Reduction of infectious epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos by non-specific protease

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.