Evaluation of virus decontamination techniques for porcine embryos produced in vitro.
The objective of this study was to explore approaches to decontaminate embryos either contaminated naturally or under experimental conditions with different viruses. Embryos were obtained from in vitro maturation and fertilisation of porcine oocytes. After 7 days of development, morula and blastocyst stages were exposed for 1 h to the following viruses: encephalomyocarditis virus (EMCV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV) at an infectivity of 100 TCID50/mL. Embryos samples were treated with different washing procedures, which all included the following standard washing solutions: PBS+0.4% BSA (five times for 10 s), Hank's+0.25% trypsin (two times for 60-90 or 120-150 s, or one time of 5 min), Hank's+0.1 mg/mL DNase 1+20 U/mL RNase One (one time of 30 min) and PBS+0.4% BSA again (five times for 10s). Two new approaches were used to improve trypsin treatment, 0.1% hyaluronidase (one time for 5 min) instead of trypsin and a pre-incubation with oviductal cells. Therefore, in the first experiment, oocytes received standard maturation treatments and in the second, they were also co-incubated with oviductal cells for the last 3 h of maturation. The effectiveness of the different washing techniques in removing viruses was evaluated by polymerase chain reaction (PCR) analysis. In the first experiment, trypsin treatment did not eliminate PRRSV, PPV, PCV, and EMCV from contaminated embryos. Surprisingly, treatment with hyaluronidase eliminated all tested viruses. In the second experiment, all viruses tested were removed from the oocytes following the different enzymatic treatments. In conclusion, in vitro embryo decontamination was more effective following exposure to oviductal secretions and hyaluronidase eliminated more virions than trypsin in washing techniques.
Bureau
Mariève
M
Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Ste-Foy, Qué., Canada G1K 7P4.
Dea
Serge
S
Sirard
Marc-André
MA
D000818:Animals; D017925:Circovirus / Q000235:genetics / Q000302:isolation & purification; D003666:Decontamination / Q000379:methods*; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000302:isolation & purification; D005307:Fertilization in Vitro / Q000662:veterinary*; D006821:Hyaluronoglucosaminidase; D028323:Parvovirus, Porcine / Q000235:genetics / Q000302:isolation & purification; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000302:isolation & purification; D013552:Swine / Q000196:embryology* / Q000821:virology*; D014357:Trypsin
D006821:Hyaluronoglucosaminidase; D014357:Trypsin