Intestinal microbiota modulation in juvenile Pacú (Piaractus mesopotamicus) by supplementation with Pyropia columbina and β-carotene

  • L. T. Rossi
  • A. Romero Sharpen
  • J. A. Zimmermann
  • C. R. Olivero
  • M. V. Zbrun
  • L. S. Frizzo
  • M. L. Signorini
  • C. Bacchetta
  • R. E. Cian
  • J. Cazenave
  • L. P. Soto
  • S. R. DragoEmail author


The aim was to evaluate the effects of diet supplementation with red seaweed Pyropia columbina or β-carotene on the intestinal microbiota of juvenile Pacú (Piaractus mesopotamicus). Three hundred and fifteen fish (body weight 12.4 ± 2.8 g) were stocked in nine 300-L tanks with 35 fishes per tank and fed for 62 days with a basal feed (BG) or the same feed added with P. columbina (35 g kg−1) (AG) or supplemented with β-carotene (225 g kg−1) (β-CG). From fecal samples of three fishes slaughtered per group at 20th, 41th, and 62th days, plate counts of bacterial populations (enterobacteria, lactic acid bacteria—LAB, total aerobes, and psychrotrophs) and DGGE were made. Enterobacteria counts were lower in AG-group than the other groups throughout the experience (p = 0.016). LAB/enterobacteria ratio was greater in fishes supplemented with AG on 20th day, showing a greater amount of beneficial bacteria than non-beneficial ones. DGGE profiles resulting from the microbiota of β-CG at 62th day were clustered and separated from the rest of the profiles with a difference of 91%. Margalef richness (p = 0.049) and Shannon diversity index (p = 0.044) were lower in β-CG than in AG at 62th day. Taxonomic characterization showed the presence of three phylum: Bacteroidetes, Firmicutes, and Fusobacteria. This is the first study in which the microbiota of juvenile Pacú fed with biofunctional diets was evaluated. Future interventions that modify the bacterial ecosystem could improve the productive performance and ensure the safety of the pacu meat intended for consumption.


Prebiotic Biofunctional diets Red seeaweeds Pacú Intestinal microbiota DGGE 



Lactic acid bacteria


denaturing gradient gel electrophoresis


Algae group


Basal group

β-CG β

Carotene group


Plate count agar medium


Violet red bile agar


de Man Rogosa and Sharpe medium


Phosphate buffered saline


Funding information

The authors are thankful to PICT-2013-1804 for the financial support and Red 318RT0549 (AQUA-CIBUS)—CYTED.

Compliance with ethical standards


All authors read and approved the final manuscript.


  1. Alishahi M, Karamifar M, Mesbah M (2015) Effects of astaxanthin and Dunaliella salina on skin carotenoids, growth performance and immune response of Astronotus ocellatus. Aquac Int 23:1239–1248CrossRefGoogle Scholar
  2. Altschul S, Gish W, Miller W, Myers E, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefGoogle Scholar
  3. Bacchetta C, Rossi AS, Cian RE, Drago SR, Cazenave J (2019) Dietary β-carotene improves growth performance and antioxidant status of juvenile Piaractus mesopotamicus. Aquac Nutr. CrossRefGoogle Scholar
  4. Beheshtipour H, Mortazavian A, Mohammadi R, Sohrabvandi S, Khosravi-Darani K (2013) Supplementation of Spirulina platensis and Chlorella vulgaris algae into probiotic fermented milks. Compr Rev Food Sci Food Saf 12:144–154CrossRefGoogle Scholar
  5. Bicudo A, Sado R, Cyrino J (2009) Dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (Holmberg, 1887). Aquac 297:151–156CrossRefGoogle Scholar
  6. Blajman J, Zbrun M, Signorini M, Zimmermann JA, Rossler E, Berisvil AP, Romero Scharpen A, Astesana D, Soto L, Frizzo L (2017) Development of cecal predominant microbiota in broilers during a complete rearing using denaturing gradient gel eletrophoresis. Anim Prod Sci 57:458–465CrossRefGoogle Scholar
  7. Cecchini F, Iacumin L, Fontanot M, Comi G, Manzano M (2012) Identification of the unculturable bacteria Candidatus arthromitus in the intestinal content of trouts using dot blot and southern blot techniques. Vet Microbiol 156:389–394PubMedCrossRefGoogle Scholar
  8. Cerezuela R, Fumanal M, Tapia-Paniagua S, Meseguer J, Moriñigo M, Esteban M (2013) Changes in intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis in gilthead sea bream (Sparus aurata L.) specimens. Fish Shellfish Immunol 34:1063–1070PubMedCrossRefGoogle Scholar
  9. Cian R, Salgado P, Drago S, González R, Mauri A (2014) Development of naturally activated edible films with antioxidant properties prepared from red seaweed Porphyra columbina biopolymers. Food Chem 146:6–14PubMedCrossRefGoogle Scholar
  10. Cian R, Drago S, Sánchez de Medina F, Martínez-Augustin O (2015) Proteins and carbohydrates from red seaweeds: evidence for beneficial effects on gut function and microbiota. Mar Drugs 13:5358–5383PubMedPubMedCentralCrossRefGoogle Scholar
  11. Cian R, Bacchetta C, Cazenave J, Drago S (2018) Extruded fish feed with high residual phytase activity and low mineral leaching increased P. mesopotamicus mineral retention. Anim Feed Sci Technol 240:78–87CrossRefGoogle Scholar
  12. Cian R, Bacchetta C, Rossi A, Cazenave J, Drago S (2019) Red seaweed Pyropia columbina as antioxidant supplement in feed for cultured juvenile Pacú (Piaractus mesopotamicus). J Appl Phycol 31:1455–1465CrossRefGoogle Scholar
  13. CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas), (2005). Marco Ético de Referencia para las Investigaciones Biomédicas en Animales de laboratorio, de granja y obtenidos de la naturaleza, Buenos Aires. Accessed 13 Jan. 2020
  14. de Agroindustria M (2012) Incremento de actividad de acuicultura en las regiones NEA. NOA y Centro, Provincia de Santa FeGoogle Scholar
  15. Del-Pozo J, Crumlish M, Ferguson H, Green D, Turnbull J (2010) A prospective longitudinal study of “Candidatus arthromitus”-associated rainbow trout gastroenteritis in the UK. Prev Vet Med 94:289–300PubMedCrossRefGoogle Scholar
  16. Dimitroglou A, Merrifield DL, Carnevali O, Picchietti S, Avella M, Daniels C, Güroy D, Davies SJ (2011) Microbial manipulations to improve fish health and production–a Mediterranean perspective. Fish Shellfish Immun 30(1):1–16CrossRefGoogle Scholar
  17. Duan Y, Zhang Y, Dong H, Wang Y, Zhang J (2017) Effect of the dietary probiotic Clostridium butyricum on growth, intestine antioxidant capacity and resistance to high temperature stress in kuruma shrimp Marsupenaeus japonicas. J Therm Biol 66:93–100PubMedCrossRefGoogle Scholar
  18. Elsayed S, Zhang K (2005) Bacteremia caused by Clostridium intestinale. J Clin Microbiol 43:2018–2020PubMedPubMedCentralCrossRefGoogle Scholar
  19. Fitzgerald C, Gallagher E, Tasdemir D, Hayes M (2011) Heart health peptides from macroalgae and their potential use in functional foods. J Agric Food Chem 59:6829–6836PubMedCrossRefGoogle Scholar
  20. Frizzo LS, Bertozzi E, Soto LP, Sequeira G, Rodríguez Armesto R, Rosmini MR (2010) Studies on translocation, acute oral toxicity and intestinal colonization of potentially probiotic lactic acid bacteria administered during calf rearing. Livest Sci 128:28–35CrossRefGoogle Scholar
  21. Gao Q, Xiao Y, Sun P, Peng S, Yin F, Ma X, Shi Z (2013) In vitro protective efficacy of Clostridium butyricum against fish pathogen infections. Indian J Microbiol 53(4):453–459PubMedPubMedCentralCrossRefGoogle Scholar
  22. Gatesoupe F (2016) Probiotics and other microbial manipulations in fish feeds: prospective update of health benefits. In: Watson, R.R. and Preddy, V.R. Probiotics, prebiotics, and synbiotics. Ed: ElsevierCrossRefGoogle Scholar
  23. Gauthier D (2015) Bacterial zoonoses of fishes: a review and appraisal of evidence for linkages between fish and human infections. Vet J 203:27–35PubMedCrossRefGoogle Scholar
  24. Ghanbari M, Kneifel W, Domig K (2015) A new view of the fish gut microbiome: advances from next-generation sequencing. Aquac 448:464–475CrossRefGoogle Scholar
  25. Gonçalves A, Gallardo-Escárate C (2017) Microbiome dynamic modulation through functional diets based on pre- and probiotics (mannan-oligosaccharides and Saccharomyces cerevisiae) in juvenile rainbow trout (Oncorhynchus mykiss). J Appl Microbiol 122:1333–1347PubMedCrossRefGoogle Scholar
  26. Gordon M (2011) Significance of dietary antioxidants for health. Int J Mol Sci 13:173–179PubMedPubMedCentralCrossRefGoogle Scholar
  27. Guerreiro I, Serra C, Oliva-Teles A, Enes P (2017) Short communication: gut microbiota of European sea bass (Dicentrarchus labrax) is modulated by short-chain fructooligosaccharides and xylooligosaccharides. Aquac Int 26:279–288CrossRefGoogle Scholar
  28. Guo Y, Pan Q, Yan S, Chen Y, Li M, Chen D, Han H, Wu B, Cai J (2017) Bdellovibrio and like organisms promoted growth and survival of juvenile abalone Haliotis discus hannai Ino and modulated bacterial community structures in its gut. Aquac Int 25:1625–1643CrossRefGoogle Scholar
  29. Hasan M, Jang W, Lee S, Kim K, Lee B, Han H, Bai S, Kong I (2018) Effect of β-glucooligosaccharides as a new prebiotic for dietary supplementation in olive flounder (Paralichthys olivaceus) aquaculture. Aquac Res 49:1310–1319CrossRefGoogle Scholar
  30. Hedblom G, Reiland H, Sylte MJ, Johnson TJ, Baumler DJ (2018) Segmented filamentous Bacteria–metabolism meets immunity. Front Microbiol 9:1991PubMedPubMedCentralCrossRefGoogle Scholar
  31. Heidrich B, Vital M, Plumeier I, Döscher N, Kahl S, Kirschner J, Ziegert S, Solbach P, Lenzen H, Potthoff A, Manns M, Wedemeyer H, Pieper D (2018) Intestinal microbiota in patients with chronic hepatitis C with and without cirrhosis compared with healthy controls. Liver Int 38:50–58PubMedCrossRefGoogle Scholar
  32. Hwang J, Islam M, Ahmed S, Mun H, Kim G, Kim Y, Yang C (2014) Seamustard (Undaria pinnatifida) improves growth, immunity, fatty acid profile and reduces cholesterol in Hanwoo steers Asian-Australas. J Anim Sci 27:1114–1123Google Scholar
  33. Janse I, Bok J, Zwart G (2004) A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J Microbiol Methods 57:279–281PubMedCrossRefGoogle Scholar
  34. Jonsson H (2013) Segmented filamentous bacteria in human ileostomy samples after high-fiber intake. FEMS Microbiol Lett 342:24–29PubMedCrossRefGoogle Scholar
  35. Kulshreshtha G, Rathgeber B, Stratton G, Thomas N, Evans F, Critchley A, Hafting J, Prithiviraj B (2014) Feed supplementation with red seaweeds, Chondrus crispus and Sarcodiotheca gaudichaudii, affects performance, egg quality, and gut microbiota of layer hens. Poult Sci 93:2991–3001PubMedCrossRefGoogle Scholar
  36. Li L, Krause L, Somerset S (2017) Associations between micronutrient intakes and gut microbiota in a group of adults with cystic fibrosis. Clin Nutr 36:1097–1104PubMedCrossRefGoogle Scholar
  37. Meidong R, Khotchanalekha K, Doolgindachbaporn S, Nagasawa T, Nakao M, Sakai K, Tongpim S (2018) Evaluation of probiotic Bacillus aerius B81e isolated from healthy hybrid catfish on growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti. Fish Shellfish Immunol 73:1–10PubMedCrossRefGoogle Scholar
  38. Miranda JM, Trigo M, Barros-Velázquez J, Aubourg SP (2018) Quality enhancement of chilled lean fish by previous active dipping in Bifurcaria bifurcata alga extract. Food Bioprocess Technol 11:1662–1673CrossRefGoogle Scholar
  39. Najdegerami E, Tokmachi A, Bakhshi F (2017) Evaluating the effects of dietary prebiotic mixture of mannan oligosaccharide and poly-β-hydroxybutyrate on the growth performance, immunity, and survival of rainbow trout, Oncorhynchus mykiss (Walbaum 1792), fingerlings. J World Aquacult Soc 48:415–425CrossRefGoogle Scholar
  40. Parma de Croux M (1990) Benzocaine (ethyl-p-aminobenzoate) as an anaesthetic for Prochilodus lineatus, Valenciennes (Pisces, Curimatidae). J Appl Ichthyol 6:189–192CrossRefGoogle Scholar
  41. Peng Y, Yu K, Mu C, Hang S, Che L, Zhu W (2017) Progressive response of large intestinal bacterial community and fermentation to the stepwise decrease of dietary crude protein level in growing pigs. Appl Microbiol Biotechnol 101:5415–5426PubMedCrossRefGoogle Scholar
  42. Pullela S (1997) Aquaculture of pacu (Piaractus mesopotamicus) and a comparison of its quality: microbiological, sensory and proximate composition. Master of science in food science and technology thesis. Virginia, EE.UUGoogle Scholar
  43. Rico RM, Tejedor-Junco MT, Tapia-Paniagua ST, Alarcón FJ, Mancera JM, López-Figueroa F, Balebona MC, Abdala-Díaz RT, Moriñigo MA (2016) Influence of the dietary inclusion of Gracilaria cornea and Ulva rigida on the biodiversity of the intestinal microbiota of Sparus aurata juveniles. Aquac Int 24(4):965–984CrossRefGoogle Scholar
  44. Rimoldi S, Terova G, Ascione C, Giannico R, Brambilla F (2018) Next generation sequencing for gut microbiome characterization in rainbow trout (Oncorhynchus mykiss) fed animal by-product meals as an alternative to fishmeal protein sources. PLoS One 13(3):e0193652PubMedPubMedCentralCrossRefGoogle Scholar
  45. Soares M, Oliveira F, Cardoso I, Urbinati E, Meldau de Campos C, Hisano H (2018) Glucan-MOS® improved growth and innate immunity in pacu stressed and experimentally infected with Aeromonas hydrophila. Fish Shellfish Immunol 73:133–140PubMedCrossRefGoogle Scholar
  46. Su Y, Yao W, Perez-Gutierrez O, Smidt H, Zhu W (2008) Changes inabundance of Lactobacillus spp. and Streptococcus suis in the stomach, jejunum and ileum of piglets after weaning. FEMS Microbiol Ecol 66:546–555PubMedCrossRefGoogle Scholar
  47. Sutthi N, Thaimuangphol W, Rodmongkoldee M, Leelapatra W, Panase P (2018) Growth performances, survival rate, and biochemical parameters of Nile tilapia (Oreochromis niloticus) reared in water treated with probiotic. Comp Clin Pathol 27:597–603CrossRefGoogle Scholar
  48. Temmerman R, Scheirlinck I, Huys G, Swings J (2003) Culture independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:220–226PubMedPubMedCentralCrossRefGoogle Scholar
  49. Tonooka T, Sakata S, Kitahara M, Hanai M, Ishizeki S, Takada M, Sakamoto M, Benno Y (2005) Detection and quantification of four species of the genus Clostridium in infant feces. Microbiol Immunol 49:987–992PubMedCrossRefGoogle Scholar
  50. Tsuchiya C, Sakata T, Sugita H (2008) Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium in the intestinal tracts of freshwater fish. Lett Appl Microbiol 46:43–48PubMedGoogle Scholar
  51. Urbinati E, Goncalves F, Takahashi L (2010) Pacu (Piaractus mesopotamicus). In: Baldisseroto B, Gomes LC (eds) Espécies nativas parapiscicultura no Brasil, second edn. UFSM, Santa Maria pp 225–246Google Scholar
  52. Wei N, Wang C, Xiao S, Huang W, Lin M, Yan Q, Ma Y (2018) Intestinal microbiota in large yellow croaker, Larimichthys crocea, at different ages. J World Aquacult Soc 49:256–267CrossRefGoogle Scholar
  53. Yin LJ (2009) Development of the prebiotics from lactic acid bacteria fermented algae hydrolysates and evaluation of their functionality. New Biotechnol 25(Suppl 1):S116–S117CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  • L. T. Rossi
    • 1
  • A. Romero Sharpen
    • 1
  • J. A. Zimmermann
    • 1
  • C. R. Olivero
    • 1
  • M. V. Zbrun
    • 1
    • 2
  • L. S. Frizzo
    • 1
    • 2
  • M. L. Signorini
    • 2
    • 3
  • C. Bacchetta
    • 4
  • R. E. Cian
    • 5
  • J. Cazenave
    • 4
  • L. P. Soto
    • 1
    • 2
  • S. R. Drago
    • 5
    Email author
  1. 1.Laboratorio de Análisis de Alimentos, ICIVET-Litoral (CONICET-UNL), Kredder 2805 (S3080HOF)EsperanzaArgentina
  2. 2.Departamento de Salud Pública, Facultad de Ciencias VeterinariasUniversidad Nacional del LitoralEsperanzaArgentina
  3. 3.Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)Instituto Nacional de Tecnología AgropecuariaRafaelaArgentina
  4. 4.INALI, UNL, CONICET, Santa Fe, Argentina. Paraje El PozoCiudad Universitaria UNLSanta FeArgentina
  5. 5.Instituto de Tecnología de Alimentos, CONICET, FIQ, UNLSanta FeArgentina

Personalised recommendations