood cells in Nile tilapia fed a diet supplemented with a combination of probiotics (Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae).

Hematological alterations occurred only in fish fed with probiotic in all feeds (100%), diverging from the results presented in the intestinal tract microbiota of the lambaris whose supply of 25% presented changes, this showed that the change in the microbiota did not guarantee an improvement/ change in immunocompetence, since the lower frequency in the supply (25%) altered the microbiota without interfering with the hematological profile of the yellowtail lambari.

The observed values of temperature (28.56±2.59°C), dissolved oxygen (5.25±1.52mg L-1), pH (7.43±0.11), total ammonia (1,37±1.32mg L-1) and nitrite (3.55±4.89mg L-1) did not differ among treatments and were suitable for the species (PORTO-FORESTI et al., 2005PORTO-FORESTI, F. et al. Biologia e criação do lambari-do-rabo-amarelo (Astyanax altiparanae). Espécies nativas para piscicultura no Brasil, v.2, p.101-116, 2005.).

Fish fed with probiotic in 100% of feeds shown an increase of 17.3% in final weight and 21.2% in weekly growth than fish fed control diet (not supplemented), while those fed with 25% of diets supplemented with probiotic did not differ between treatments. The survival, feed efficiency and yield did not show significant differences among treatments (Table 4). The higher frequency in the probiotic supply suggested higher concentrations of microorganisms, so this result corroborates with GOBI et al. (2018GOBI, Narayanan et al. Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus. Fish & Shellfish Immunology, 2018. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S105046481730815X >. Accessed: May 11, 2018. doi: 10.1016/j.fsi.2017.12.066.
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) that observed higher final weight in tilapia (O. mossambicus) fed with the highest concentration of probiotic (Bacillus licheniformis Dahb1).

Table 4
Growth performance of yellowtail lambari (A. bimaculatus) supplemented with probiotic (Lactobacillus spp.), offered on different frequencies.

One of the benefits of probiotic use is the better absorption of nutrients by animals (FERREIRA et al., 2015FERREIRA, A.H.C. et al. Probiótico na alimentação de pós-larvas de tilápias-do-nilo submetidas a desafio sanitário. Revista Brasileira de Saúde e Produção Animal, v.16, n.2, p.430-439, 2015. Available from: <Available from: http://revistas.bvs-vet.org.br/rbspa/article/view/27033 >. Accessed: Jan. 18, 2018.
http://revistas.bvs-vet.org.br/rbspa/art...
). This effect was observed in Nile tilapia autochthone probiotic, after been reared for 35 days, shown higher final weight and weekly growth (JATOBÁ & MOURIÑO, 2015JATOBÁ, A.; MOURIÑO, J.L.P. Efeito do Lactobacillus plantarum no trato intestinal de alevinos de Oreochromis niloticus. Ciência Animal Brasileira, v.16, n.1, p.45-53, 2015. Available from: <Available from: http://189.126.110.61/cab/article/view/25259 >. Accessed: Jan. 19, 2018. doi: 10.1590/1089-68916i127789.
http://189.126.110.61/cab/article/view/2...
), as well as Nile tilapia fed commercial probiotic mix in the diet administered for 6 weeks (STANDEN et al., 2015STANDEN, B.T. et al. Modulation of the intestinal microbiota and morphology of tilapia, Oreochromis niloticus, following the application of a multi-species probiotic. Applied Microbiology and Biotechnology, v.99, n.20, p.8403-8417, 2015. Available from: <hAvailable from: ttps://link.springer.com/article/10.1007/s00253-015-6702-2 >. Accessed: Jan. 24, 2018. doi: 10.1007/s00253-015-6702-2.
ttps://link.springer.com/article/10.1007...
).

Effect of probiotics on specific growth rate and weekly gain are divergent, it is possible to find authors who prove their efficacy, improving up to 15% of the feed efficiency of animals fed with autochthonous probiotic (JATOBÁ & MOURIÑO, 2015JATOBÁ, A.; MOURIÑO, J.L.P. Efeito do Lactobacillus plantarum no trato intestinal de alevinos de Oreochromis niloticus. Ciência Animal Brasileira, v.16, n.1, p.45-53, 2015. Available from: <Available from: http://189.126.110.61/cab/article/view/25259 >. Accessed: Jan. 19, 2018. doi: 10.1590/1089-68916i127789.
http://189.126.110.61/cab/article/view/2...
; YAMASHITA et al., 2017YAMASHITA, M.M. et al. Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis. Aquaculture Nutrition, v. 23, n. 6, p. 1235-1243, 2017. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1111/anu.12498/full >. Accessed: Jan. 23, 2018. doi: 10.1111/anu.12498.
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), as well as others did not observed improvement in these zootechnical variables for Nile tilapia (O. niloticus) fed with probiotic (Saccharomyces cervisiae), prebiotic (mananoligosaccharide) and symbiotic (S. cervisiae + nanaoligosaccharide) (SCHWARZ et al., 2016SCHWARZ, K.K. et al. Probiótico, prebiótico e simbiótico na nutrição de alevinos de tilápia do nilo Oreochromis niloticus. Archives of Veterinary Science, v.21, n.2, 2016. Available from: <Available from: http://revistas.ufpr.br/veterinary/article/view/44179/29568 >. Accessed: Jan. 23, 2018. doi: 10.5380/avs.v21i2.44179.
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), this divergence may be related to the specificity between host-host, justifying the best results of the autochthonous bacteria. Probiotic bacteria capable of acting in several (more than one) species are especially attractive to aquaculture, because they can be used in integrated and polyculture systems, such as Bacillus licheniformis Dahb1 which demonstrated beneficial effects for P. hypophthalmus and O. mossambicus (GOBI et al., 2016GOBI, Narayanan et al. GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus. Fish & Shellfish Immunology, v. 52, p. 230-238, 2016. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S1050464816300857 >. Accessed: May 11, 2018. doi: 10.1016/j.fsi.2016.03.006.
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; 2018); and L. plantarum for O. niloticus and Litopenaeus vannamei (JATOBÁ et al., 2011).

The apparent feed efficiency (AFE)/feed conversion (FC) are parameters that alleviate the ratio of feed consumption with produced biomass; however, whether the probiotic used does not improve the absorption or attractiveness of the diet, as well as does not promote fast growth, the relation consumption/weight gain will not be affected (TELLI et al., 2014TELLI, G.S. et al. Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities. Fish & Shellfish Immunology, v.39, n.2, p.305-311, 2014. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464814001740 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2014.05.025.
http://www.sciencedirect.com/science/art...
; STANDEN et al., 2015STANDEN, B.T. et al. Modulation of the intestinal microbiota and morphology of tilapia, Oreochromis niloticus, following the application of a multi-species probiotic. Applied Microbiology and Biotechnology, v.99, n.20, p.8403-8417, 2015. Available from: <hAvailable from: ttps://link.springer.com/article/10.1007/s00253-015-6702-2 >. Accessed: Jan. 24, 2018. doi: 10.1007/s00253-015-6702-2.
ttps://link.springer.com/article/10.1007...
). However, several authors observed greater weight gain and weekly growth in conjunction with better AFE and/or FC (JATOBÁ et al., 2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
; CORNÉLIO et al., 2013CORNÉLIO, F.H.G. et al. Growth, digestibility and resistance to pathogen infection in Nile tilapia fed with probiotics. Pesquisa Agropecuária Brasileira, v.48, n.8, p.863-870, 2013. Available from: <https://seer.sct.embrapa.br/index.php/pab/article/view/11483/12285>. Accessed: Jan. 18, 2018. doi: 10.1590/S0100-204X2013000800008.
https://doi.org/https://seer.sct.embrapa...
; JATOBÁ & MOURIÑO 2015; SCHWARZ et al., 2016SCHWARZ, K.K. et al. Probiótico, prebiótico e simbiótico na nutrição de alevinos de tilápia do nilo Oreochromis niloticus. Archives of Veterinary Science, v.21, n.2, 2016. Available from: <Available from: http://revistas.ufpr.br/veterinary/article/view/44179/29568 >. Accessed: Jan. 23, 2018. doi: 10.5380/avs.v21i2.44179.
http://revistas.ufpr.br/veterinary/artic...
; YAMASHITA et al., 2017YAMASHITA, M.M. et al. Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis. Aquaculture Nutrition, v. 23, n. 6, p. 1235-1243, 2017. Available from: <Available from: http://onlinelibrary.wiley.com/doi/10.1111/anu.12498/full >. Accessed: Jan. 23, 2018. doi: 10.1111/anu.12498.
http://onlinelibrary.wiley.com/doi/10.11...
), despite Lactobacillus spp. (same strain) to increase the villus length and number of villi in the intestinal mucosa of lambari of the yellow tail (JATOBÁ et al., 2017JATOBÁ, A. et al. Selection of autochtone probiotic for Astyanax bimaculatus. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v.69, n.6, p.1645-1652, 2017. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0102-09352017000601645&script=sci_arttext >. Accessed: Jan. 19, 2018. doi: 10.1590/1678-4162-9158.
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), in this research no better absorption of nutrients was observed.

The changes of the intestinal tract microbiota associated with the increase in the number of total leukocytes, monocytes and lymphocytes are indicative of an improvement in the animal immunity, justifying the higher survival rate of fish fed probiotic diets in relation to fish that did not eat it, or ate in inadequate quantity and/or frequency, when challenged experimentally or not (JATOBÁ et al., 2011JATOBÁ, A. et al. Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp. Fish Physiology and Biochemistry, v.37, n.4, p.725-732, 2011. Available from: <Available from: https://link.springer.com/article/10.1007/s10695-011-9472-5 >. Accessed: Jan. 19, 2018. doi: 10.1007/s10695-011-9472-5.
https://link.springer.com/article/10.100...
; MELLO et al., 2013MELLO, H. et al. Efeitos benéficos de probióticos no intestino de juvenis de Tilápia-do-Nilo. Pesquisa Veterinária Brasileira, p.724-730, 2013. Available from: <Available from: https://repositorio.unesp.br/bitstream/handle/11449/76301/2s2.084881530200.pdf?sequence=1&isAllowed=y >. Accessed: Jan. 19, 2018. doi: 10.1590/S0100-736X2013000600006.
https://repositorio.unesp.br/bitstream/h...
; TELLI et al., 2014TELLI, G.S. et al. Dietary administration of Bacillus subtilis on hematology and non-specific immunity of Nile tilapia Oreochromis niloticus raised at different stocking densities. Fish & Shellfish Immunology, v.39, n.2, p.305-311, 2014. Available from: <Available from: http://www.sciencedirect.com/science/article/pii/S1050464814001740 >. Accessed: Jan. 23, 2018. doi: 10.1016/j.fsi.2014.05.025.
http://www.sciencedirect.com/science/art...
).

Experiment performed with Nile tilapia fed with L. plantarum reported that the supply of probiotic in 50% of feeds guarantee better growth performance, (JATOBÁ et al., 2018JATOBÁ, A. et al. Action time and feed frequency of Lactobacillus plantarum for Nile tilapia. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, v. 70, n. 1, p. 327-332, 2018. Available from: <Available from: http://www.scielo.br/scielo.php?pid=S0102-09352018000100327&script=sci_arttext >. Accessed: May 11, 2018. doi: 10.1590/1678-4162-9870.
http://www.scielo.br/scielo.php?pid=S010...
), unlike that observed in this study, for lambari yellow tail, Lactobacillus spp. used should be offered in all feedings to guarantee the probiotic effect. This divergence may be related to animal physiology, since the digestive tract of Nile tilapia is longer than lambari yellow tail, allowing longer residence time of the probiotic microorganisms in the digestive tract.

CONCLUSION:

Probiotic (Lactobacillus spp.) offered in 25% of feeds (or more) alters the yellowtail lambari microbiota (A. bimaculatus); however, must be offer in 100% of feeds to improve the hematology profile and productive performance. The frequency of probiotic (Lactobacillus spp.) supply influences the microbiota, hematology and growth performance of yellowtail lambari (A. bimaculatus).

ACKNOWLEDGEMENTS

The authors of this study acknowledge (CNPq) Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico, process 460362/2014-3) and (FAPESC) Fundação de Amparo a Pesquisa do Estado de Santa Catarina, process 2015TR1703 for funding; GUABI for financing the diets used; and Luiz Sergio Moreira for technical support

REFERÊNCIAS:

  • 0
    CR-2018-0042.R1

AUTHORS’ CONTRIBUTIONS

  • AUTHORS’ CONTRIBUTIONS

    Adolfo Jatobá and Klayton Natan Moraes conceived and designed experiments. All authors performed the experiments, Emanuel Fernandes Rodrigues, Leandro Marcolino Vieira, Marina Oliveira Pereira carried out the lab analyses. Adolfo Jatobá supervised and coordinated the animal experiments, provided clinical data and performed statistical analyses of experimental data. Adolfo Jatobá e Marina Oliveira Pereira prepared the draft of the manuscript. All authors critically revised the manuscript and approved of the final version.

Publication Dates

  • Publication in this collection
    2018

History

  • Received
    23 Jan 2018
  • Accepted
    22 May 2018
  • Reviewed
    17 Sept 2018
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
E-mail: cienciarural@mail.ufsm.br