title: Quality controls for bovine viral diarrhea virus-free IVF embryos.
abstract: Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.
authors.affiliation: College of Veterinary Medicine, Auburn University, AL 36849-5519, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Stringfellow
authors.forename: K P
authors.initials: KP
authors.lastname: Riddell
authors.forename: P K
authors.initials: PK
authors.lastname: Galik
authors.forename: P
authors.initials: P
authors.lastname: Damiani
authors.forename: M D
authors.initials: MD
authors.lastname: Bishop
authors.forename: J C
authors.initials: JC
authors.lastname: Wright
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D001755:Blastocyst / Q000821:virology; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000517:prevention & control / Q000635:transmission*; D002417:Cattle / Q000196:embryology* / Q000821:virology; D002970:Cleavage Stage, Ovum; D003470:Culture Media; D046508:Culture Techniques; D001908:Diarrhea Viruses, Bovine Viral / Q000276:immunology / Q000302:isolation & purification*; D004624:Embryo Transfer / Q000662:veterinary; D004622:Embryo, Mammalian / Q000502:physiology / Q000821:virology*; D005314:Embryonic and Fetal Development; D005307:Fertilization in Vitro / Q000662:veterinary*; D009028:Morula / Q000821:virology; D009865:Oocytes / Q000821:virology; D011786:Quality Control; D014357:Trypsin / Q000494:pharmacology
chemical_list: D000914:Antibodies, Viral; D003470:Culture Media; D014357:Trypsin