Resumo

Frozen bovine semen used in the in vitro fertilization (IVF) process can be a potential source of microorganisms that can prevent or disturb embryo development and cause issues with the sanitary certification for bovine embryo commercialization and export. Therefore,the aim of this work is to introduce a novel tool for the fast identification of the pathogens on the frozen semen based on the mass spectra of their ribosomal proteins analyzed by matrix-assisted desorption/ionization-mass spectrometry (MALDI-MS).Thirty bovine semen samples, which were aliquots of commercial sealed straws daily used in the commercial IVF routine at In Vitro Brasil Ltd were used for this work. Fifty microliters of semen were incubated in 10 ml of brain heart infusion broth (BHI) for 24 h at 37°C.If turbidity was observed, the bacterial cultures were submitted to bacterial extraction and mass spectrometric analysis according to Barreiro et al. (2010). The mass spectra were obtained using an AUTOFLEX MALDI TOF/TOF and were analyzed with the database library MALDI Biotyper 3.0 software (Bruker Daltonik, Germany) at default settings. For each sample, the result was given by means of a log score with a maximum value of 3.0. In this study, only scores higher than 2.0 were considered, which provide confident species identification. The bacteria identified were Citrobacter freundii, Stenotrophomonas maltophilia, Enterobacter cloacae complex, Candida parapsilosis,and Enterococcus mundtii. Note that all the identified bacteria consistently match with the most common contaminants reported in literature for bovine frozen semen(A. Bielanskia et al.2003). The capability of the technique to identify the bacteria without the ribosomal extraction, i.e. of bacteria pellets diluted in water and acetonitrile was successful for the pellet of S. maltophilia, C. freundii and E. cloacae complex with scores higher than 2.3, indicating a very high probability of the identification of the bacterial genus and the species. This can be explained by considering the capability of the mass spectrometric matrix to lyse the membrane of the bacteria and directly extract and then ionize the ribosomal proteins. In order to exclude the presence of a mixing of bacteria in the pellet the colonies were properly isolated. The results matched with the ones obtained before the isolation. In order to confirm the MALDI MS identification, the isolated bacterial from the bovine semen were also submitted to sequencing of region 16SrRNA. In conclusion,MALDI-MS technique was successfully applied for the identification of pathogens in the bovine semen used in the in vitro fertilization process. This robust and fast approach is able to detect early contaminations and allows to prevent economical losses in bovine IVF process.