Study of osteopontin protein from canine seminal plasma

• Grant number: 13/25831-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2014
• End date: May 31, 2015
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Fabiana Ferreira de Souza
• Grantee: Leticia de Andrade Ramos
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

In view of the concern about the stray dogs population control, the search for a antigen to produce a contraceptive vaccine has been constant. Thus, the intention of this study will be to identify the osteopontin, a protein related to fertility in different species, in dogs seminal plasma by the technic of western-blot and based on this information submit the marked sample to mass spectrometry, in order to sequence the amino acid residues. Furthermore, the objective is also performing the immunolocalization of this protein at the canine spermatic cell. For this purpose, will be used seminal plasma of 5 dogs, with fertility confirmed by the history of recent coverage. The semen will be collected by digital manipulation of the penis, the sperm quality will be evaluated and the seminal plasma separated by two centrifugations (800xg, 10 minutes at ambient temperature and 10.000xg, 30 minutes, at 4ºC). The total protein concentration will be measured by the Bradford method and the samples will submitted to one-dimensional electrophoresis and western-blot using anti-OPN. The marked bands at the western-blot will be cropped and the amino acids sequencing will be made by mass spectrometry, in a partnership with the Università degli Studi di Bari, Italy. It is expected to identify the homology between the bovine osteopontin and the marked protein by anti-OPN at the dog seminal plasma sample and localize this protein at the dogs sperm.