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KMID : 0900920000240010089
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Korean journal of Animal Reproduction
2000 Volume.24 No. 1 p.89 ~ p.95
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Optimization of One-Step Dilution Method of Vitrified Bovine IVM/IVF/IVC Blastocysts
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Lee K.-S.
Kim E.-Y.
Nam H.-K.
Park S.-Y.
Park E.-M.
Yoon S.-H.
Park S.-P.
Chung K.-S.
Lim J.-H.
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Abstract
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This study was to establish an effective dilution technique in a vitrification of bovine blastocysts for the field trial. For vitrification, blastocysts were exposed in glycerol (G) and ethylene glycol (EG) mixture in m-DPBS supplemented with 10% FBS. Blastocysts were first exposed to 10% (v/v) G for 5 min, and subsequently were transferred to 10% G plus 20% EG (v/v) for 5 min. Finally, embryos were transferred to 25% G plus 25% EG (v/v) for 30 see and were placed in nitrogen vapor for 3 min, and then were plunged into LN_2. At thawing, the straw containing blastocysts was placed in air for 10 sec, and then plunged into a water bath at 25^{circ}C until all ice had disappeared. They were placed in 25^{circ}C and 36^{circ}c water according to treatment group for different time. Also, in vitro survival was assessed by the re-expansion and hatched rates at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) In the survival rates of vitrified bovine blastocysts according to different dilution time at thawing, the data of 1 min group (86.6, 56.6%) were higher than those of other treatment groups (2 min; 93.5, 35.4%, 2.5 min; 76.9, 30.7%, 3 min; 88.8, 36.1% and 3.5 min; 83.7, 8.1%). 2) When the in vitro survival of vitrified groups according to different developmental stage was examined at 48 h after thawing using 1 min dilution method, the hatching rates of fast developed embryos (expanded blastocyst: 81.3%: early hatching blastocyst: 86.2%) were higher than that of delayed developed one (early blastocyst: 46.6%). 3) In addition, when the in vitro survival of vitrified groups according to different embryo age was compared, the hatched rates at 48 h after thawing of Day 7 (66.6%) and Day 8 embryos (60.0%) were significantly higher than that of Day 9 embryos (22.7%) (P＜0.05). These results demonstrate that vitrified bovine IVM/IVF/IVC blastocysts can be successfully survived in vitro using one-step dilution (1 min) method.
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KEYWORD
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Bovine blastocyst, Vitrification, One-step dilution, In vitro survival
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