155 EMBRYONIC APOPTOSIS AFTER BTV-8 INFECTION IN BOVINE HATCHED IN VITRO PRODUCED BLASTOCYSTS

The recent blue tongue virus serotype-8 (BTV-8) epidemic in central Western Europe has been associated with field fertility problems (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). Previous research clearly showed that in vitro produced bovine hatched blastocysts are susceptible for BTV-8 infection (Vandaele et al. 2010 Reprod. Fertil. Dev. 22, 254 abst.). The aim of the present study was to investigate the effect of a BTV-8 infection on the occurrence of apoptosis in embryos in order to gain a clear insight into the role BTV-8 might play in early embryonic death. Immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h post-insemination and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum (tested negative for BTV antibodies) at 38.5°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. At 8.5 dpi, 4 to 8 hatched embryos were placed in 800 μL of minimum essential medium (MEM), containing 103.8 to 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/3, VAR, Brussels, Belgium) and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air. Two groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After inoculation, embryos were washed according to IETS guidelines with the exception that they were not ZP-I and cultured in new SOF. At 48 and 72 h post-inoculation (hpi), half of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 and apoptosis with a double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France) in combination with TUNEL (Roche Diagnostics, Mannheim, Germany). All mock-inoculated embryos were negative for BTV-8 virus antigen. At 48 and 72 hpi, respectively, 45 and 38% of the embryos were BTV-8 positive in all embryonic cells, and all remaining embryos had at least some BTV-8 blastomeres. The overall apoptotic cell ratio in infected embryos (17.9 ± 1.14% at 48 hpi and 15.3 ± 1.16% at 72 hpi) was significantly higher than in noninfected, mock-inoculated embryos (10.4 ± 0.57% at 48 hpi and 4.3 ± 0.30% at 72 hpi) (P < 0.01). Furthermore, total cell number was substantially lower in infected embryos (103 ± 14.2 at 48 hpi and 120 ± 17.5 at 72 hpi) compared with noninfected, mock-inoculated embryos (258 ± 47.4 at 48 hpi and 348 ± 64.1 at 72 hpi) (P < 0.05). This study indicates that embryonic apoptosis after BTV-8 infection results in embryonic arrest and early embryonic death and thus might be involved in herd fertility problems during a BTV epidemic. The first author is supported by Research Foundation Flanders (Grant number G.0210.09).