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Identification and production of antibodies against sex-specific protein of bovine sperm

Grant number: 13/23351-3
Support Opportunities:Scholarships in Brazil - Doctorate
Effective date (Start): March 01, 2014
Effective date (End): August 31, 2017
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:José Antonio Dell'Aqua Junior
Grantee:Caroline Scott
Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil


The sexing of sperm , to obtain semen doses with a high percentage of gametes carrying the X or Y chromosome ,makes more relevant the role of artificial insemination on maximizing genetic progress between generations ; productivity and economic return in some livestock activities , in assisted reproduction in mammals , and genetic counseling of the human species . The objectives of this project are to study a more appropriate method for solubilization of plasma membrane proteins of spermatozoa ; separated by electrophoresis 2D - DIGE membrane proteins of sperm containing the X or Y chromosomes of cattle to select proteins sex - specific , identify the amino acid residues of the protein of interest by mass spectrometry , to produce sex-specific antibodies by immunization heterologous (chicken ), and verify the efficiency of binding of these antibodies to bovine sperm . Will be tested three protocols solubilization of proteins in sperm samples of conventional semen : 1 ) Nonidet P - 40 , 2 ) modified RIPA , 3) urea and thiourea , with or without the freezing and thawing of samples in liquid nitrogen and sonication . The protocol that separetes a greater number of spots is selected for sample preparation sexed semen . Will be used commercial doses of sexed sperm to separation by 2D - DIGE electrophoresis using probes Cy2 , Cy3 and Cy5 . The differential spots will be excised from the gels and mass spectrometry inoculation laying hens for the production of antibodies from the egg yolk. The antibodies will be purified by liquid chromatography and tested for binding efficiency at 50% of cells from a non-sexed semen using flow cytometry , which verifies the binding of anti- protein of interest. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
SCOTT, CAROLINE; DE SOUZA, FABIANA F.; ARISTIZABAL, VIVIANA H. V.; HETHRINGTON, LOUISE; KRISP, CHRISTOPH; MOLLOY, MARK; BAKER, MARK A.; DELL'AQUA JUNIOR, JOSE ANTONIO. Proteomic profile of sex-sorted bull sperm evaluated by SWATH-MS analysis. Animal Reproduction Science, v. 198, p. 121-128, . (13/23351-3, 15/25638-3, 15/25698-6)

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