title: Risk assessment of porcine reproductive and respiratory syndrome virus (PRRSV) transmission via somatic cell nuclear transfer (SCNT) embryo production using oocytes from commercial abattoirs.
abstract: Somatic cell nuclear transfer (SCNT) technology has become a powerful tool for reproductive biology to preserve and propagate valuable genetics for livestock. Embryo production through SCNT involves enucleation of the oocyte and insertion of a somatic donor cell into the oocyte. These procedures lead to a few small openings on the zona pellucida that may elevate risk of viral infection for the produced SCNT embryos. The oocytes used for SCNT are mainly obtained from abattoirs where viral contamination is almost inevitable. Therefore, a systematic evaluation of risk of disease transmission through SCNT embryo production is necessary prior large scale implementation of this technology in the livestock industry. The objective of the current study was to evaluate the risk of disease transmission via SCNT embryo production and transfer by testing for the presence of porcine reproductive and respiratory syndrome virus (PRRSV) throughout the process of SCNT embryo production. The presence of PRRSV in each step of SCNT embryo production, from donor cells to pre-implantation SCNT embryo culture, was carefully examined using a real-time PCR assay with a sensitivity of five copies per-reaction. All 114 donor cell lines derived from pig skin tissue over a period of 7 years in our facility tested negative for PRRSV. Out of the 68 pooled follicular fluid samples collected from 736 ovaries, only four (5.9%) were positive indicating a small amount of viral molecule present in the oocyte donor population. All 801 Day 7 SCNT embryos produced in four separate trials and over 11,571 washed oocytes obtained in 67 batches over 10 months tested negative. These oocytes were collected from multiple abattoirs processing animals from areas with high density of pig population and correspond to a donor population of over 5828 individuals. These results indicate that the oocytes from abattoirs were free of PRRSV infection and therefore could be safely used for in vitro embryo production. Additionally, the established SCNT embryo production system, including donor cell testing, oocytes decontamination, and pathogen free embryo reconstruction and culturing, bears no risk of PRRSV transmission.
authors.affiliation: Viagen, Inc., 12357-A Riata Trace Parkway, Austin, TX 78727, USA. Keqin.gregg@gmail.com
authors.forename: K
authors.initials: K
authors.lastname: Gregg
authors.forename: T
authors.initials: T
authors.lastname: Xiang
authors.forename: S S
authors.initials: SS
authors.lastname: Arenivas
authors.forename: E
authors.initials: E
authors.lastname: Hwang
authors.forename: F
authors.initials: F
authors.lastname: Arenivas
authors.forename: S-H
authors.initials: SH
authors.lastname: Chen
authors.forename: S
authors.initials: S
authors.lastname: Walker
authors.forename: A
authors.initials: A
authors.lastname: Picou
authors.forename: I
authors.initials: I
authors.lastname: Polejaeva
mesh_terms: D000818:Animals; D005260:Female; D015571:Follicular Fluid / Q000821:virology; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000821:virology*; D019318:Porcine Reproductive and Respiratory Syndrome / Q000635:transmission*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification*; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D018570:Risk Assessment; D012680:Sensitivity and Specificity; D013552:Swine
chemical_list: D012313:RNA