194 THE EFFECT OF OVIDUCTAL INCUBATION OF OOCYTES AND EMBRYOS ON ADHERENCE OF BOVINE VIRAL DIARRHEA VIRUS

Abstract

The zona pellucida (ZP) plays a major role as a protective shell against infection of embryonic cells and as a carrier of infectious agents in the spread of livestock diseases through embryo transfer practices. It has been demonstrated that pathogenic agents are more likely to adhere to the surface of ZP of IVF embryos than to that of in vivo fertilized embryos. It has been suggested that divergent conditions for the production of these two types of embryos may lead to changes in their morphology and to differences in the interaction of the ZP with pathogens. The objective of this study was to investigate whether or not experimentally induced changes in hardening ZP (HZP) affect adherence of BVDV to the ZP of oocytes and embryos produced in vitro. To induce HZP, the oocytes or IVF embryos in groups of 30–50 were incubated in the ligated, ampullar part of the oviduct at 38°C in 5%CO₂, 5%O₂ and 90% nitrogen for 5 h. Following incubation, a proportion of oocytes/embryos was exposed to 1% pronase to determine HZP (the time of ZP lysis), while the remaining oocytes and embryos were incubated with 10⁶ TCID₅₀/mL of either noncytopathic (NY-1) or cytopathic (NADL) strain of BVDV at 38°C for 3 h. Subsequently, oocytes and embryos were washed according to the method recommended by IETS and then tested for the presence of BVDV (virus isolation and PCR tests). At the end of experiments the oviductal tissues were tested by PCR and proven free of BVDV. For immature and matured oocytes and embryos not exposed to the oviduct, the ZP dissolution times were 3.6 ± 0.24 (mean ± SEM, n = 20), 3.8 ± 0.24, and 4.0 ± 1.24 min, respectively (Chi-square test; P > 0.05). Corresponding times for those incubated in the oviduct were 393 ± 47, 431.0 ± 50, and 467 ± 61 min, respectively (P > 0.05). There was no difference between the number of virus-positive oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). Lower, but not significant, differences in percentages of samples associated with the infectious cytopathic strain of BVDV as compared to a noncytopathic strain were detected (P > 0.05). It was concluded that the modification in proteolytic resistance properties of ZP during in vitro oviductal incubation did not influence the adherence of BVDV to ZP of oocytes or IVF embryos. Further studies are warranted to determine why IVF embryos are more prone to the adherence of pathogenic agents than are in vivo fertilized embryos.