154 EVALUATION OF THE EFFECTIVENESS OF TREATMENT WITH TRYPSIN IN MURINES EMBRYOS EXPERIMENTALLY EXPOSED TO BOVINE HERPES VIRUS TYPE-1 (BoHV-1) BY THE NESTED-PCR TECHNIQUE

Abstract

The cryopreservation of bovine embryos has allowed an increase in their commercialization causing concern about the transmission of pathogens through the production and transfer of in vitro embryos. The International Society of Embryo Transfer (IETS) suggests the treatment of embryos with trypsin or antibiotics in alternating washes with culture medium in order to remove and/or inactivate infectious agents that may interfere with the final product. The aim of this study was to evaluate the effectiveness of trypsin treatment in the elimination and/or removal of bovine herpes virus type-1 (BoHV-1), Colorado strain, in murine embryos. Viral detection was made by n-PCR and cytopathic effect in Madin Darby bovine kidney (MDBK) cells. Six- to 8-wk-old female mice (Swiss) were superovulated and mated with fertile males of the same strain. After 24 h, the zygotes (n = 262) were divided into 3 groups: control group submitted to sequential wash (C_SW), the group exposed to the virus (30 μL; 10^6.5 virus mL^–1) and submitted to sequential wash (E_SW), and the group exposed to the virus and submitted to the trypsin treatment (E_TT). All the groups of zygotes and the last sequential wash drops were tested by nested-PCR and inoculated in MDBK cells to allow observation of cytopathic effects. All groups except for C_SW showed positive results for the nested-PCR for both zygotes and for the last wash drops. A cytopathic effect was observed in all groups except for C_SW, demonstrating the viability of the virus after treatment. These results demonstrated that trypsin treatment was not effective in eliminating or removal of BoHV-1. The need for such studies is fundamentally important when considering the potential risk of pathogen transmission by reproductive biotechnologies.