| Abstrakt: |
Introduction and Objective: Alpha-lipoic acid (α-LA) synthesized enzymatically in the mitochondrion from octanoic acid. Endogenous levels of α-lipoic acid have been reported in the micro molar range. Alpha-lipoic acid can easily pass through biological membranes due to its high lipophilicity and is converted into dihydro lipoic acid (DHLA) in cells. Alpha-lipoic acid and DHLA have been described as an ideal antioxidant couple, due to their very high protection ability against oxidative stress through multiple pathways. Alpha-lipoic acid and DHLA have metal chelating activity, acts as a coenzyme in glucose metabolism, and can produce other antioxidants such as ascorbate, vitamin E, and glutathione (GST). Alpha-lipoic acid has anti-inflammatory properties and its supplementation to broiler diets improves the expression of inflammation-related genes in the spleen. On the other hand, trivalent chromium has been recognized as an essential trace element for humans and other animals, which is part of the glucose tolerance factor (GTF). Chromium is essential for the metabolism of carbohydrates, proteins and lipids. Chromium may be present in the diet in the form of inorganic compounds or organic complexes. After absorption, chromium circulates in a free state and binds to transferrin or other plasma proteins (protein β-globulin). Trivalent chromium seems to play a role in the structure and expression of genetic information in animals. Chromium binding to nucleic acids is stronger than other metal ions. Chromium protects RNA against thermal denaturation. It is also clear that chromium is concentrated in the cell nucleus. By binding to chromatin, chromium participates in gene expression and increases the start sites and thus increases RNA synthesis. This increase is due to the induction of protein bound to the nucleus and the activation of nuclear chromatin, as a result, chromium effects gene function. Chromium supplementation has shown a positive effect on immune response of broiler chickens. The aim of this research was to investigate the simultaneous effect of dietary chromium propionate and alpha-lipoic acid supplementation on the expression of cytokines interleukin 1, 2, 4, and 10, as well as IFN-γ, TNF, and TGF β4 genes in the spleen tissue of broiler chickens. Material and Methods: For this purpose, an experiment was performed with 144 Ross 308 broiler chicks in complete randomized design (CRD) with a 3×2 factorial arrangement of treatments including six treatments, 3 replicates and 8 chickens in each replicate. There were 6 dietary treatments: three doses of chromium propionate (0, 750 and 1500 μg / kg) combined with two levels of alpha-lipoic acid (0 and 300 mg/kg) supplemented to the basal diets. At the end of the experimental period (42 days old), two chickens from each pen were randomly selected and killed. The sample was taken from the spleen and immediately frozen in liquid nitrogen and then stored at -80°C. The quantity and purity of the extracted RNA was checked using a Nanodrop device. In order to confirm the specific amplification of the desired genes in this study using primers, the PCR reaction was first performed. The expression of cytokine profile genes were quantified by quantitative real time PCR. In this way, 28S was used as a house-keeping gene to normalize the gene expression data. To analyze the obtained data, the method of difference in threshold degree changes (ΔΔCT) was used. The method of investigating gene expression changes in this research was the 2-ΔΔCT method (comparative threshold) and the ratio of gene expression (28s). Results: The results of electrophoresis confirmed the expression of genes in the spleen cells of broiler chickens and they appeared as one band on 2% agarose gel. Electrophoresis of polymerase chain reaction products showed fragments of 86, 51, 82, and 88bp for interleukin 1, 2, 4, and 10, respectively. No bands were formed on agarose gel for IFN-γ, TNF and TGF β4 genes. The results showed that alpha-lipoic acid at the level of 300 mg/kg significantly decreased interleukin 1 gene expression than the control group (0 mg/kg). Also, supplementing the basal diet with the chromium propionate at the level of 1500 μg/kg significantly increased interleukin 1 and 4 genes expression compared to the control group and the chromium propionate at the level of 750 μg/kg. While the addition of alpha lipoic acid and chromium propionate had no effect on the expression of interleukin 2 and 10 genes. Conclusion: Adding 300 mg/kg alpha lipoic acid to the diet can improve the immune response by reducing the expression of IL 1 gene in broilers. In addition, the addition of high amounts of chromium propionate (1500 μg) leads to a decrease in immunity (by increasing the expression of IL 1 and 4 genes). While the addition of chromium propionate up to the level of 750 μg/kg does not affect safety. Considering the results, it is recommended to add alpha lipoic acid at the level of 300 mg to the broiler diet. Addition of chromium propionate up to the level of 750 μg is fine if it has beneficial effects on yield and production characteristics. [ABSTRACT FROM AUTHOR] |
