Three experiments were conducted to investigate the effect of 1 -, 2-and 7-step saturation with cryoprotectant on vitrification of germinal vesicle porcine oocytes to M-II stage. In Experiment I non-cooled cumulus-oocyte complexes (COCs) were equilibrated in ethylene glycol (EG) using a single-step or step-wise protocol, EG was removed by single-step or step-wise procedure with all manipulations being performed at 42°C. In Experiment II non-cooled COCs were equilibrated in ethylene glycol (EG)

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... sing the single-step or step-wise protocol with further EG removal by the single-step or step-wise procedure and pretreatment with Cytochalasin B. In experiment III, performed taking into account the results of Experiments I and II, a total of 177 COCs were divided into three treatment and one control groups. All treatment COCs were vitrified after pretreatment with Cytochalasin B (5|ig/ml at 38.5°C for 10-15 min), direct plunging into liquid nitrogen, thawing for 5 sec in a waterbath at 50°C, removal of the cryoprotectant by exposure to 1M, 0.75M, 0.5M, 0.25M for 4, 2, 2, and 2 min respectively at 40-42°C. Group 1 COCs (n=52) were cryopreserved after exposition to 40% EG for 4 min. Group 2 COCs (n = 47) were exposed to 25% EG for 5 min and then to 40% EG for 30 sec prior to vitrification. Group 3 COCs (n = 48) were equilibrated over 13 min using a series of EG concentrations 5, 10, 15, 20,25, 30, and 40% EG for 5,2,2,2, 1, 0.5, and 0.5 min, respectively. Group 4 COCs (n =30) were control oocytes (not exposed to EG and not frozen). After thawing and passage through the sucrose series (treatment) or without treatment (control) COCs were cultured for 48 h in TCM-199 with 10% heat-inactivated follicular fluid and lug/ml FSH in 5% C0 2 at 39°C. The developmental capacity (progression to M-II stage) as assessed by aceto-orcein staining was 0,4.2, 29.2, and 86.6%, respectively (P, 2 < 0.01; P M 34 2 4 <0.001).