110 VITRIFICATION DEVICES AFFECT DEVELOPMENTAL COMPETENCE AND BIOCHEMICAL PROPERTIES OF IVM OVINE OOCYTES

Abstract

This study was designed to evaluate the effects of 3 different vitrification devices on the developmental ability and on the maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of ovine oocytes in vitro-matured. Cumulus-oocytes complex (COCs) were in vitro-matured in TCM-199 supplemented with 10% FCS, 10 ¼L/mL of FSH/LH, and 1 ¼g/mL estradiol at 39°C and 5% CO₂ atmosphere. For vitrification, oocytes were incubated in HEPES-buffered TCM-199 containing 20% FCS supplemented with DMSO (7.5%) and ethylene glycol (7.5%). After 3 min, oocytes were loaded into the same medium containing 0.5 m sucrose, 16.5% DMSO, and 16.5% EG, and then immersed into N₂ using open pull straw (OPS; Vajta et al. 1998 Mol. Reprod. Dev.), cryoloop (CL; Lane et al. 1999 Nat. Biotechnol.), or cryotop (CT; Kuwayama and Kato 2000 J. Assist. Reprod. Genet.). After warming a part of oocytes were fertilized and cultured in vitro up to blastocyst stage in standard conditions (Leoni et al. 2005 Anim. Reprod. Sci., in press). The fertilization (23.8%, 31.6%, and 36.8% vs. 61.5%) and blastocyst rates (0, 12.5, and 0% vs. 50.0%) were lower for oocytes vitrified in OPS, CL, and CT, compared with control group. Control and vitrified IVM oocytes were valued for MPF and MAPK activity at 0 and 2 h after warming. In both post-warming experimental groups, the MAPK activity did not differ from control group. Immediately post-warming, MPF activity was lower in the vitrified groups compared with control oocytes (P < 0.01). If 100% is assigned to MPF activity in the control oocytes, those in the OPS, CL, and CT groups were, respectively, 63.3%, 65.5%, and 26.2%. After warming and culture for 2 h in standard condition, the activity of MPF was restored to values similar to control oocytes (87.0 and 76.8%, respectively) in the OPS and CL groups, whereas it was at the similar value in the CT group. To evaluate if the lowered MPF activity could cause parthenogenetic activation, the vitrified-warmed oocytes were cultured in SOF + 2% oestrus sheep serum in 5% O₂, 5% CO₂; after 27 h of culture, the oocytes were fixed and stained with propidium iodide to evaluate chromatin configuration. Results showed significantly higher parthenogenetic activation rates in the CT group compared with OPS and CL groups (54.5% vs. 22.6 and 27.4%, respectively). Our results indicate that the success of cryopreservation of the ovine oocyte is still very limited. The use of different vitrification devices not only modifies the ability to survive cryopreservation and developmental competence of oocytes but is also associated with important molecular alterations in the warmed oocyte cytoplasm. This work was supported by Cofin 2003.