TIMING OF BLASTOCOELIC CAVITY RE-EXPANSION REPRESENTS A QUALITY INDEX OF IN VITRO PRODUCED OVINE BLASTOCYSTS.

There is general consensus that the quality of in vitro-produced embryos is inferior to that of their in vivo counterparts and that along with other factors this may be responsible for the poorer cryosurvival and the lower pregnancy rates reported for these embryos. The feasibility to accurately select viable embryos would be valuable for improving pregnancy rates and avoiding futile transfer attempts. The aim of our study was to assess whether in vitro-produced ovine embryo quality could be determined by the timing blastocoelic cavity re-expansion after vitrification, thawing, and in vitro culture. Embryos were produced in vitro from ovine ovaries collected at the local slaughterhouse following a standard protocol developed for ovine oocytes, as previously described (Berlinguer et al. 2005 Reprod. Fertil. Dev. 17, 201). Blastocysts were then vitrified/warmed according to a simple method (Leoni et al. 2002 Cryobiology 45, 204-212) and cultured in vitro in TCM-199 supplemented with 10% FCS for 72 h in 5% CO2 in air at 39°C. Vitrified embryos were divided into two groups: (A) expanded within 8 h of in vitro culture after warming; (B) expanded during 8 to 16 h of in vitro culture after warming. Of the 338 vitrified/warmed embryos, 173 (51.1%) showed a re-expanded blastocoelic cavity after 8 h of in vitro culture, whereas 58 (17.1%) re-expanded during 8 to 16 h of in vitro culture. We also analyzed by semiquantitative RT-PCR a panel of genes expressed during pre-implantation embryo development (glucose transporter, HPS-10, e-caderin, poly(A)polimerase, and ubiquitine); our results showed that these genes, apart from ubiquitine which showed no difference in the two groups, were more expressed in Group A blastocysts compared to Group B. Group A blastocysts showed higher hatching rates (67.6%) than Group B blastocysts (43.1%; P < 0.01). The total cell number calculated for the hatched blastocysts after staining with Hoechst 33342 was significantly higher in Group A (140.7 ± 8.3, n = 42) than Group B (102.2 ± 8.4, n = 27; P < 0.01). Pregnancy rate (detected by ultrasonography 30 days after embryo transfer) after laparoscopic embryo transfer to 12 synchronized recipient Sarda sheep was 40% (6/15) in Group A and 13.4% (2/15) in Group B. The results indicated that timing of blastocoelic cavity re-expansion after vitrification/warming and in vitro culture can be considered as a reliable index of in vitro-produced ovine embryo quality, and in addition, a better prediction for improved survival rate after transfer to synchronized recipients.