Effect of Chitosan on Nitric Oxide Content and Inducible Nitric Oxide Synthase Activity in Serum and Expression of Inducible Nitric Oxide Synthase mRNA in Small Intestine of Broiler Chickens

  • Li, H.Y. (College of Animal Science and Veterinary Science, Inner Mongolia Agricultural University) ;
  • Yan, S.M. (College of Animal Science and Veterinary Science, Inner Mongolia Agricultural University) ;
  • Shi, B.L. (College of Animal Science and Veterinary Science, Inner Mongolia Agricultural University) ;
  • Guo, X.Y. (College of Animal Science and Veterinary Science, Inner Mongolia Agricultural University)
  • Received : 2008.12.26
  • Accepted : 2008.03.18
  • Published : 2009.07.01


The present study was conducted to determine the effects of chitosan on nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity in serum, and relative expression of iNOS mRNA in the duodenum, jejunum, and ileum of broiler chickens. A total of 240 one-day-old Arbor Acre mixed-sex broiler chickens were randomly allotted to six dietary treatments with five replicates in each treatment and eight chickens in each replicate. The broiler chickens in the six treatments were fed the basal diet supplemented with 0 (control), 0.05, 0.2, 0.5, 1.0 or 2.0 g/kg chitosan. The trial lasted for 42 days. The results showed that dietary chitosan enhanced NO content and iNOS activity in serum as well as iNOS mRNA expression in the duodenum and ileum of broiler chickens in a quadratic dose-dependent manner (p<0.05), and improved jejunum iNOS mRNA expression in a quadratic dose-dependent manner (p<0.10) with increasing addition of chitosan. Chicks fed a diet containing 0.5-1.0 g/kg chitosan had higher NO content and iNOS activity in serum as well as small-intestinal iNOS mRNA expression compared with birds given the control diet, but positive effects of chitosan tended to be suppressed when addition of chitosan in the diet was increased to 2.0 g/kg. These results implied that there was a threshold level of chitosan inclusion beyond which progressive reductions in serum NO content and small intestinal iNOS expression occured, and the regulation of chitosan on immune functions in chickens is probably associated with activated expression of iNOS and NO secretion.


Chitosan;Broiler Chickens;Nitric Oxide;Inducible Nitric Oxide Synthase;mRNA Expression


Supported by : National Natural Science Foundation


  1. Alspaugh, J. A. and D. L. Granger. 1991. Inhibition of cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis. Infect. Immun. 59:2291-2296
  2. Chou, T. C., E. Fu and E. C. Shen. 2003. Chitosan inhibits prostaglandin E2 formation and cyclooxyenase-2 induction in lipopolysaccharide-treated RAW 264.7 macrophages. Biochem. Biophys. Res. Commun. 308(2):403-407
  3. Deng, X. Z., X. J. Li, P. Liu, S. L.Yuan, J. J. Zang, S. Y. Li and X. S. Piao. 2008. Effect of chito-oligosaccharide supplementation on immunity in broiler chickens. Asian-Aust. J. Anim. Sci. 21(11):1651-1658
  4. James, S. L. and J. Glavin. 1989. Macrophage cytotoxicity against schistosomula of schistosoma mansoni involves argininedependent production of reactive nitrogen intermediates. J. Immunol. 143:4208-4212
  5. Liew, F. Y., S. Millott, C. Parkison, R. M. J. Palmer and S. Moncada. 1990. Macrophage killing of leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J. Immunol. 144:4794-4797
  6. Livak, K. J. and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(- Delta Delta C(T)) Method. Methods 25:402-408
  7. Nishimura, K., S. Nishimura, H. Seo, N. Nishi, S. Tokura and I. Azuma. 1986. Macrophage activation with multi-porous beads prepared from partially deacetylated chitin. J. Biomed. Mater. Res. 20(9):1359-1372
  8. Razdan, B. A. and D. Pettersson. 1996. Hypolipidemic, gastrointestinal and related responses of broiler chickens to chitosan of different viscosity. Br. J. Nutr. 76 (3):387-398
  9. Yin, Y. L., Z. R. Tang, Z. H. Sun, Z. Q. Liu, T. J. Li, R. L. Huang, Z. Ruan, Z. Y. Deng, B. Gao, L. X. Chen, G. Y. Wu and S. W. Kim. 2008. Effect of galacto-mannan-oligosaccharides or chitosan supplementation on cytoimmunity and humoral immunity in early-weaned piglets. Asian-Aust. J. Anim. Sci. 21(5):723-731
  10. Zaharoff, D. A., C. J. Rogers, K. W. Hance, J. Schlom and J. W. Greiner. 2007. Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Vaccine 25(11):2085-2094
  11. Lin, A. W., C. C Chang and C. C. Mccormick. 1996. Molecular cloning and expression of an avian macrophage nitric-oxide synthase cDNA and the analysis of the genomic 5'-anking region. J. Biol. Chem. 271:11911-11919
  12. Seferian, P. G. and M. L. Martinez. 2000. Immune stimulating activity of two new chitosan containing adjuvant formulations. Vaccine 19(6):661-668
  13. Morris, S. M. and T. R. Billiar. 1994. New insights into the regulation of inducible nitric oxide synthase. Am. J. Physiol. 266:829-839
  14. Zhang, H. J., Y. M. Guo, Y. Yang and J. M. Yuan. 2006. Dietary conjugated linoleic acid enhances spleen PPAR-gamma mRNA expression in broiler chicks. Br. Poult. Sci. 47(6):726-733
  15. Granger, D. L., J. B. Hibbs, J. R. Perfect and D. T. Durack. 1988. Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J. Clin. Investig. 81:1129-1136
  16. Nose, M., K. Terawaki, K. Oguri, Y. Ogihara, K. Yoshimatsu and K. Shimomura. 1998. Activation of macrophages by crude polysaccharide fractions obtained from shoots of glycyrrhiza glabra and hairy roots of glycyrrhiza uralensis in vitro. Biol. Pharm. Bull. 21(10):1110-1112
  17. Zhong, W., Y. M. Guo, J. M. Yuan and B. K. Zhang. 2008. Effect of dietary $\beta$-1,3/1,6-glucan supplementation on growth performance, immune response and plasma prostaglandin E2, growth hormone and ghrelin in weanling piglets. Asian-Aust. J. Anim. Sci. 21(5):707-714
  18. Moncada, S. and E. A. Higgs. 2006. Nitric oxide and the vascular endothelium. Handb Exp. Pharmacol. 176(1):213-254
  19. Wang, J. W., Y. Chen and M. Zhou. 1999. Effect of protein-bound polysaccharide on inhibition of LDL oxidation induced by macrophages and their relation with iNOS gene expression. Chinese. J. First Military Surgeon University 19(4):25-28
  20. Suk, Y. O. 2004. Interaction of breed-by-chitosan supplementation on growth and feed efficiency at different supplementing ages in broiler chickens. Asian-Aust. J. Anim. Sci. 17(12):1705-1711
  21. Moilanen, E., B. Whittle and S. Moncada. 1999. Nitric oxide as a factor in inflammation. In Inflammation: Basic principles and clinical correlates (Ed. J. I. Gallin, R. Snyderman). Philadelphia. Lippincott Williams & Wilkins 787-800
  22. Shimizu, T., K. Kinugawa, Y. Sugishita, K. Sugishita, K. Harada, H. Matsui, O. Kohmoto, T. Serizawa and T. Takahashi. 1998. Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes. Cardiovasc. Res. 38:405-413
  23. Huang, D. N., G. Hou and Q. F. Zhu. 1999. Effects of lentinan on nitric oxide production in mouse peritoneal macrophages and its mechanism. Chinese J. Basic Medical Science and Clinics 19(3):43-47
  24. Korhonen, R., A. Lahti, H. Kankaanranta and E. Moilanen. 2005. Nitric oxide production and signaling in inflammation. Curr. Drug. Targets. Inflamm. Allergy. 4:471-479
  25. Nathan, C. F. and J. B. Hibbs. 1991. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr. Opin. Immunol. 3:65-70
  26. Moncada, S., R. M. J. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43(2):109-142
  27. Peluso, G., O. Petillo, M. Ranieri, M. Santin, L. Ambrosio, D. Calabro, B. Avallone and G. Balsamo. 1994. Chitosanmediated stimulation of macrophage function. Biomaterials 15(15):1215-1220
  28. Tropea, A., F. Tiberi, F. Minici, M. Orlando, M. F. Gangale, F. Romani, F. Miceli, S. Catino, S. Mancuso, M. Sanguinetti, A. Lanzone and R. Apa. 2007. Ghrelin affects the release of luteolytic and luteotropic factors in human luteal cells. J. Clin. Endocrinol. Metab. 92(8):3239-3245
  29. Hou, G., D. N. Huang and Q. F. Zhu. 2000. Effects of grifola polysaccharide on nitric oxide production in mouse peritoneal macrophages and its mechanism. Chinese Geraeology 20(4):233-235
  30. Denis, M. 1991. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell. Immunol. 132:150-157
  31. Yu, Z., L. Zhao and H. Ke. 2004. Potential role of nuclear factorkappa B in the induction of nitric oxide and tumor necrosis factor-alpha by oligochitosan in macrophages. Int. Immunopharmacol. 4(2):193-200
  32. Razdan, A., D. Pettersson and J. Pettersson. 1997. Broiler chicken bodyweights, feed intakes, plasma lipid and small-intestinal bile acid concentrations in response to feeding of chitosan and pectin. Br. J. Nutr. 78(2):283-291
  33. Bredt, D. S. and S. H. Snyder. 1992. Nitric oxide, a novel neuronal messenger. Neuron. 8:3-11
  34. Porporatto, C., I. D. Bianco, C. M. Riera and S. G. Corres. 2003. Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages. J. Biochem. Biophys. Res. Commun. 304(2):266-272
  35. Shi, B. L., X. S. Piao, D. F. Li and Z. G. Chen. 2005. Alleviating action of chitosan on immunological stress in broilers. Chinese J. Anim. Sci. 41(6):6-8
  36. Shi, B. L., D. F. Li, X. S. Piao and S. M. Yan. 2005a. Effects of chitosan on growth performance and energy and protein utilisation in broiler chickens. Br. Poult. Sci. 46(4):516-519
  37. Xing, Z. and K. A. Schat. 2000. Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus. J. Virol. 74(8):3605-3612

Cited by

  1. Effects of Chitosan on Body Weight Gain, Growth Hormone and Intestinal Morphology in Weaned Pigs vol.26, pp.10, 1970,
  2. Effects of Chitosan on the Secretion of Cytokines and Expression of Inducible Nitric Oxide Synthase mRNA in Peritoneal Macrophages of Broiler Chicken vol.57, pp.4, 2014,
  3. Chitosan and its oligosaccharide derivatives (chito-oligosaccharides) as feed supplements in poultry and swine nutrition vol.99, pp.1, 2014,
  4. Effect of Chitosan Supplemented Diet on Survival, Growth, Feed Utilization, Body Composition & Histology of Sea Bass (Dicentrarchus labrax) vol.03, pp.04, 2015,
  5. Effect of Dietary Supplementation of Chitosan on Blood Biochemical Profile of Laying Hens vol.16, pp.9, 2017,
  6. Feeding broilers with thyme essential oil loaded in chitosan nanoparticles: an efficient strategy for successful delivery pp.1466-1799, 2018,