تأثیر سطوح مختلف اسید آمینه متیونین بر عملکرد سیستم ایمنی و بیان ژن اینترفرون گاما در جوجه‌های گوشتی

نوع مقاله : علمی پژوهشی- ژنتیک و اصلاح دام و طیور

نویسندگان

1 موسسه تحقیقات علوم دامی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی. کرج. ایران.

2 گروه فیزیولوژی ورزشی، دانشکده علوم ورزشی، دانشگاه مازندران، بابلسر، ایران.

3 گروه علوم دامی، دانشکده کشاورزی، دانشگاه جیرفت، جیرفت، ایران.

4 مؤسسه تحقیقات علوم دامی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی

5 گروه فیزیولوژی، دانشکده دامپزشکی، دانشگاه شیراز، ایران.

چکیده

متیونین اولین اسید آمینه محدود­­کننده­ای است که نقش مهمی در متابولیسم پروتئین و عملکرد سیستم ایمنی در جوجه­ها دارد. اینترفرون گاما (IFNg) یکی از اجزا گروه سایتوکاین­های ایمنی اختصاصی و عامل مهم فعال­کننده­ی ماکروفاژها    می­باشد. هدف از مطالعه حاضر، بررسی تأثیر سطوح مختلف اسید آمینه متیونین بر عملکرد سیستم ایمنی و بیان ژن IFNg در جوجه­های گوشتی سویه آرین می­باشد. به این­ منظور در این مطالعه با استفاده از480 قطعه جوجه در دوره رشد  (14-28روزگی) در قالب طرح کاملاً تصادفی با 6 سطح اسید آمینه متیونین (29/0، 36/0، 43/0، 50/0، 57/0 و 64/0)، 4 تکرار و 20 جوجه در هر تکرار انجام شد. شاخص­های مورد مطالعه شامل بررسی ایمنی همورال، اندازه­گیری سلول­های خونی و میزان بیان ژن IFNg بود. به منظور بررسی بیان ژن IFNg  ابتدا کل RNA از بافت کبد استخراج و پس از ساخت cDNA، میزان بیان ژن با استفاده از روش Real­-­time­ ­PCR اندازه­گیری شد. نتایج حاصل از بررسی بیان ژن IFNg در سطوح مختلف اسید آمینه متیونین اختلاف معنی­داری را بین تیمار­ها نشان داد (05/0p≤)، بطوریکه بیان ژن  IFNg با افزایش سطوح مصرف متیونین، از 29/0 درصد به 43/0 به طور معنی‌داری نسبت به گروه کنترل افزایش ولی تفاوت معنی­داری بین سطوح متیونین 5/0، 57/0 و 64/0درصد مشاهده نشد. شاید یکی از دلایل افزایش بیان ژن IFNg بکارگیری سطوح مناسب متیونین در تیمارهای آزمایشی و بهره­مندی از مزایای سطوح مناسب متیونین به منظور افزایش عملکرد سیستم ایمنی در پرنده باشد، هرچند که تحقیق حاضر در شرایط عادی پرورش و بدون چالش­ عوامل بیماری­زا انجام شده است. 

کلیدواژه‌ها


عنوان مقاله [English]

Effect of Different Levels of Methionine on Immune Function and IFN- Gene Expression in Broilers Chickens

نویسندگان [English]

  • Hamid Reza Seyedabadi 1
  • Khadijeh Nasiri 2
  • Zahra Roudbari 3
  • Seyed Abdoullah Hosseini 4
  • Aboulfazl Akbari 5
1 Assistance Professor of Animal Science Research Institute of Iran (ASRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
2 Department of Exercise Physiology, Faculty of Sport Science, University of Mazandaran, Babolsar, Iran
3 Department Animal Science, University of Jiroft, Jiroft, Iran
4 Animal Nutrition Department, Animal Science Research Institute, Karaj, Iran.
5 Department of Exercise Physiology, Faculty of Sport Science, University of Mazandaran, Babolsar, Iran
چکیده [English]

Introduction: Essential amino acids comprise 10 to 13% of the poultry diet. Methionine is the first limiting amino acid that plays important roles in protein metabolism and immune functions in chickens. Previous studies have shown that the appropriate level of methionine in the diet increases the growth and it is essential for enhancing the immune response. Methionine is also requirement to increase the function of the T cells produced from the thymus. Methionine has beneficial effects on the immune system and improves both humoral and cellular immune responses. Interferon-gamma (IFN-g) is one of the components of group-specific immune cytokines and an important activator of macrophages. IFN-g is known as cytokine which it is critical for innate and adaptive immunity against viral, some bacterial and protozoa infections. The aim of present study was to investigate effect of different diet levels of methionine on immune system and IFNγ gene expression in broiler chickens.
 
 Materials and Methods: This study was conducted in a completely randomized design with six experimental groups with 4 replicates and 20 observations in each replicate. The difference was in the levels of dietary methionine in the growth period, which included experimental groups 0.29, 0.36, 0.43, 0.51, 0.57 and 0.64%. The antibody produced against the sheep's red blood cell, white blood cell differential counts and the volume percentages of red blood cells were determined. In order to determine the IFNγ gene expression, the whole RNA was extracted from the liver tissue of different treatment chickens. Then, cDNA was synthesized and the expression of the IFNγ gene was evaluated using Real Time PCR. In this study, design of primers (GAPDH and IFNγ) was performed using primer premier software version 5 to evaluate IFNγ gene expression in broiler chickens. Real-time PCR was performed using SYBER Green qPCR Master Mixes (Thermo) in Lightcyclear 96 (Roche). Melting curve of IFNg and GAPDH gene productions were drawn using Real Time PCR for broiler chickens. The relative gene expression was quantified by the 2- ΔΔct method. The results were analyzed by GLM method of SAS software. Tukey post hoc test was used to compare the means of the experimental groups at the significant level of 0.05.
 
Results and Discussion: The results showed that response to sheep's red blood cell, immunoglobulin G, immunoglobulin M, white blood cells, heterophile, lymphocyte and heterophile to lymphocyte ratio were not affected by different levels of methionine, but the number of red blood cells was affected by different levels of methionine, so that the highest number of red blood cells associated to methionine level was 0.29% and the lowest value was 0.57%(p≤0.05). The result of the absorption measurement of the extracted RNA samples at a wavelength of 280/260 was in a range of 1.8 to 1.9 mm, indicating the desired quality of extracted RNA. The result of melting curve of Real Time PCR and PCR products on agarose gel showed that the IFNγ and GAPDH genes were amplified in the liver tissue. The observation of band at 259 bp for the IFNγ and at 264 bp for the GAPDH gene for all samples indicates the correctness of the test and the amplification of the desired fragments. The expression results showed that there was a significant increase in IFNγ gene expression with increasing methionine levels from 0.29% to 0.43% and higher levels (p≤0.05). However, there was no significant difference between the levels of methionine 0.43 to 0.64%. Regarding the fact that the present study was carried out under normal conditions without disease challenges, etc., different levels of methionine not effect on the immune system. IFN-γ gene is a type of cytokine. Cytokines do not exist as precursor molecules, and their production begins with transcription of the genes. This transcription activity is usually temporary and mRNA coding for cytokines is unstable and if the immune system is stimulated by the pathogen, Innate immune is the first hostile defense way. After detecting pathogens, host cell receptors such as Toll-like and nucleotide oligomers that include receptors are able to transmit a variety of signals, and subsequently cytokine gene expression networks begin to function until the innate immune responses begin.
 
Conclusion: The results showed that IFN-γ gene expression was significantly increased by increasing methionine levels from 0.29% upwards. Perhaps one of the reasons for increased IFN2 gene expression in this study is the application of appropriate methionine levels in experimental treatments and the benefits of appropriate methionine levels in the diet to enhance immune function in the bird, although the present study was conducted under normal growing conditions and without the challenge of pathogens.
 

کلیدواژه‌ها [English]

  • Broiler chickens
  • Gene expression
  • Immune system
  • IFNγ gene
  • Adams, S.C., Z. Xing, J. Li, and C. J. Cardona. 2009. Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells. Molecular immunology, 46:1744-1749.
  • Bateman, A., D. A. Roland and M. Bryant .2008. Optimal methionine + cysteine / lysine ratio for first cycle of egg production in commercial leghorns. International Journal of Poultry Science, 7: 932-939.
  • Bhanja, S. K., M. Sudhagar, A. Goel, N. Pandey, M. Mehra, S. K. Agarwal, and A. Mandal. 2014. Differential expression of growth and immunity related genes influenced by in ovo supplementation of amino acids in broiler chickens. Czech Journal of Animal Science, 59:399-408.
  • Chamruspollert, M., G. M. Pesti, and R. I. Bakalli. 2004. Influence of Temperature on the Arginine and Methionine Requirements of Young Broiler Chicks. The Journal of Applied PoultryResearch, 13:628-638.
  • De Souza Khatlab, A., A. P. Del Vesco, A. R. de Oliveira Neto, R. P. Fernandes, and E. Gasparino. Dietary supplementation with free methionine or methionine dipeptide mitigates intestinal oxidative stress induced by Eimeria spp. challenge in broiler chickens. Journal of Animal Science and Biotechnology, 10(1):58.
  • Grass, W. B., and H. S. Siegel. 1983. Evaluation of the heterophile/lymphocyte ratio as a measure of stress in chickens. Avian Disease, 27:972-979.
  • Kogut, M. H., L. Rothwell, and P. Kaiser. 2005. IFN-γ priming of chicken heterophils upregulates the expression of proinflammatory and Th1 cytokine mRNA following receptor-mediated phagocytosis of Salmonella enterica serovar enteritidis. Journal of Interferon & Cytokine Research, 25:73-81.
  • Lai, A., G. Dong, D. Song, T. Yang, and X. Zhang. 2018. Responses to dietary levels of methionine in broilers medicated or vaccinated against coccidia under Eimeria tenella-challenged condition. BMC Veterinary Research, 14:140.
  • Li, P., Y. L. Yin, D. Li, S. W. Kim, and G. Wu. 2007. Amino acids and immune function. British Journal of Nutrition, 98: 237-252.
  • Liu, Z., A. Bateman, M. Bryant, A. Abebe, and D. Roland. 2004. Estimation of bioavailability of DL-methionine hydroxy analogue relative to DL-methionine in layers with exponential and slope-ratio model. Poultry Science, 83:1580-1586.
  • Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 25: 402-408.
  • Maroufyan, E., A. Kasim, G. Yong Meng, M. Ebrahimi, L. Teck Chwen, P. Mehrbod, B. Kamalidehghan, and A. Soleimani Farjam. 2013. Effect of dietary combination of methionine and fish oil on cellular immunity and plasma fatty acids in infectious bursal disease challenged chickens. The Scientific World Journal, 2013(1):531397.
  • Métayer, S., I. Seiliez, A. Collin, S. Duchêne, Y. Mercier, P. A. Geraert, and S. Tesseraud. 2008. Mechanisms through which sulfur amino acids control protein metabolism and oxidative status. The Journal of nutritional biochemistry, 19:207-215.
  • Mirzaaghatabar, F., A. A. Saki, P. Zamani, H. Aliarabi, and H. R. Hemati Matin. 2011. Effect of different levels of diet methionine and metabolisable energy on broiler performance and immune system. Food and Agricultural Immunology, 22:93-103.
  • Nafisi Bahabadi, M., S. Dadgar, F. Lakzaei, Z. Mohajeri, and Abdolahi. 2016. The effect of subacute concentrations of Butachlor herbicide on some blood parameters in rainbow trout (Oncorhynchus mykiss). Iranian Scientific Fisheries Journal, 25:151-160. (In Persian)
  • Ohta, Y., and T. Ishibashi. 1995. Effect of dietary glycine on reduced performance by deficient and excessive methionine in broilers. Japanese Poultry Science, 32:81-89.
  • Schroder, K., P. J. Hertzog, T. Ravasi, and D. A. Hume. 2004. Interferonγ: an overview of signals, mechanisms and functions. Journal of leukocyte biology, 75:163-189.
  • Shini, S., X. Li, and W. L. Bryden. 2005. Methionine requirement and cell-mediated immunity in chicks. Asia Pacific journal of clinical nutrition, 14:
  • Wegmann, T., and O. Smithies. 1966. A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion, 6: 67-75.
  • Wu, B.Y., H. M. Cui, P. Xi, J. Fang, W. Cui, and X. D. Liu. 2012. Effect of methionine deficiency on the thymus and the subsets and proliferation of peripheral blood T-cell, and serum IL-2 contents in broilers. Journal of Integrative Agriculture, 11:1009-1019.
CAPTCHA Image