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.
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