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 vitr o produced bovine embryos than to zonae of in vivo derived embryos. Current ly, the International Embryo Transfer Society recommends that all in vivo d erived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contra st to in vivo derived embryos, this method is not effective for disinfectin g in vitro produced embryos. Our hypothesis was that a more potent, non-spe cific 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 defin ed in vitro conditions. Zygotes were washed according to the procedure outl ined by the International Embryo Transfer Society, replacing trypsin with t he experimental protease. Experimental incubations were with 0.1% (4 units/ ml) protease for 0, 30, 45, 60 and 75 s intervals. Embryos were able to wit hstand exposure to this enzymatic treatment for only 45 s before their deve lopmental potential was significantly reduced; 60 s exposure was detrimenta l (P < 0.05). Oocytes were exposed to epizootic hemorrhagic disease virus s erotype 2 (EHDV-2, 10(6) TCID50/ml) during in vitro maturation. Resulting z ygotes were washed according to the International Embryo Transfer Society p rocedure and either exposed to trypsin or protease. Exposure to EHDV-2 prev ented cumulus expansion and markedly reduced embryonic development (P < 0.0 5). There were no differences in development among virus exposed groups rec eiving no treatment or treatment with trypsin or protease. However. proport ions of infected embryos were reduced after protease treatment versus posit ive controls and trypsin treated embryos. (C) 2001 Elsevier Science B.V. Al l rights reserved.