Cryopreservation of Immature Porcine Oocytes Using Open Pulled Straw Vitrification

Abstract

The aims of this study were to investigate the meiotic competence of immature porcine oocytes following open pulled straw (OPS) vitrification and warming and also to test the efficiency of the pretreatment of immature oocytes with CB prior to OPS vitrification. Experiment 1, cumulus oocyte complexes (COCs) were randomly assigned to OPS vitrification, cryoprotectant (CPA) toxicity test and non-CPA/non-vitrified controls. Experiment 2, the COCs were treated with 7.5 μg/ml CB for 30 min. The oocytes were either immediately fixed or further incubated in a CB-free medium for 5 h, non-CB treated COCs served as controls. Experiment 3, CB-pretreated COCs were vitrified. The COCs exposed to CB and vitrification solutions and non-CPA, non-CB treated oocytes served as controls. In all cases, all the COCs were matured in vitro for 40-44 h. After IVM, the oocytes were examined for the stages of nuclear maturation. Vitrifying and warming significantly reduced the capability of the immature porcine oocytes to resume and reach metaphase (M II) (21.7%), compared unfavorably with the control (81.8%, p<0.05). This, however, was not the result from the cryoprotactants per se, since the number of CPA-treated oocytes reaching MII stage did not significantly differ from the control (76% vs. 81.8%, p>0.05). While cytochalasin B depolymerized actin cytoskeleton, this effect was completely reversible during 5 h of culture. Pretreatment with CB before the vitrification of immature porcine oocytes significantly yielded greater MII rates compared with non-CB oocytes (34.9% vs. 21.7%). In conclusion, porcine immature oocytes can be cryopreserved by OPS vitrification but it adversely affects the meiotic competence. Cytochalasin B reversibly depolymerizes actin microfilaments and improves the cryopreservability of porcine immature oocytes.