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Effect of Sucrose Concentration on Survival After Frozen-thawed of Bovine IVF Blastocysts in Ethylene Glycol Based Freezing Medium for Slow-Cooling

소 체외수정란의 Slow Freezing을 위해서 Ethylene Glycol 동결보호제에 Sucrose 첨가 농도에 의한 동결효율

  • 조상래 (축산연구소) ;
  • 김현종 (축산연구소) ;
  • 최창용 (축산연구소) ;
  • 진현주 (축산연구소) ;
  • 손동수 (축산연구소) ;
  • 최선호 (축산연구소)
  • Published : 2006.12.31

Abstract

The present study was undertaken to investigate the post-thawed survivability of bovine embryo depending on different dose of ethylene glycol and sucrose. Ovaries were collected at local slaughterhouse and the cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured at 39°C in an atmosphere of 5% CO2 incubator. For conventional slow-freezing, d 7 or 8 expanded blastocysts were collected. Embryos were equilibrated in 1.5 M and 1.8 M ethylene glycol(EG) with 0.1 M and 0.3 M sucrose in Dulbecco's phosphate-buffered saline(D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25ml-straw and placed directly into cooling chamber of programmable freezer precooled to 󰠏7°C, after 2 min, the straw was seeded, maintained at 󰠏7°C for 8 min, and then cooled to 󰠏35°C at 0.3°C/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 sec and exposed to 37°C water for 20 sec. Straws were then removed from 37°C water. Rates of blastocyst survive and hatching were evaluated at 24 to 72 h post-warming. No difference of the survivability was shown between 1.5 M and 1.8 M EG (71 and 70%, respectively). Addition of 0.1 M sucrose to 1.5 M and 1.8 M ethylene glycol in the freezing solution did not differ significantly embryo survival (74 and 77%, respectively), whereas survival rates was higher(89%) in freezing solution contained 0.3M sucrose to 1.8M EG compared with 0.3M sucrose to 1.5M EG group(71%). However, there was no difference in the overall total cell number between the two groups (122±1.8 vs 131±1.4, respectively). In conclusion, the results suggest that 0.3 M sucrose in 1.8 M EG may be optimal condition for freezing and thawing methods with in vitro produced embryos and may be applied to on-farm conditions for embryo transfer.

Keywords

Bovine;In vitro;Slow-freezing;Ethylene glycol;Sucrose

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