Vitrification of GV-porcine oocytes by 1-, 2-, and 7-step saturation in ethylene glycol
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Department of Animal Reproduction, Institute for Animal Science, RO.Kulinichi, 312120 Kharkov, Ukraine
Department of Animal Biology, Valencia University, Dr. Moliner 50, 46100 Burjassot, Catalonia
Department of Human Reproduction, Institute for Problems of Cryobiology and Cryomedicine, Perejaslavs'ka 23, Kharkov, Ukraine
Publication date: 1998-03-25
J. Anim. Feed Sci. 1998;7(2):219–230
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) using 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 µg/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 1 µg/ml FSH in 5% CO 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 (P1-2 < 0.01; P 2-3, 3-4, 2-4 <0.001).
Ultrastructure of Centrifuged Bovine Oocyte-Cumulus Complexes after Pre-treatment with Cytoskeletal Relaxant
V. Isachenko, J. L. Alabart, H. W. Michelmann, N. Bezugly, E. Isachenko, C. Soler, F. Nawroth
Anatomia, Histologia, Embryologia