Abstract: 268

ENZYMATIC REMOVAL OF EPIZOOTIC HEMORRHAGIC DISEASE VIRUS (EHDV-2) FROM IN VITRO PRODUCED BOVINE ZYGOTES.

MB Dinkins¹ , DE Stallknecht² , BG Brackett^{1 *}
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA ¹
Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, The University of Georgia, Athens, GA ²

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced (IVP) bovine embryos than to zonae of in vivo derived embryos. In contrast to in vivo embryos, trypsin treatment of IVP embryos is not effective in viral elimination from zonae. Our working hypothesis was that treatment of IVP embryos with protease (type XIV, Sigma) could effectively remove EHDV-2 without severely compromising embryonic viability. Bovine oocytes were matured (IVM), fertilized, and cultured in completely defined in vitro conditions. For virus exposure, oocytes were incubated with EHDV-2 (10⁶ TCID₅₀/ml) during IVM. Presumptive zygotes (PZs) were washed 5X in PBS + 0.3% polyvinyl alcohol (PVA), 1X in 0.1% protease XIV (4 units/ml) or 0.25% trypsin, and an additional 5X in PBS-PVA. Embryos exposed (as oocytes) to EHDV-2 were taken at the morula (M) stage for virus isolation. PZs were able to withstand protease treatment for 45 sec without compromising (p>0.05, chi-square) the proportions that cleaved (C, 67.7%) and became blastocysts (B, 18.5%) compared to controls (C, 77.3%; B, 25.8%). Exposure to EHDV-2 prevented cumulus expansion and reduced (p<0.05) development (C, 43.4%; M, 14.2%) when compared to controls (C, 76.8%; M, 47.0%). Proportions of infected embryos were reduced after a 45 sec protease treatment (20.0%) versus positive controls (63.6%) and a 60 sec trypsin treatment (66.7%). Data show EHDV-2 exposure during IVM to be detrimental to embryonic development. Also, IVP zygotes can withstand exposure to protease type XIV in conditions that reduced (p<0.05) infectious EHDV-2 associated with their zonae pellucidae. (Ares Advanced Technology, Inc., Randolph, MA, Genex/CRI, Ithaca, NY, and Shapiro Packing Co., Augusta, GA are gratefully acknowledged.)

    This abstract is being presented on Monday, August 2 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.