The Effect of Intra Amniotic Injection of Zinc-Methionine and Nano Zinc-Methionine on Metallothionein Gene Expression in the Broiler chickens

Document Type : Research Paper


1 Ph.D. Student, Department of Animal Science, Faculty of Agriculture Science, University of Guilan, Rasht, Iran

2 Associate Professor, Department of Animal Science, Faculty of Agriculture Science, University of Guilan, Rasht, Iran

3 Assistant Professor, Department of Animal Science, Faculty of Agriculture Science, University of Guilan, Rasht, Iran


The effects of intra amniotic injection of zinc-methionine and nano zinc-methionine on metallothionein mRNA expression of chicken’s liver and intestine were studied. Four hundred fertiled eggs (Ross 308 strain) were allocated to 4 treatments, 4 replicates/ treatment and 25 eggs per replicate using a CRD (Complete Random Design) design. Experimental treatments were: 1- no injection (negative control), 2- injection of 1 ml 0.9% saline solution (positive control), 3- injection of 1 ml 25% zinc- methionine solution and 4- injection of 1 ml 25% nano-zinc- methionine solution. On day 17 of incubation, eggs were injected using insulin syringes. Results showed that in Ovo injection lead to no significant effect on hatchability. The injection of zinc-methionine and nano zinc-methionine caused significant (P<0.01) increase in chicks weight at hatch, and a week after that. A similar result was obtained for liver and intestine samples. Groups treated with zinc-methionine and nano zinc-methionine showed significant (P<0.01) increase in metallothionein mRNA expression in liver and intestine and remained significant (P<0.01) at day 7 after hatch. Experimental treatments significantly (P<0.01) increased the liver and intestine metallothionein content at day 1 and 7 after hatch. Conclusion is that in ovo injection of zinc-methionine and nano zinc-methionine lead to increase body weight at first 3weeks of broiler rearing.


  1. Allan, A. K., Hawksworth, G. M., Woodhouse, L. & Beattie, J. (2000). Lymphocyte Metallothionein mRNA responds to marginal zinc intake in human volunteers. British Journal of Nutrition, 84(5), 747-756.
  2. Andrews, G. K. (2000). Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochemical. Pharmacology Journal, 59, 95-104.
  3. Cao, J., Henry, P. R., Guo, R., Holwerda, R. A., Toth, J. P., Littell. R.C., Miles, R.D. & Ammerman, C. B. (2000). Chemical characteristics and relative bioavailability of supplemental organic zinc sources for poultry and ruminant. Journal of Animal Science, 78(8), 2039-2054.
  4. Carpene, E., Andreani G. & Isani, G. (2007). Metallothionein functions and structural characteristics. Journal of Trace Elements in Medicine and Biology, 21(1), 35-39.
  5. Cousins, R. J. & Lee-Ambrose, L. M. (1992). Nuclear zinc uptake and interactions and metallothionein gene expression are influenced by dietary zinc in rats. Journal of Nutrition, 122, 56-64.
  6. Coyle, P., Philcox, J. C. & Rofe, A. M. (1995). Hepatic zinc in metallothionein-null mice following zinc challenge: in vivo and in vitro studies. Biochemical. Pharmacology Journal, 309, 25-31.
  7. Coyle, P., Philcox, J. C., Careya, L. C. & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular. Life Science, 59, 627-647.
  8. Chimienti, F., Devergnas, S., Favier, A. & Seve, M. (2004). Identification and Cloning of a -Cell–Specific Zinc Transporter, ZnT-8, Localized Into Insulin Secretory Granules. Diabetes, 53, 2330-2337.
  9. Davis, S. R. & Cousins R. J. (2000). Metallothionein Expression in Animals: A Physiological Perspective on Function. Journal of Nutrition, 130, 1085-1088.
  10. Desouky Mahmoud, M. A. (2012). Metallothionein is up-regulated in molluscan responses to cadmium, but not aluminum, exposure. The Journal of Basic and Applied Zoology, 65, 139-143.
  11. Dufner, J. B., Langmad, S. J., Wang, F., Eide, D. & Andrews, G. K. (2003). Structure, Function, and Regulation of a Subfamily of Mouse ZincTransporter. Genesis journal, 278(50), 50142-50150.
  12. Eide D. J. (2006). Zinc transporters and the cellular trafficking of zinc. Biochimical et BiophysicaActa, 1763, 711-722.
  13. Fernando, L. P., Wei, D. & Andrews, G. K. (1989) Structure and expression of chicken metallothionein. Journal of Nutrition, 119, 309-318.
  14. Fleet, J. C., Golemboski, K. A., Dietert, R. R., Andrews, G. K. & McCormick, C. C. (1990) Induction of hepatic metallothionein by intraperitoneal metal injection: an associated inflammatory response. American journal of Physiology Gastrointestinal and Liver Physiology, 258, 926-933.
  15. Gielda, L. M. & DiRita, V. J. (2012). Zinc Competition among the Intestinal Microbiota. American society for microbiology, 3(4), 217-223.
  16. Huang, Y. L., Lu, L., Li, S. F., Luo, X. G. & Liu, B. (2007). An Optimal Dietary Zinc Level of Broiler Chicks Fed a Corn-Soybean Meal Diet1. Poultry Science, 86, 2582-2589.
  17. Huang, Y. L., Lu, L., Li, S. F., Luo, X. G. & Liu, B. (2009). Relative bioavailabilites of organic zinc sources with different chelation strengths for broilers fed a conventional corn-soybean meal diet. Journal of Animal Science, 87, 2038-2046.
  18. Hudson, B. P., Fairchild, B. D. Wilson, J. L. Dozier, W. A. & Buhr, R. J. (2004). Breeder Age and Zinc Source in Broiler Breeder Hen Diets on Progeny Characteristics at Hatching. Journal of Applied PoultryResearch, 13, 55-64.
  19. Isani, G. & Carpenè, E. (2014). Metallothioneins, Unconventional Proteins from Unconventional Animals: A Long Journey from Nematodes to Mammals. Biomolecules, 4, 435-457.
  20. Kaler, P. & Prasad, R. (2007). Molecular cloning and functional characterization of novel zinc transporter rZip10 (Slc39a10) involved in zinc uptake across rat renal brush-border membrane. American journal of Physiology Renal Physiology, 292, 217-2299.
  21. Linde, A. R. & Garcia-Vazquez, E. (2006). A simple assay to quantify metallothionein helps to learn about bioindicators and environmental health. Biochemical Molcular Biology Education, 34(5), 360-363.
  22. Livak, K. J. & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta CT) method. Methods, 25: 402-208.
  23. Lowry, O.H., Rosebrogh, N.J., Farr, A.L. & Randall, R.J. (1951). Protein measurement with the Folin Phenol reagent. Journal of Biological Chemistry, 193(1), 265-275.
  24. Martinez, N. M., Hill, G. M., Link, J. E., Raney, N. E., Tempelmam, R. J. & Ernst, C. W. (2004). Pharmacological zinc and phytase supplementation enhance metallothionein abundance and protein concentration in newly weaned pigs. Journal of Nutrition, 134(3), 538-544.
  25. McCormick, C. C. (1984). Induction and Accumulation of Metallothionein in Liver and Pancreas of Chicks Given Oral Zinc: A Tissue Comparison. Journal of Nutrition, 114, 191-203.
  26. Miles, R. D. (2000). Trace Minerals and Avian Embryo Development. Ciência Animal Brasileira, 2(1), 1-10.
  27. NRC. (1994). National Research Council. Nutrient requirements of poultry. National Academy of Science Washington (DC), USA.
  28. Oh, S.H., Nakaue, H., Deagen, J. T., Whanger, P. D. & Arscott, G. H. (1979). Accumulation and Depletion of Zinc in Chick Tissue Metallothioneins. Journal of Nutrition, 109, 1720-17299.
  29. Otsuka, F. (2001). Molecular Mechanism of the Metallothionein Gene Expression Mediated by Metal-Responsive Transcription Factor 1. Journal of Health Science, 47(6), 513-519.
  30. Ramesh, S. A., Shin, R., Eide, D. J. & Schachtman, D. P. (2003). Differential Metal Selectivity and Gene Expression of Two Zinc Transporters from Rice. Plant Physiology, 133(1), 126-1344.
  31. Richards, J. D., Zhao, J., Harrell, R. J., Atwell, C. A. & Dibner, J. J. (2010). Trace Mineral Nutrition in Poultry and Swine.Asian-Australasian journal of Animal Science, 23(11), 1527-1534.
  32. Richards, J., Shirley, R., Winkelbauer, P., Atwell, C., Wuelling, C., Wehmeyer, M. & Buttin, P., (2007). Bioavailibity of zinc source in chicken determined via real time polymerase chain reaction (RT-PCR) assay for metallothnionein. 16th European symposium on poultry nutrition. In: Proceeding of 16th European symposium on poultry nutrition, 26-30 Aug. France, pp. 71-74.
  33. Richards, M. P. (1989). Recent Developments in Trace Element Metabolism and Function: l: Role of Metallothionein in Copper and Zinc Metabolism. Journal.of Nutrition, 119, 1062 -1070.
  34. Richards, M. P. (1997). Trace mineral metabolism in the avian embryo. Poultry Science, 76,152-164.
  35. Sakulsak, N. (2012). Metallothionein: an overview on its metal homeostatic regulation in mammals. International Journal of Morphoogyl, 30(3), 1007-1012.
  36. Sandoval, M., Henry, P. R., Ammerman, C. B., Miles, R. D. & Littell, R. C. (1997). Relative bioavailability of supplemental inorganic zinc sources for chicks. Journal of Animal Science, 75, 3195-3205.
  37. Sandoval, M., Henry, P. R. Luo, X. G. Littell, R. C. Miles, R. D. & Ammerman, C. B. (1998). Performance and Tissue Zinc and Metallothionein Accumulation in Chicks Fed a High Dietary Level of Zinc. Poultry Science, 77, 1354-1363.
  38. SAS. (2003). Version 9.1 SAS. STAT user’s guide. Statistical Analysis Systems Institute, Cary, NC, U.S.A.
  39. Shen, S. F., Wang, R. L. Lu, L., Li, S. F., Liu, S. B., Xie, J. J., Zhang, L. Y., Wang, M. L. & Luo, X.G. (2013). Effect of intravenously injected zinc on tissue zinc and metallothionein gene expression of broilers. British Poultry Science, 54(3), 381-390
  40. Suhy, D. A., Simon, K. D. Linzer, D. I. H. & O’Halloran, T. V. (1999). Metallothionein Is Part of a Zinc-scavenging Mechanism for Cell Survival under Conditions of Extreme Zinc Deprivation. The Journal of Biological Chemistry, 274(14), 9183-9192.
  41. Sullivan V. K., Burnett, F. R. & Cousins, R. J. (1998(. Metallothionein Expression Is Increased in Monocytes and Erythrocytes of Young Men during Zinc Supplementation. Journal of Nutrition, 128, 707-713.
  42. Tako, E., Ferket, P. R. & Uni, Z. (2005(. Changes in chicken intestinal zinc exporter mRNA expression and small intestinal functionality following intra-amniotic zinc-methionine administration. Journal of Nutritional Biochemistry, 16, 339-346.
  43. Trayhurn, P., Duncan, J. S., Wood, A. M. & Beattie, J. H. (2000). Metallothionein gene expression and secretion in white adipose tissue. American journal of Physiology Regulatory Integrative Comparative Physiology, 279, 2329-2335.
  44. Wei, D. Y. & Andrews, G. K. (1988). Molecular cloning of chicken metallothionein. Deduction of the complete amino acid sequence and analysis of expression using cloned cDNA. Nucleic Acids Research, 16(2), 537-555.