- Email: [email protected]
Prevention of bovine viral diarrhea virus transmission during in vitro fertilization including oocytes with follicular epithelial cells from a persistently infected heifer
253
Theriogenology
PREVENTION OF BOVINE VIRAL DIARRHEA VIRUS TRANSMISSION DURING IN VITRO FERTILIZATION INCLUDING OOCYTES WITH FOLLICULAR EPITHELIAL CELLS FROM A PERSISTENTLY INFECTED HEIFER T. Tsuboi, T. Imada, K. Katsuda and M. Eguchi National Institute of Animal Health, Tsukuba, lbaraki 305, Japan About 1% of cattle are persistently infected with bovine viral diarrhea virus (BVD V ) , with infection rates closer to 10% in some areas. Thus, there is a possibility that cattle persistently infected with BVDV will be slaughtered, and that oocytes with follicular epithelial (FE: cumulus) cells from the infected cattle will be used for in vitro fertilization (IVF). In this study, we attempted to determine the danger of using oocytes with FE cells from persistently infected cattle and we investigated prevention measures. Experiment (Exp.) 1 : One oocyte with FE cells derived from a heifer persistently infected with BVDV was mixed with 10 oocytes with FE cells from healthy cattle. Oocytes were matured, fertilized and cultured by previously described methods (Tsuboi et al., Vet. Microbial., 1996). The transmission of BVDV to normal oocytes with FE cells in the IVF system was examined. Exp.2 : 100 oocytes with FE cells from healthy cattle were exposed to non-cytopathogenic BVDV strain No.12 (106 TClD56/ml). Oocytes were cultured as in Exp.1. Fetal bovine serum (FBS) either without neutralizing antibody (negative group) or with a titer of 8 (positive group) were used in the IVF system. The effect of FBS on BVDV titer was determined. The survival of BVDV was investigated after culturing in medium only without both FE cells and embryos at 39°C up to 264 hr. In Exp.l,BVDV was isolated from development medium and FE cells of the mixture group, which were collected 9 days after adding sperm to matured oocytes (Table 1). No virus was isolated from embryos in 3 out of 4 replicates (Table 1). In the nonmixture group, no BVDV was isolated from the medium, FE cells and embryos (Table 1). Development into blastocysts was observed in both groups. In Exp.2, the BVDV titer of the maintenance medium of the negative group was 103.53 to 10693 TCID56/ 0.25ml and that of embryos was 10 2 53TCID59/0.25ml. However, in the positive group, the virus titer of maintenance medium and embryos showed a marked decrease compared to the negative group. BVDV was inactivated by 72 hr when cultured with medium alone. Table 1. BVDV isolation from development(D) Mixture qroup Rep#3 Rep#l Rep#2 2.75 D.medium 2.75a 3.75 (1.5 Embryos Xl .5 cl.5 2.25 FE cells 2.0 2.25 a) logTClD56/0.lml drop
medium, embryos and FE cells. Control qroup Rep#4 (Non-M. group) 3.0 -0.5 1.75 -0.5 2.5 -0.5
Results indicate that mixture of oocytes with FE cells from persistently infected cattle could result in virus transmission, but that using BVDV-positive FBS and removing FE cells were effective ways to prevent BVDV transmission,
Theriogenology
PREVENTION OF BOVINE VIRAL DIARRHEA VIRUS TRANSMISSION DURING IN VITRO FERTILIZATION INCLUDING OOCYTES WITH FOLLICULAR EPITHELIAL CELLS FROM A PERSISTENTLY INFECTED HEIFER T. Tsuboi, T. Imada, K. Katsuda and M. Eguchi National Institute of Animal Health, Tsukuba, lbaraki 305, Japan About 1% of cattle are persistently infected with bovine viral diarrhea virus (BVD V ) , with infection rates closer to 10% in some areas. Thus, there is a possibility that cattle persistently infected with BVDV will be slaughtered, and that oocytes with follicular epithelial (FE: cumulus) cells from the infected cattle will be used for in vitro fertilization (IVF). In this study, we attempted to determine the danger of using oocytes with FE cells from persistently infected cattle and we investigated prevention measures. Experiment (Exp.) 1 : One oocyte with FE cells derived from a heifer persistently infected with BVDV was mixed with 10 oocytes with FE cells from healthy cattle. Oocytes were matured, fertilized and cultured by previously described methods (Tsuboi et al., Vet. Microbial., 1996). The transmission of BVDV to normal oocytes with FE cells in the IVF system was examined. Exp.2 : 100 oocytes with FE cells from healthy cattle were exposed to non-cytopathogenic BVDV strain No.12 (106 TClD56/ml). Oocytes were cultured as in Exp.1. Fetal bovine serum (FBS) either without neutralizing antibody (negative group) or with a titer of 8 (positive group) were used in the IVF system. The effect of FBS on BVDV titer was determined. The survival of BVDV was investigated after culturing in medium only without both FE cells and embryos at 39°C up to 264 hr. In Exp.l,BVDV was isolated from development medium and FE cells of the mixture group, which were collected 9 days after adding sperm to matured oocytes (Table 1). No virus was isolated from embryos in 3 out of 4 replicates (Table 1). In the nonmixture group, no BVDV was isolated from the medium, FE cells and embryos (Table 1). Development into blastocysts was observed in both groups. In Exp.2, the BVDV titer of the maintenance medium of the negative group was 103.53 to 10693 TCID56/ 0.25ml and that of embryos was 10 2 53TCID59/0.25ml. However, in the positive group, the virus titer of maintenance medium and embryos showed a marked decrease compared to the negative group. BVDV was inactivated by 72 hr when cultured with medium alone. Table 1. BVDV isolation from development(D) Mixture qroup Rep#3 Rep#l Rep#2 2.75 D.medium 2.75a 3.75 (1.5 Embryos Xl .5 cl.5 2.25 FE cells 2.0 2.25 a) logTClD56/0.lml drop
medium, embryos and FE cells. Control qroup Rep#4 (Non-M. group) 3.0 -0.5 1.75 -0.5 2.5 -0.5
Results indicate that mixture of oocytes with FE cells from persistently infected cattle could result in virus transmission, but that using BVDV-positive FBS and removing FE cells were effective ways to prevent BVDV transmission,