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Comparing and improving techniques for progesterone and estradiol assays using bovine plasma samples from previous experiments that evaluated the influence of these steroids on endometrial thickness

Grant number: 18/09266-7
Support type:Scholarships abroad - Research Internship - Master's degree
Effective date (Start): August 20, 2018
Effective date (End): February 19, 2019
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Roberto Sartori Filho
Grantee:Jéssica Cristina Lemos Motta
Supervisor abroad: Milo Wiltbank
Home Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
Local de pesquisa : University of Wisconsin-Madison (UW-Madison), United States  
Associated to the scholarship:17/16701-9 - Influence of circulating concentrations of estradiol and progesterone on endometrial thickness and LH and FSH concentrations after GnRH treatment, BP.MS

Abstract

Radioimmunoassay (RIA) is widely used for hormone concentrations analyses and it presents high specificity and sensitivity. However, this method requires the use of radioactive labels, which can be harmful to the operators and requires adequate disposal of the reagents in order to avoid radioactive pollution. Further, the reagents employed in this method present a short shelf life and its importation is a laborious, expensive and slow process. Therefore, it is relevant to study and to improve alternative methods that could replace the RIA when necessary. In order to do that, in the first experiment a total of 400 plasma samples will be analyzed for progesterone concentration by RIA (ImmuChem Progesterone CT, 07-270105, MP Biomedicals, Santa Ana, CA). Then half of the same samples (n = 200) will be submitted to chemiluminesce assay (Immulite commercial kit) and the other half to Enzyme-linked immunosorbent assay (ELISA). A second experiment will be developed attempting to solve another problem related to RIA. Although this assay is efficient in analyze hormone concentrations, for bovine estradiol there is no longer any reliable kit available. Thus, the following experiment will test a "hand-made" RIA for bovine estradiol analyses (n = 600 samples), using E2 125I (Cat 07-138226 - MP Biomedicals) and Anti-E2 (Cat 07-138216 MP), adapting previous protocol available in the literature. All samples that will be used in this project are from three previous experiments developed in Brazil and financed by FAPESP (2017/16701-9), using nonlactating Holstein cows as a model. Finally, simultaneously to the progesterone and estradiol assays, other samples (n = 600) from these experiments will be also analyzed for FSH and LH by RIA.