US 12,121,024 B2
Method for vitrification and thawing of oocyte of canine and frozen-thawed oocyte produced using the same
Min Kyu Kim, Daejeon (KR); Yoon Seok Nam, Nonsan-si (KR); Kang sun Park, Sejong-si (KR); Gook Bin Ji, Incheon (KR); and Ji Hye Lee, Deajeon (KR)
Assigned to MKBIOTECH. CO. LTD, Daejeon (KR); and THE INDUSTRY & ACADEMIC COOPERATION IN CHUNGNAM NATIONAL UNIVERSITY (IAC), Daejeon (KR)
Appl. No. 16/616,488
Filed by MKBIOTECH. CO. LTD, Daejeon (KR); and THE INDUSTRY & ACADEMIC COOPERATION IN CHUNGNAM NATIONAL UNIVERSITY (IAC), Daejeon (KR)
PCT Filed Jul. 18, 2019, PCT No. PCT/KR2019/008861
§ 371(c)(1), (2) Date Nov. 24, 2019,
PCT Pub. No. WO2020/213789, PCT Pub. Date Oct. 22, 2020.
Claims priority of application No. 10-2019-0045801 (KR), filed on Apr. 19, 2019.
Prior Publication US 2021/0352888 A1, Nov. 18, 2021
Int. Cl. C12N 5/075 (2010.01); A01N 1/02 (2006.01)
CPC A01N 1/0284 (2013.01) [C12N 5/0609 (2013.01)] 2 Claims
 
1. A method for vitrification and thawing of an oocyte of a canine, the method comprising:
a process of collecting a mature oocyte from a canine to form a collected mature oocyte (step 1);
a process of performing a vitrification of the collected oocyte (step 2),
wherein the step of performing the vitrification includes steps in the following sequence:
a step of preparing a mixed liquid, an equilibrium solution, an vitrification solution (step 2-1);
a step of denuding a cumulus cell of the collected oocyte to form a denuded mature oocyte (step 2-2);
a step of adding the denuded mature oocyte into the mixed liquid to form a first mixture, wherein the mixed liquid is pre-equilibrated to a room temperature (step 2-3);
a step of sequential treating the first mixture to form a second mixture in the following order (step 2-4):
adding the equilibrium solution to the first mixture;
further adding the mixed liquid to the first mixture,
further adding the equilibrium solution to the first mixture, and
further adding the mixed liquid to the first mixture;
a step of further adding the equilibrium solution to the second mixture to form a third mixture (step 2-5);
a step of transferring the third mixture into the vitrification solution to form a fourth mixture (step 2-6); and
a step of placing the fourth mixture on an oocyte freezing tool and removing vitrification solution by a glass pipet therefrom and then immediately storing the occyte freezing tool in liquid nitrogen to form a vitrification-frozen mature oocyte (step 2-7),
wherein,
the mixed liquid includes a mixture of 300-1000 parts by weight of modified HTF medium-HEPES with gentamicin (mHTF) and 100 parts by weight of 10 to 30% serum substitute supplement (SSS),
the equilibrium solution contains 6.5 to 8.5% (v/v) ethylene glycol, 6.5 to 8.5% (v/v) dimethyl sulfoxide, 10 to 30% dextran serum supplement, and a HEPES butter solution containing 25 to 45 μg/ml of gentamicin sulface, and
the vitrification solution contains 5 to 25% (v/v) ethylene glycol, 5 to 25% (v/v) dimethyl sulfoxide, 10 to 30% dextran serum supplement, 0.3 to 0.6 M of sucrose, and a HEPES butter solution containing 25 to 45 μg/mL gentamicin sulfate.