274 ASSESSMENT OF TRYPSIN AND ANTIBIOTIC TREATMENT EFFECTIVENESS IN IN VITRO-MATURED OOCYTES EXPERIMENTALLY EXPOSED TO LEPTOSPIRA INTERROGANS SEROVAR GRIPPOTYPHOSA

Abstract

Techniques of production and transfer of embryos is safe as long as it follows the control regulations defined by the manual of the International Embryo Transfer Society (IETS) for treating oocytes with trypsin, antibiotics, and TCM-199 medium. The aim of this work was to evaluate the effectiveness of treatments, established by IETS, in bovine oocytes experimentally exposed to Leptospira interrogans serovar Grippotyphosa and to assist implementation of quality control standards on in vitro embryo production. The oocytes were obtained through follicular puncture of ovaries derived from the slaughterhouse. They were selected and divided into 4 groups: the control group and groups exposed to 5, 10, and 30 μL of an L. interrogans strain at 4.7 × 10⁵ μL^-1; and 4 additional groups exposed to the same concentrations of another L. interrogans strain at 6.3 × 10⁵ μL^-1, in which the gene for virulence is not expressed. The groups were kept in maturation medium (TCM-199 medium, 0.5 (iLof FSH, 50IU mL^-1 hCG, 1μL mL^-1 17-βiestradiol) and incubated at 38°C, 5% CO₂, and 95% humidity for 24 h. All the groups were separately subjected to the treatments with antibiotic, trypsin, and TCM-199 medium after maturation. The treatment involved 10 drops (each 200 μL), with 8 drops of TCM-199 medium and 2 drops of antibiotic (penicillin 10000 IU mL^-1 and streptomycin 10 mg mL^-1) or trypsin 0.25%; exposed to trypsin and antibiotic for 120 s. For the sequential washes, all drops contained TCM-199 medium. The analysis for presence of the pathogen by dark-field optical microscopy showed that in the groups exposed to L. interrogans and subjected to antibiotic washes, the effectiveness was 50% (100/200) for the group exposed to 5 μL, 40% (80/200) for that exposed to 10 μL, and 22.5% (45/200) for that exposed to 30 μL. We found the same results after the trypsin washes. After the washings with TCM-199 medium, the groups infected with 5 and 10 μL presented 100% of effectiveness; however, for the group infected with 30 μL, the washings were not effective. For the groups exposed to L. interrogans that did not express virulence, after the washings with antibiotics as well as with trypsin, the results showed no effectiveness in all of them (n = 200). Yet, after washings with TCM-199, the group exposed to 5 μL showed 28.5% (57/200) of effectiveness, whereas in those exposed to 10 and 30 μL, the medium washes were not effective. Complementary studies are being made with ultramicrotome cut and polymerase chain reaction for more reliable conclusions, to confirm the results. With such results, we conclude that the quality control regulations established by IETS for IVP could be reviewed and possibly redefined, because the effectiveness of the treatment may depend not only on the pathogen species, but also its virulence as well as its concentration and the action of the treatments on the type of pathogen.

We thank Vitrocel/Embriolife for supporting the laboratory.