247 USING THE ACIDIC ORGANIC BUFFER MES TO CLEAN IVP BOVINE EMBRYOS FROM FOOT AND MOUTH DISEASE VIRUS

Abstract

The risk of transmitting foot and mouth disease virus (FMDV) by in vivo-produced embryos is extremely small. In vitro produced (IVP) bovine embryos carry a greater risk of transmitting FMDV. IVP Day 7 embryos, exposed to FMDV cannot be freed of the virus by washing (Marquant-LeGuienne et al. 1998 Theriogenology 50, 109–116). Similarly, IVP embryos exposed to FMDV during IVM and IVF cannot be freed of FMDV by washing (Jooste et al. 2003 Theriogenology 59, 443). The aim of this study was to test if bovine IVP embryos, exposed to FMDV during IVM and IVF, can be rendered free of infective FMDV by treating them with 2-(Morpholino)ethanesulfonic acid (MES) at a pH of 5.5. MES is an organic buffer with pKa 6.1 (MES, Sigma, South Africa, M2933). Four groups of 300 and two groups of 320 oocytes were obtained from ovaries from a local abattoir. Oocytes were matured, fertilized and cultured as described earlier (Jooste et al. 2003 Theriogenology 59, 443). Virus was added at a concentration of 2 × 10⁶ TCID₅₀ in all treatments. Treatment with MES was between 30 and 60 s in all cases. After exposure to MES, oocytes were washed 5 times in IVM, IVF, or IVC medium. In all groups, virus detection was attempted by PCR and on pig kidney cell monolayers. Group 1 was not exposed to MES and not spiked with FMDV (control). In Group 2, FMDV was added during IVM and IVF. FMDV detection was attempted after the denuded presumptive zygotes were exposed to MES. Group 3 was treated as group 2, except that presumptive zygotes in this group were cultured up to Day 7 before virus detection was attempted. In Group 4, COCs were treated with MES after IVM and viral detection attempted. Group 5 was treated as group 4, but COCs were subjected to IVF. FMDV was added again during IVF. Denuded presumptive zygotes were again treated with MES and viral detection was attempted. Group 6 was treated as group 5, except that virus detection was not attempted after denuding, but presumptive zygotes were cultured up to Day 7 before virus detection was attempted. No FMDV could be demonstrated in the control group. All denuded oocytes/embryos that were treated with MES for 30–60 s were negative for the presence of infective FMDV. FMDV could be detected in all COCs. Treatment with MES is ineffective in rendering COCs free from infective FMDV. Treatment with MES did not negatively affect the blastocyst rate (chi-square test, P < 0.05) in this trial. Blastocyst rates were 8.3% (28/300), 10.3% (31/300), and 9.3% (28/300) for Group 1 (control), Group 3, and Group 6, respectively; the low blastocyst rate was ascribed to the production of embryos in a non-designated IVF laboratory. It thus appears that FMDV is protected by the cumulus cells. It is not clear if the virus infects the cumulus cells per se or if it is protected in the intercellular spaces. It also appears that FMDV does not penetrate the zona pellucida and if the surface of the zona pellucida is exposed to an acidic environment, FMDV can be inactivated.