Viral contamination of embryos cryopreserved in liquid nitrogen

Despite the worldwide application of embryo-freezing technology as the mean s of preserving germplasm of mammalian species, there is no information ava ilable on the possible transmission of infectious agents to cryopreserved e mbryos via contaminated liquid nitrogen (LN). Recently, it has been reporte d that new methods of cryopreservation which employ ultrarapid freezing or vitrification require direct contact between the freezing medium containing oocytes or embryos and liquid phase nitrogen (LPN). As models for human an d animal viral pathogens three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV), and bovine immunodeficiency virus (BIV) , were employed to study the potential for their transmission by experiment ally contaminated LN to embryos frozen and stored in open freezing containe rs. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME2SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25 ml or modified ope n pulled straws or in plastic cryovials and then plunged into contaminated LPN. After 3-5 weeks of storage in LN, embryos were thawed and sequentially washed and only those with intact ZP were pooled together and tested in ba tches of three for viral contamination. From this pool of 83 batches, 13 of 61 (21.3%) batches exposed to BVDV and BHV-1 tested positive for viral ass ociation while all 22 batches exposed to BIV in unsealed containers tested negative. All control embryos vitrified in sealed cryovials and straws were free from viral contamination. (C) 2000 Academic Press.