Susceptibility of Bovine Naked 2- and 4-Cell Embryos and Hatched Blastocysts Produced In Vitro to Infection with Noncytopathogenic Bovine Viral Diarrhea Virus.

Abstract

ABSTRACT. To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2-and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10 6 TCID 50 /ml of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2-and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2-and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus.-KEY WORDS: bovine embryo, hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.J. Vet. Med. Sci. 61(8): 943-945, 1999 culture. The blastocysts that hatched naturally from the ZP were collected, and used. These naked embryos were exposed to NCP BVDV No. 12 strain, 10 6 TCID50/ml. After 1 hr of culture at 37°C under 5% CO2, the embryos were washed by transferring them through six changes of wash medium. The washed naked embryos were cultured for 24 hr in a 0.1-ml drop covered with mineral oil (E. R. Squibb & Sons, Inc., U.S.A.) in a small Petri dish (35 × 10 mm: Nunc, Denmark). Eighty 2-and 4-cell embryos were transferred to the development medium. Forty embryos were immediately collected as 0-hr 2-and 4-cell embryos, and development medium also was collected as 0-hr 2-and 4-cell medium. Using the same method, eight hatched blastocysts were transferred to the development medium and four blastocysts were immediately collected as 0-hr hatched blastocysts, and the development medium was collected as 0-hr hatched blastocyst medium. Fresh development medium was added to culture the remaining 2-and 4-cell embryos and hatched blastocysts. After 24 hr of culture, the media were collected, and the embryos were washed by transferring them through three changes of wash medium, then suspended in 1 ml of minimum essential medium supplemented with 0.2% bovine serum albumin and antibiotics, and sonicated for 30 sec. Each control group unexposed to virus also was prepared for observation of 2-and 4-cell embryos and hatched blastocyst development, and virus isolation from these embryos. The titers of NCP BVDV in the medium and embryo samples were determined by a micro-interference test using bovine fetal muscular (BFM) cells and the cytopathogenic Nose strain [6]. The virus titers were calculated and expressed as TCID50/0.1 ml and TCID50/all embryos for medium and embryo samples, respectively. The BFM cells and FBS used were confirmed to be free of BVDV. The experiments were repeated four times.F...