Probiotics and Antimicrobial Proteins

, Volume 11, Issue 3, pp 957–965 | Cite as

Evaluation of the Impacts of Long-Term Enriched Artemia with Bacillus subtilis on Growth Performance, Reproduction, Intestinal Microflora, and Resistance to Aeromonas hydrophila of Ornamental Fish Poecilia latipinna

  • Nasrollah AhmadifardEmail author
  • Vahid Rezaei Aminlooi
  • Amir Tukmechi
  • Naser Agh


The present study investigated the effect of enriched Artemia with Bacillus subtilis on growth performance, reproductive factors, proximate composition, intestinal microflora, and resistance to Aeromonas hydrophila of ornamental fish, Poecilia latipinna. Using a completely randomized design, the experiment included three groups. The first group was fed with commercial food without any probiotic. The second group was fed with unenriched Artemia, and the last group consumed long-time enriched Artemia with Bacillus subtilis. The bacteria B. subtilis with a density of 1 × 105 CFU mL−1 was added daily to Artemia culture medium. The total microflora and Bacillus subtilis counts were significantly increased in enriched Artemia compared to the unenriched group (P < 0.05). In fish fed groups, growth factors did not show any significant difference (P > 0.05). The maximum relative fecundity (28.65 ± 2.52 egg number g−1), fry production (62.93 ± 4.6 individual per female), and fry survival (70.97 ± 1.56%) obtained in the third group were found to be significantly more than those in the first and the second groups. Moreover, intestinal bacterial count for Bacillus revealed that the higher concentration of bacteria was significantly related to the third group (6.24 ± 0.11 log CFU g−1) (P < 0.05). Maximum protein and fat contents were observed in fish fed with Bacillus-enriched Artemia; however, no significant difference was found between control and unenriched Artemia groups (P > 0.05). The highest amount of ash was observed in fish fed with commercial food without any probiotic (P < 0.05). At the end of the feeding period, each of the three groups along with positive group (oxytetracycline 100 mg kg−1 of commercial food) was exposed to A. hydrophila (BCCM5/LMG3770) bacteria intraperitoneally. Based on the results, the lowest cumulative mortality was significantly found in group three (68.75 ± 3.6%) and positive group (62.5 ± 7.0%) compared to control and unenriched Artemia groups (P < 0.05). Hence, B. subtilis with a concentration of 1 × 105 CFU mL−1 during the period of Artemia culturing can improve the reproductive parameters, intestinal microflora, and resistance to pathogenic bacteria of Poecilia latipinna.


Aeromonas hydrophila Artemia Bacillus subtilis Poecilia latipinna Probiotics Reproduction 


  1. 1.
    Langroudi HE, Mousavi SH, Falahatkar B, Moradkhani Z (2009) Effect of diets containing artemia enriched with unsaturated fatty acids and vitamin C on angel fish Pterophyllum scalare propagation. Int Aquat Res 1:67–72Google Scholar
  2. 2.
    Wang Y-B, Li J-R, Lin J (2008) Probiotics in aquaculture: challenges and outlook. Aquaculture 281(1–4):1–4. CrossRefGoogle Scholar
  3. 3.
    Merrifield DL, Dimitroglou A, Foey A, Davies SJ, Baker RT, Bøgwald J, Castex M, Ringø E (2010) The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302(1):1–18. CrossRefGoogle Scholar
  4. 4.
    Holzapfel WH, Haberer P, Snel J, Schillinger U (1998) Overview of gut flora and probiotics. Int J Food Microbiol 41(2):85–101. CrossRefPubMedGoogle Scholar
  5. 5.
    Gatesoupe F-J (1994) Lactic acid bacteria increase the resistance of turbot larvae, Scophthalmus maximus, against pathogenic Vibrio. Aquat Living Resour 7(4):277–282. CrossRefGoogle Scholar
  6. 6.
    Balcázar JL, De Blas I, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114(3):173–186. CrossRefPubMedGoogle Scholar
  7. 7.
    Nayak S (2010) Probiotics and immunity: a fish perspective. Fish Shellfish Immun 29(1):2–14. CrossRefGoogle Scholar
  8. 8.
    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):1–16. CrossRefGoogle Scholar
  9. 9.
    Tukmechi A, Bandboni M (2014) Effects of Saccharomyces cerevisiae supplementation on immune response, hematological parameters, body composition and disease resistance in rainbow trout, Oncorhynchus mykiss (Walbaum, 1792). J Appl Ichthyol 30(1):55–61. CrossRefGoogle Scholar
  10. 10.
    Azimirad M, Meshkini S, Ahmadifard N, Hoseinifar SH (2016) The effects of feeding with synbiotic (Pediococcus acidilactici and fructooligosaccharide) enriched adult Artemia on skin mucus immune responses, stress resistance, intestinal microbiota and performance of angelfish (Pterophyllum scalare). Fish Shellfish Immun 54:516–522. CrossRefGoogle Scholar
  11. 11.
    Ghosh S, Sinha A, Sahu C (2007) Effect of probiotic on reproductive performance in female livebearing ornamental fish. Aquac Res 38(5):518–526. CrossRefGoogle Scholar
  12. 12.
    González-Félix ML, Gatlin DM III, Urquidez-Bejarano P, de la Reé-Rodríguez C, Duarte-Rodríguez L, Sánchez F, Casas-Reyes A, Yamamoto FY, Ochoa-Leyva A, Perez-Velazquez M (2018) Effects of commercial dietary prebiotic and probiotic supplements on growth, innate immune responses, and intestinal microbiota and histology of Totoaba macdonaldi. Aquaculture 491:239–251. CrossRefGoogle Scholar
  13. 13.
    Yi Y, Zhang Z, Zhao F, Liu H, Yu L, Zha J, Wang G (2018) Probiotic potential of Bacillus velezensis JW: antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus. Fish Shellfish Immun 78:322–330. CrossRefGoogle Scholar
  14. 14.
    Tseng D-Y, Ho P-L, Huang S-Y, Cheng S-C, Shiu Y-L, Chiu C-S, Liu C-H (2009) Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20. Fish Shellfish Immun 26(2):339–344. CrossRefGoogle Scholar
  15. 15.
    Liu K-F, Chiu C-H, Shiu Y-L, Cheng W, Liu C-H (2010) Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae. Fish Shellfish Immun 28(5):837–844. CrossRefGoogle Scholar
  16. 16.
    Balcázar JL, Rojas-Luna T (2007) Inhibitory activity of probiotic Bacillus subtilis UTM 126 against Vibrio species confers protection against vibriosis in juvenile shrimp (Litopenaeus vannamei). Curr Microbiol 55(5):409–412. CrossRefPubMedGoogle Scholar
  17. 17.
    Ghosh S, Sinha A, Sahu C (2008) Dietary probiotic supplementation in growth and health of live-bearing ornamental fishes. Aquac Nutr 14(4):289–299. CrossRefGoogle Scholar
  18. 18.
    Lin S, Mao S, Guan Y, Luo L, Luo L, Pan Y (2012) Effects of dietary chitosan oligosaccharides and Bacillus coagulans on the growth, innate immunity and resistance of koi (Cyprinus carpio koi). Aquaculture 342:36–41. CrossRefGoogle Scholar
  19. 19.
    He S, Liu W, Zhou Z, Mao W, Ren P, Marubashi T, Ringø E (2011) Evaluation of probiotic strain Bacillus subtilis C-3102 as a feed supplement for koi carp (Cyprinus carpio). J Aquac Res Dev S1:005:1–7. CrossRefGoogle Scholar
  20. 20.
    Makridis P, Bergh Ø, Skjermo J, Vadstein O (2001) Addition of bacteria bioencapsulated in Artemia metanauplii to a rearing system for halibut larvae. Aquac Int 9(3):225–235. CrossRefGoogle Scholar
  21. 21.
    Gatesoupe F-J (1991) Managing the dietary value of Artemia for larval turbot, Scophthalmus maximus; the effect of enrichment and distribution techniques. Aquac Eng 10(2):111–119. CrossRefGoogle Scholar
  22. 22.
    Negm RK, Cobcroft JM, Brown MR, Nowak BF, Battaglene SC (2014) Performance and skeletal abnormality of striped trumpeter Latris lineata larvae and post larvae fed vitamin A enriched Artemia. Aquaculture 422:115–123. CrossRefGoogle Scholar
  23. 23.
    Soltanian S, Dhont J, Sorgeloos P, Bossier P (2007) Influence of different yeast cell-wall mutants on performance and protection against pathogenic bacteria (Vibrio campbellii) in gnotobiotically-grown Artemia. Fish Shellfish Immun 23(1):141–153. CrossRefGoogle Scholar
  24. 24.
    Gomez-Gil B, Herrera-Vega MA, Abreu-Grobois FA, Roque A (1998) Bioencapsulation of two different Vibrio species in nauplii of the brine shrimp (Artemia franciscana). Appl Environ Microbiol 64(6):2318–2322PubMedPubMedCentralGoogle Scholar
  25. 25.
    Sorgeloos P (1986) Manual for the culture and use of brine shrimp Artemia in aquaculture. State University of Ghent, Faculty of Agriculture, Ghent 319 pGoogle Scholar
  26. 26.
    Coutteau P, Lavens P, Sorgeloos P (1990) Baker’s yeast as a potential substitute for live algae in aquaculture diets: Artemia as a case study. J World Aquacult Soc 21(1):1–9. CrossRefGoogle Scholar
  27. 27.
    Niu Y, Defoirdt T, Baruah K, Van de Wiele T, Dong S, Bossier P (2014) Bacillus sp. LT3 improves the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged with Vibrio campbellii by enhancing the innate immune response and by decreasing the activity of shrimp-associated vibrios. Vet Microbiol 173(3):279–288. CrossRefPubMedGoogle Scholar
  28. 28.
    Tukmechi A, HRR A, Manaffar R, Sheikhzadeh N (2011) Dietary administration of beta-mercapto-ethanol treated Saccharomyces cerevisiae enhanced the growth, innate immune response and disease resistance of the rainbow trout, Oncorhynchus mykiss. Fish Shellfish Immun 30(3):923–928. CrossRefGoogle Scholar
  29. 29.
    Utiswannakul P, Sangchai S, Rengpipat S (2011) Enhanced growth of black tiger shrimp Penaeus monodon by dietary supplementation with Bacillus (BP11) as a probiotic. J Aquac Res Dev.
  30. 30.
    Mahious A, Gatesoupe F, Hervi M, Metailler R, Ollevier F (2006) Effect of dietary inulin and oligosaccharides as prebiotics for weaning turbot, Psetta maxima (Linnaeus, C. 1758). Aquac Int 14(3):219. CrossRefGoogle Scholar
  31. 31.
    Sneath PH (1986) Endospore-forming Gram-positive rods and cocci. In: Bergey’s manual of systemic bacteriology, vol 2. Williams & Wilkins, pp 1104–1207Google Scholar
  32. 32.
    Cappuccino N, Mackay R, Eisner C (2002) Spread of the invasive alien vine Vincetoxicum rossicum: tradeoffs between seed dispersability and seed quality. Am Midl Nat 148(2):263–270.[0263:SOTIAV]2.0.CO;2Google Scholar
  33. 33.
    Chitra G, Krishnaveni N (2013) Effect of probiotics on reproductive performance in female livebearing ornamental fish Poecilia sphenops. Int J Pure Appl Zool 1(3):235–245Google Scholar
  34. 34.
    Misra CK, Das BK, Mukherjee SC, Pattnaik P (2006) Effect of long term administration of dietary β-glucan on immunity, growth and survival of Labeo rohita fingerlings. Aquaculture 255:82–94. CrossRefGoogle Scholar
  35. 35.
    AOAC (1990) In: Horwitz W (ed) Official methods of analyses, 15th edn. Association of Official Analytical Chemists Inc., Arlington 445pGoogle Scholar
  36. 36.
    Douillet PA, Langdon CJ (1994) Use of a probiotic for the culture of larvae of the Pacific oyster (Crassostrea gigas Thunberg). Aquaculture 119(1):25–40. CrossRefGoogle Scholar
  37. 37.
    Ghosh K, Sen SK, Ray AK (2003) Supplementation of an isolated fish gut bacterium, Bacillus circulans, in formulated diets for rohu, Labeo rohita, fingerlings. Isr J Aquac 55(1):13–21. Google Scholar
  38. 38.
    Carnevali O, Zamponi MC, Sulpizio R, Rollo A, Nardi M, Orpianesi C, Silvi S, Caggiano M, Polzonetti AM, Cresci A (2004) Administration of probiotic strain to improve sea bream wellness during development. Aquac Int 12(4–5):377–386. CrossRefGoogle Scholar
  39. 39.
    Abdel-Tawwab M, Abdel-Rahman AM, Ismael NE (2008) Evaluation of commercial live bakers’ yeast, Saccharomyces cerevisiae as a growth and immunity promoter for Fry Nile tilapia, Oreochromis niloticus (L.) challenged in situ with Aeromonas hydrophila. Aquaculture 280(1–4):185–189. CrossRefGoogle Scholar
  40. 40.
    El-Rhman AMA, Khattab YA, Shalaby AM (2009) Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immun 27(2):175–180. CrossRefGoogle Scholar
  41. 41.
    Avella MA, Gioacchini G, Decamp O, Makridis P, Bracciatelli C, Carnevali O (2010a) Application of multi-species of Bacillus in sea bream larviculture. Aquaculture 305(1–4):12–19. CrossRefGoogle Scholar
  42. 42.
    Nandi A, Banerjee G, Dan SK, Ghosh K, Ray AK (2018) Evaluation of in vivo probiotic efficiency of Bacillus amyloliquefaciens in Labeo rohita challenged by pathogenic strain of Aeromonas hydrophila MTCC 1739. Probiotics Antimicrob Proteins 10(2):391–398. CrossRefPubMedGoogle Scholar
  43. 43.
    Adorian TJ, Jamali H, Farsani HG, Darvishi P, Hasanpour S, Bagheri T, Roozbehfar R (2018) Effects of probiotic bacteria Bacillus on growth performance, digestive enzyme activity, and hematological parameters of Asian sea bass, Lates calcarifer (Bloch). Probiotics Antimicrob Proteins.
  44. 44.
    Sun Y-Z, Yang H-L, Ma R-L, Lin W-Y (2010) Probiotic applications of two dominant gut Bacillus strains with antagonistic activity improved the growth performance and immune responses of grouper Epinephelus coioides. Fish Shellfish Immun 29(5):803–809. CrossRefGoogle Scholar
  45. 45.
    Avella MA, Olivotto I, Silvi S, Place AR, Carnevali O (2010b) Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish. Am J Physiol-Reg 298(2):359–371. CrossRefGoogle Scholar
  46. 46.
    Patra S, Mohamed K (2003) Enrichment of Artemia nauplii with the probiotic yeast Saccharomyces boulardii and its resistance against a pathogenic Vibrio. Aquac Int 11(5):505–514. CrossRefGoogle Scholar
  47. 47.
    Verschuere L, Rombaut G, Huys G, Dhont J, Sorgeloos P, Verstraete W (1999) Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. Appl Environ Microbiol 65(6):2527–2533PubMedPubMedCentralGoogle Scholar
  48. 48.
    Jamali H, Imani A, Abdollahi D, Roozbehfar R, Isari A (2015) Use of probiotic Bacillus spp. in rotifer (Brachionus plicatilis) and Artemia (Artemia urmiana) enrichment: effects on growth and survival of Pacific white shrimp, Litopenaeus vannamei, larvae. Probiotics Antimicrob Prot 7(2):118–125. CrossRefGoogle Scholar
  49. 49.
    Ringø E, Birkbeck T (1999) Intestinal microflora of fish larvae and fry. Aquac Res 30(2):73–93. CrossRefGoogle Scholar
  50. 50.
    Kesarcodi-Watson A, Kaspar H, Lategan MJ, Gibson L (2008) Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274(1):1–14. CrossRefGoogle Scholar
  51. 51.
    Picchietti S, Fausto AM, Randelli E, Carnevali O, Taddei AR, Buonocore F, Scapigliati G, Abelli L (2009) Early treatment with Lactobacillus delbrueckii strain induces an increase in intestinal T-cells and granulocytes and modulates immune-related genes of larval Dicentrarchus labrax (L.). Fish Shellfish Immun 26(3):368–376. CrossRefGoogle Scholar
  52. 52.
    Dehghan M, Jafariyan H, Rezai MH, Amoozagar MA, Sahandi J (2011) Potential of the brine shrimp (Artemia urniana) enrichment with two species of Bacillus and yeast (Saccharomyces cerevisiae). World J Fish Marine Sci 3(6):523–528Google Scholar
  53. 53.
    Gatesoupe F-J (2008) Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments. J Mol Microbiol Biotechnol 14(1–3):107–114. CrossRefPubMedGoogle Scholar
  54. 54.
    Hajibeglou A, Sudagar M (2010) Effect of dietary supplementation with probiotic on reproductive performance of female livebearing ornamental fish. Res J Anim Sci 4(4):103–107Google Scholar
  55. 55.
    Dahlgren B (1980) The effects of three different dietary protein levels on the fecundity in the guppy, Poecilia reticulata (Peters). J Fish Biol 16(1):83–97. CrossRefGoogle Scholar
  56. 56.
    Goldin BR, Gorbach SL (1992) Probiotics for humans. In: Probiotics. Springer, pp 355–376Google Scholar
  57. 57.
    Avella MA, Place A, Du S-J, Williams E, Silvi S, Zohar Y, Carnevali O (2012) Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems. PLoS One 7(9):e45572. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Munro P, Barbour A, Blrkbeck T (1994) Comparison of the gut bacterial flora of start-feeding larval turbot reared under different conditions. J Appl Microbiol 77(5):560–566. CrossRefGoogle Scholar
  59. 59.
    Kennedy BS, Tucker JW Jr, Neidig CL, Vermeer GK, Cooper VR, Jarrell JL, Sennett DG (1998) Bacterial management strategies for stock enhancement of warmwater marine fish: a case study with common snook (Centropomus undecimalis). B Mar Sci 62(2):573–588Google Scholar
  60. 60.
    Wooster GA, Bowser PR, Brown SB, Fisher JP (2000) Remediation of Cayuga syndrome in landlocked Atlantic Salmon Salmo salar using egg and sac-fry bath treatments of thiamine-hydrochloride. J World Aquac Soc 31(2):149–157. CrossRefGoogle Scholar
  61. 61.
    Hornung M, Miller L, Peterson R, Marcquenski S, Brown S (1998) Efficacy of various treatments conducted on Lake Michigan salmonid embryos in reducing early mortality syndrome. Early life stage mortality syndrome in fishes of the Great Lakes and Baltic Sea. In: McDonald G, Fitzsimons JD, Honeyfield DC (eds) Am Fish Soc. Bethesda. pp 124–134Google Scholar
  62. 62.
    Vine NG, Leukes WD, Kaiser H (2006) Probiotics in marine larviculture. FEMS Microbiol Rev 30(3):404–427. CrossRefPubMedGoogle Scholar
  63. 63.
    Nadella RK, Prakash RR, Dash G, Ramanathan SK, Kuttanappilly LV, Mothadaka MP (2018) Histopathological changes in giant freshwater prawn Macrobrachium rosenbergii (de Man 1879) fed with probiotic Bacillus licheniformis upon challenge with Vibrio alginolyticus. Aquac Res 49(1):81–92. CrossRefGoogle Scholar
  64. 64.
    Gerard J, Lloyd R, Barsby T, Haden P, Kelly MT, Andersen RJ (1997) Massetolides A– H, antimycobacterial cyclic depsipeptides produced by two pseudomonads isolated from marine habitats. J Nat Prod 60(3):223–229. CrossRefPubMedGoogle Scholar
  65. 65.
    Jafaryan H, Mehdi TM, Mohammad MN (2010) The effects of probiotic bacillus for promotion of growth and feeding parameters in beluga (Huso huso) larvae via feeding by bioencapsulated Artemia. AACL Bioflux 3(4):273–280Google Scholar
  66. 66.
    Allam HYH (2007) Physiological effects of some additives on growth, blood constituents and immunity in Nile tilapia (Oreochromis niloticus). PhD thesis. Fac of Agric, Assiut Univ, EgyptGoogle Scholar
  67. 67.
    Carvalho A, Escaffre A-M, Teles AO, Bergot P (1997) First feeding of common carp larvae on diets with high levels of protein hydrolysates. Aquac Int 5(4):361–367. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Fisheries, Faculty of Natural ResourcesUrmia UniversityUrmiaIran
  2. 2.Department of Microbiology, Faculty of Veterinary MedicineUrmia UniversityUrmiaIran
  3. 3.Department of Artemia, Urmia Lake Research InstituteUrmia UniversityUrmiaIran

Personalised recommendations