Metalloproteomic study of hepatic tissue and plasma of broilers supplemented with copper and manganese

Abstract

Copper and manganese are considered essential microminerals for broiler chickens and are responsible for a wide range of functions critical to maintaining animal health and performance. The main sources of supplementation of these minerals are from inorganic salts, known to have low bioavailability due to the occurrence of antagonisms in the gastrointestinal tract. On the other hand, hydroxychlorides are a potential source of copper and manganese supplementation, due to the greater proportion of covalent bonds that reduce the chances of negative interactions with other nutrients. Metalloproteomic tools can be used to evaluate the bioavailability of different sources of supplementation, as well as elucidation of the biological roles, essentiality and toxicity of copper and manganese in broiler chickens. In this sense, the general objective of the work proposal will be to identify possible biomarkers associated with the deficiency, excess and bioavailability of copper and manganese, from two sources of supplementation (sulfates and hydroxychlorides), using proteomic/metalloproteomic strategies in samples of hepatic tissue and plasma of broiler chickens. The treatments will be determined from the 2x3+2 factorial scheme, being two levels of copper (15 and 150 ppm) and three levels of manganese (40, 80 and 120 ppm), in which the hydroxychlorides will be used as a source of supplementation, and two more control groups will be used where the sulfates with levels according to the recommendation (15 ppm Cu and 80 ppm Mn) and above the recommendation (150 ppm Cu and 120 ppm Mn) for broilers will be used. Blood samples will be collected at 42 and 43 days of age to obtain plasma and hepatic tissue respectively. After the collection, the procedures of extraction of the protein fraction of the samples to obtain the proteome by two-dimensional electrophoresis (2D-PAGE) of polyacrylamide gel will be performed. Subsequently, procedures for the extraction of copper and manganese from protein spots by Graphite Furnace Atomic Absorption Spectrometry (GFAAS) will be performed, and the procedures for preparation of the spots to characterize the proteins associated with these minerals by mass spectrometry in sequence with electrospray ionization (ESI-MS/MS). Then, we will analyze the expression of these proteins and bioinformatics studies using software Blast2GO, Cytoscap and DAVID to identify functional levels, metabolic pathways and protein-protein interactions. The identification of biomarkers responsive to concentrations of copper to manganese in the hepatic tissue and plasma of broiler chickens can yield more reliable responses on the bioavailability, essentiality and toxicity of these minerals. (AU)