Journal of Microbiology

, Volume 55, Issue 9, pp 694–702 | Cite as

Effect of dietary copper level on the gut microbiota and its correlation with serum inflammatory cytokines in Sprague-Dawley rats



In China’s swine industry, copper is generally supplemented above the National Research Council (NRC) requirement (2012) because of its antimicrobial properties and the potential for growth promotion. Yet few are concerned about whether this excess supplementation is necessary. In this study, the 16S rRNA pyrosequencing was designed and used to investigate the effect of dietary copper level on the diversity of the fecal microbial community and the correlation of copper level with the serum level of inflammatory cytokines in Sprague-Dawley rat models. The results showed that the diet containing a high level of Cu (120 and 240 mg/kg) changed the microbial richness and diversity of rat feces associated with the increased copper content in the rat ileac and colonic digesta. Furthermore, a Pearson’s correlation analysis indicated that an accumulation of unabsorbed copper in the chyme was correlated with the microbial composition of the rat feces, which was linked with TNF-α in serum. The results suggest that dietary copper level may have a direct impact on circulating inflammatory cytokines in the serum, perhaps inducing an inflammatory response by altering the microbial composition of rat feces. Serum TNF-α could be the chief responder to excessive copper exposure.


copper inflammatory cytokines gut microbiota 16S rRNA pyrosequencing 


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Supplementary material

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Supplementary data Table S1. Effect of dietary copper level on serum biochemical parameters of SD rats (Mean±SD)


  1. Amato, K.R., Yeoman, C.J., Kent, A., Righini, N., Carbonero, F., Estrada, A., Gaskins, H.R., Stumpf, R.M., Yildirim, S., Torralba, M., et al. 2013. Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. ISME J. 7, 1344–1353.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ansteinsson, V., Refsnes, M., Skomedal, T., Osnes, J.B., Schiander, I., and Lag, M. 2009. Zinc- and Copper-induced interleukin-6 release in primary cell cultures from rat heart. Cardiovasc. Toxicol. 9, 86–94.CrossRefPubMedGoogle Scholar
  3. Armstrong, T.A., Cook, D.R., Ward, M.M., Williams, C.M., and Spears, J.W. 2004. Effect of dietary copper source (cupric citrate and,cupric sulfate) and concentration on growth performance and fecal copper excretion in weanling pigs. J. Anim. Sci. 82, 1234–1240.CrossRefPubMedGoogle Scholar
  4. Bailey, J.D., Ansotegui, R.P., Paterson, J.A., Swenson, C.K., and Johnson, A.B. 2001. Effects of supplementing combinations of inorganic and complexed copper on performance and liver mineral status of beef heifers consuming antagonists. J. Anim. Sci. 79, 2926–2934.CrossRefPubMedGoogle Scholar
  5. Bailey, M.T., Dowd, S.E., Galley, J.D., Hufnagle, A.R., Allen, R.G., and Lyte, M. 2011. Exposure to a social stressor alters the structure of the intestinal microbiota: Implications for stressor-induced immunomodulation. Brain Behav. Immun. 25, 397–407.CrossRefPubMedGoogle Scholar
  6. Boente, R.F., Ferreira, L.Q., Falcao, L.S., Miranda, K.R., Guimaraes, P.L.S., Santos, J., Vieira, J.M.B.D., Barroso, D.E., Emond, J.P., Ferreira, E.O., et al. 2010. Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains. Anaerobe 16, 190–194.CrossRefPubMedGoogle Scholar
  7. Dewar, M.L., Arnould, J.P., Dann, P., Trathan, P., Groscolas, R., and Smith, S. 2013. Interspecific variations in the gastrointestinal microbiota in penguins. Microbiologyopen 2, 195–204.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Dias, R.S., Lopez, S., Montanholi, Y.R., Smith, B., Haas, L.S., Miller, S.P., and France, J. 2013. A meta-analysis of the effects of dietary copper, molybdenum, and sulfur on plasma and liver copper, weight gain, and feed conversion in growing-finishing cattle. J. Anim. Sci. 91, 5714–5723.CrossRefPubMedGoogle Scholar
  9. Dziarski, R., Park, S.Y., Kashyap, D.R., Dowd, S.E., and Gupta, D. 2016. Pglyrp-regulated gut microflora Prevotella falsenii, Parabacteroides distasonis and Bacteroides eggerthii enhance and Alistipes finegoldii attenuates colitis in mice. PLoS One 11, 1–24.CrossRefGoogle Scholar
  10. Felske, A., Wolterink, A., van Lis, R., De Vos, W.M., and Akkermans, A.D.L. 1999. Searching for predominant soil bacteria: 16S rDNA cloning versus strain cultivation. FEMS Microbiol. Ecol. 30, 137–145.CrossRefPubMedGoogle Scholar
  11. Frank, D.N., Amand, A.L.S., Feldman, R.A., Boedeker, E.C., Harpaz, N., and Pace, N.R. 2007. Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc. Natl. Acad. Sci. USA 104, 13780–13785.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Fry, R.S., Ashwell, M.S., Lloyd, K.E., O’Nan, A.T., Flowers, W.L., Stewart, K.R., and Spears, J.W. 2012. Amount and source of dietary copper affects small intestine morphology, duodenal lipid peroxidation, hepatic oxidative stress, and mRNA expression of hepatic copper regulatory proteins in weanling pigs. J. Anim. Sci. 90, 3112–3119.CrossRefPubMedGoogle Scholar
  13. Gong, J.H., Forster, R.J., Yu, H., Chambers, J.R., Sabour, P.M., Wheatcroft, R., and Chen, S. 2002. Diversity and phylogenetic analysis of bacteria in the mucosa of chicken ceca and comparison with bacteria in the cecal lumen. FEMS Microbiol. Lett. 208, 1–7.CrossRefPubMedGoogle Scholar
  14. Gowanlock, D.W., Mahan, D.C., Jolliff, J.S., Moeller, S.J., and Hill, G.M. 2013. Evaluating the NRC levels of Cu, Fe, Mn, and Zn using organic minerals for grower-finisher swine. J. Anim. Sci. 91, 5680–5686.CrossRefPubMedGoogle Scholar
  15. Hojberg, O., Canibe, N., Poulsen, H.D., Hedemann, M.S., and Jensen, B.B. 2005. Influence of dietary zinc oxide and copper sulfate on the gastrointestinal ecosystem in newly weaned piglets. Appl. Environ. Microbiol. 71, 2267–2277.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Huang, Y.L., Ashwell, M.S., Fry, R.S., Lloyd, K.E., Flowers, W.L., and Spears, J.W. 2015. Effect of dietary copper amount and source on copper metabolism and oxidative stress of weanling pigs in short-term feeding. J. Anim. Sci. 93, 2948–2955.CrossRefPubMedGoogle Scholar
  17. Jondreville, C., Revy, P.S., and Dourmad, J.Y. 2003. Dietary means to better control the environmental impact of copper and zinc by pigs from weaning to slaughter. Livest. Prod. Sci. 84, 147–156.CrossRefGoogle Scholar
  18. Jost, T., Lacroix, C., Braegger, C.P., Rochat, F., and Chassard, C. 2014. Vertical mother-neonate transfer of maternal gut bacteria via breastfeeding. Environ. Microbiol. 16, 2891–2904.CrossRefPubMedGoogle Scholar
  19. Konstantinov, S.R. 2005. Ph.D. thesis. Lactobacilli in the porcine intestine: from composition to functionality. Wageningen University, Wageningen, Netherlands.Google Scholar
  20. Kumar, V., Kalita, J., Bora, H.K., and Misra, U.K. 2016. Relationship of antioxidant and oxidative stress markers in different organs following copper toxicity in a rat model. Toxicol. Appl. Pharmacol. 293, 37–43.CrossRefPubMedGoogle Scholar
  21. Lin, Z.M., Ning, H.F., Bi, J.G., Qiao, J.F., Liu, Z.H., Li, G.H., Wang, Q.S., Wang, S.H., and Ding, Y.F. 2014. Effects of nitrogen fertilization and genotype on rice grain macronutrients and micronutrients. Rice Science 21, 233–242.CrossRefGoogle Scholar
  22. Liu, J.H., Zhang, M.L., Zhang, R.Y., Zhu, W.Y., and Mao, S.Y. 2016. Comparative studies of the composition of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of lactating dairy cows. Microb. Biotechnol. 9, 257–268.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lu, L., Wang, R.L., Zhang, Z.J., Steward, F.A., Luo, X.G., and Liu, B. 2010. Effect of dietary supplementation with copper sulfate or tribasic copper chloride on the growth performance, liver copper concentrations of broilers fed in floor pens, and stabilities of vitamin E and phytase in feeds. Biol. Trace. Elem. Res. 138, 181–189.CrossRefPubMedGoogle Scholar
  24. Luo, X.G., Ji, F., Lin, Y.X., Steward, F.A., Lu, L., Liu, B., and Yu, S.X. 2005. Effects of dietary supplementation with copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and oxidation stability of vitamin E in feed. Poult. Sci. 84, 888–893.CrossRefPubMedGoogle Scholar
  25. Ma, Y.L., Zanton, G.I., Zhao, J., Wedekind, K., Escobar, J., and Vazquez-Anon, M. 2015. Multitrial analysis of the effects of copper level and source on performance in nursery pigs. J. Anim. Sci. 93, 606–614.CrossRefPubMedGoogle Scholar
  26. Mattie, M.D., McElwee, M.K., and Freedman, J.H. 2008. Mechanism of copper-activated transcription: activation of AP-1, and the JNK/SAPK and p38 signal transduction pathways. J. Mol. Biol. 383, 1008–1018.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mavromichalis, I., Hancock, J.D., Kim, I.H., Senne, B.W., Kropf, D.H., Kennedy, G.A., Hines, R.H., and Behnke, K.C. 1999. Effects of omitting vitamin and trace mineral premixes and(or) reducing inorganic phosphorus additions on growth performance, carcass characteristics, and muscle quality in finishing pigs. J. Anim. Sci. 77, 2700–2708.CrossRefPubMedGoogle Scholar
  28. Mei, S.F., Yu, B., Ju, C.F., Zhu, D., and Chen, D.W. 2010. Effect of different levels of copper on growth performance and cecal ecosystem of newly weaned piglets. Int. J. Mol. Sci. 9, 378–381.Google Scholar
  29. Mori, H., Maruyama, F., Kato, H., Toyoda, A., Dozono, A., Ohtsubo, Y., Nagata, Y., Fujiyama, A., Tsuda, M., and Kurokawa, K. 2014. Design and experimental application of a novel non-degenerate universal primer set that amplifies prokaryotic 16S rRNA genes with a low possibility to amplify eukaryotic rRNA genes. DNA Res. 21, 217–227.CrossRefPubMedGoogle Scholar
  30. Munoz, C., Lopez, M., Olivares, M., Pizarro, F., Arredondo, M., and Araya, M. 2005. Differential response of interleukin-2 production to chronic copper supplementation in healthy humans. Eur. Cytokine Netw. 16, 261–265.PubMedGoogle Scholar
  31. Namkung, H., Gong, J., Yu, H., and De Lange, C.F.M. 2006. Effect of pharmacological intakes of zinc and copper on growth performance, circulating cytokines and gut microbiota of newly weaned piglets challenged with coliform lipopolysaccharides. Can. J. Anim. Sci. 86, 511–522.CrossRefGoogle Scholar
  32. Novotny, J., Pistl, J., and Kovac, G. 2003. Effects of supplementation of organic-bound trace elements on blood and tissues-Micromineral profile and immune parameters of piglets. Acta Vet.-Beogr. 53, 11–18.CrossRefGoogle Scholar
  33. Pang, Y., Patterson, J.A., and Applegate, T.J. 2009. The influence of copper concentration and source on ileal microbiota. Poult. Sci. 88, 586–592.CrossRefPubMedGoogle Scholar
  34. Pereira, T.C., Campos, M.M., and Bogo, M.R. 2016. Copper toxicology, oxidative stress and inflammation using zebrafish as experimental model. J. Appl. Toxicol. 36, 876–881.CrossRefPubMedGoogle Scholar
  35. Petta, S., Gastaldelli, A., Rebelos, E., Bugianesi, E., Messa, P., Miele, L., Svegliati-Baroni, G., Valenti, L., and Bonino, F. 2016. Pathophysiology of non alcoholic fatty liver disease. Int. J. Mol. Sci. 17, 1–26.CrossRefGoogle Scholar
  36. Rajilic-Stojanovic, M., Shanahan, F., Guarner, F., and De Vos, W.M. 2013. Phylogenetic analysis of dysbiosis in ulcerative colitis during remission. Inflamm. Bowel Dis. 19, 481–488.CrossRefPubMedGoogle Scholar
  37. Reeves, P.G., Nielsen, F.H., and Fahey, G.C.Jr. 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.PubMedGoogle Scholar
  38. Sanchez, D., Miguel, M., and Aleixandre, A. 2012. Dietary fiber, gut peptides, and adipocytokines. J. Med. Food 15, 223–230.CrossRefPubMedGoogle Scholar
  39. Satokari, R., Fuentes, S., Mattila, E., Jalanka, J., De Vos, W.M., and Arkkila, P. 2014. Fecal transplantation treatment of antibioticinduced, noninfectious colitis and long-term microbiota followup. Case. Rep. Med. 2014, 1–7.CrossRefGoogle Scholar
  40. Shelton, J.L., Southern, L.L., LeMieux, F.M., Bidner, T.D., and Page, T.G. 2004. Effects of microbial phytase, low calcium and phosphorus, and removing the dietary trace mineral premix on carcass traits, pork quality, plasma metabolites, and tissue mineral content in growing-finishing pigs. J. Anim. Sci. 82, 2630–2639.CrossRefPubMedGoogle Scholar
  41. Singh, K.K., Kumar, M., Kumar, P., Gupta, M.K., Jha, D.K., Kumari, S., Roy, B.K., and Kumar, S. 2012. “Free” copper: a new endogenous chemical mediator of inflammation in birds. Biol. Trace. Elem. Res. 145, 338–348.CrossRefPubMedGoogle Scholar
  42. Song, J., Li, Y.L., and Hu, C.H. 2013. Effects of copper-exchanged montmorillonite, as alternative to antibiotic, on diarrhea, intestinal permeability and proinflammatory cytokine of weanling pigs. Appl. Clay Sci. 77-78, 52–55.CrossRefGoogle Scholar
  43. Turnlund, J.R., Jacob, R.A., Keen, C.L., Strain, J.J., Kelley, D.S., Domek, J.M., Keyes, W.R., Ensunsa, J.L., Lykkesfeldt, J., and Coulter, J. 2004. Long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men. Am. J. Clin. Nutr. 79, 1037–1044.PubMedGoogle Scholar
  44. Veum, T.L., Carlson, M.S., Wu, C.W., Bollinger, D.W., and Ellersieck, M.R. 2004. Copper proteinate in weanling pig diets for enhancing growth performance and reducing fecal copper excretion compared with copper sulfate. J. Anim. Sci. 82, 1062–1070.CrossRefPubMedGoogle Scholar
  45. Walter, R.M., Uriuhare, J.Y., Olin, K.L., Oster, M.H., Anawalt, B.D., Critchfield, J.W., and Keen, C.L. 1991. Copper, zinc, manganese, and magnesium status and complications of diabetes-mellitus. Diabetes Care 14, 1050–1056.CrossRefPubMedGoogle Scholar
  46. Wang, M.Q., Du, Y.J., Wang, C., Tao, W.J., He, Y.D., and Li, H. 2012. Effects of copper-loaded chitosan nanoparticles on intestinal microflora and morphology in weaned piglets. Biol. Trace. Elem. Res. 149, 184–189.CrossRefPubMedGoogle Scholar
  47. Wu, X.Z., Zhang, T.T., Guo, J.G., Liu, Z., Yang, F.H., and Gao, X.H. 2015. Copper bioavailability, blood parameters, and nutrient balance in mink. J. Anim. Sci. 93, 176–184.CrossRefPubMedGoogle Scholar
  48. Xia, M.S., Hu, C.H., and Xu, Z.R. 2005. Effects of copper bearing montmorillonite on the growth performance, intestinal microflora and morphology of weanling pigs. Anim. Feed. Sci. Technol. 118, 307–317.CrossRefGoogle Scholar
  49. Xue, J., Li, H., Deng, X., Ma, Z., Fu, Q., and Ma, S. 2015. L-Menthone confers antidepressant-like effects in an unpredictable chronic mild stress mouse model via NLRP3 inflammasome-mediated inflammatory cytokines and central neurotransmitters. Pharmacol. Biochem. Behav. 134, 42–48.CrossRefPubMedGoogle Scholar
  50. Yang, T.H., Yuan, T.H., Hwang, Y.H., Lian, I.B., Meng, M., and Su, C.C. 2015. Increased inflammation in rheumatoid arthritis patients living where farm soils contain high levels of copper. J. Formos. Med. Assoc. 15, 1–6.Google Scholar
  51. Yu, S.G., Vandenberg, G.J., and Beynen, A.C. 1995. Copper-metabolism in analbuminemic rats fed a high-copper diet. Comp. Biochem. Phys. A 110, 259–266.CrossRefGoogle Scholar
  52. Zhou, W., Kornegay, E.T., van Laar, H., Swinkels, J.W., Wong, E.A., and Lindemann, M.D. 1994. The role of feed consumption and feed efficiency in copper-stimulated growth. J. Anim. Sci. 72, 2385–2394.CrossRefPubMedGoogle Scholar

Copyright information

© The Microbiological Society of Korea and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Feng Zhang
    • 1
  • Weijiang Zheng
    • 1
  • Rong Guo
    • 1
  • Wen Yao
    • 1
    • 2
  1. 1.College of Animal Science and TechnologyNanjing Agricultural UniversityNanjingP. R. China
  2. 2.Key Lab of Animal Physiology and BiochemistryMinistry of AgricultureNanjingP. R. China

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