89EFFECT OF PRE-EQUILIBRATION PROCEDURES ON THE DEVELOPMENT POTENTIAL OF VITRIFIED BOVINE OOCYTES AFTER IVF

Abstract

This experiment was designed to improve oocyte developmental potential after droplet vitrification and IVF by different equilibration procedures as reported by Papis K. et al. (2000 Theriogenology 54, 651–658). Bovine oocyte-cumulus complexes were collected from slaughterhouse ovaries, and matured in vitro for 24 hours in TCM199 medium supplemented with 7.5% FBS, 0.5μgmL−1 FSH, 5μgmL−1 LH and 2μgmL−1 estrodial under 5% CO2 in humidified air at 39°C. Oocytes were then partially stripped of most expanded cumulus cells with only 3–5 inner layers left by a short exposure to 0.1% hyaluronidase and careful pipetting. Oocytes were randomly assigned to the following pre-equilibration treatments (39°C): Group 1, oocytes were pre-equilibrated in medium 1 consisting of holding medium (HEPES-buffered TCM199 supplemented with 20% FBS)+10% ethylene glycol (EG) (v/v)+10% dimethylsulphoxide (DMSO) (v/v) for 30–45s; Group 2, oocytes were pre-equilibrated in medium 1 for 3min; and Group 3, oocytes were pre-equilibrated in medium 2 (holding medium+3% EG+3% DMSO) for 20min. Oocytes were then equilibrated in 1.0mL of vitrification medium (holding medium+20% EG and 20% DMSO) as described by Vajta G et al. (1998 Mol. Reprod. Dev. 51, 53–58). Each droplet contained 8–10 oocytes in about 2μL vitrification medium and was dropped into liquid nitrogen immediately after 25–30s exposure to the vitrification solution. Vitrified oocytes were subsequently warmed by transfer into 3mL holding medium containing 0.25M sucrose (39°C). After standard BO IVF procedure, presumptive zygotes were cultured in CR1-aa medium supplemented with 6mgmL−1 BSA at 39°C in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 for eight days. Non-vitrified oocytes were used as a control. Data were analyzed with General Linear Model, SPSS 11.0. As shown in Table 1, there was a significant low survival rate (P<0.05) in Group 3 compared to other treatments. The cleavage rates of Groups 1 and 2 were significantly (P<0.05) higher than that of Group 3, but lower than that in the Control. Furthermore, blastocyst rate on Day 8 in Group 2 was significantly (P<0.05) higher than in the other two experimental groups, but still lower than the Control. This study suggests that the developmental capacity of vitrified oocytes can be improved by a duration of equilibration for 3 minutes in holding medium plus 10% EG and 10% DMSO via droplet vitrification. Table 1 In vitro development of vitrifield bovine oocytes following warming and IVF