Method for vitrification and thawing of oocyte of canine and frozen-thawed oocyte produced using the same

Patent metadata

Field	Value
Publication number	US-12121024-B2
Application number	US-201916616488-A
Country	US
Kind code	B2
Filing date	Jul 18, 2019
Priority date	Apr 19, 2019
Publication date	Oct 22, 2024
Grant date	Oct 22, 2024

Abstract

The present invention relates to a method for vitrification and thawing of oocytes of animals for somatic cell cloning. More specifically, the present disclosure relates to a method for vitrification and thawing of canine oocytes, and to thus produced frozen-thawed oocytes. In a conventional approach of the vitrification-frozen oocyte production for the dog, an estrous cycle may not coincide with an experimental schedule. However, the method for vitrification and thawing of the canine oocyte according to the present disclosure and the resulting frozen-thawed oocyte allows an experimental schedule to coincide with the estrous cycle, resulting in high nuclear transfer and fertilization effects.

First claim

The invention claimed is:

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.

2. The method of claim 1 , wherein the method further comprises a step of thawing the vitrification-frozen oocyte (step 3), after step 2-7,
wherein the step of thawing the vitrification-frozen oocyte (step 3) includes steps in the following sequence:
a step of preparing a thawing solution, a dilution solution, two washing solutions, and a recovery medium (step 3-1),
a step of heating the thawing solution to 35 to 40° C. in an incubator (step 3-2);
a step of heating the recovery medium to 35 to 40° C. in the incubator (step 3-3);
a step of withdrawing the oocyte freezing tool from the liquid nitrogen, heating the oocyte freezing tool, inputting the oocyte freezing tool into the thawing solution, separating the mature oocyte from the oocyte freezing tool, and recovering the separated mature oocyte (step 3-4);
a step of transferring the separated mature oocyte into the dilution solution equilibrated to a room temperature to form a treated mature ooctye (step 3-5);
a step of sequentially transferring the treated mature oocyte into each of the two washing solutions to form a washed mature oocyte, wherein each of the two washing solutions is pre-equilibrated to a room temperature state (step 3-6); and
a step of transferring the washed mature oocyte to the heated recovery medium for recovery culture and then culturing the washed mature oocyte in the recovery medium (step 3-7),
wherein,
the thawing solution contains 0.5 to 1.5 M of sucrose, 10 to 30% dextran serum supplement, and a HEPES butter solution containing 25 to 45 μg/ml of gentamicin sulfate,
the dilution solution contains 0.3 to 0.7 M of sucrose, 10 to 30% dextran serum supplement, and a HEPES butter solution containing 25 to 45 μm/ml of gentamicin sulface,
the washing solution contains 15 to 25% dextran serum supplement and a HEPES butter solution containing 25 to 45 μg/ml gentamicin sulfate, and
the recovery medium includes a mixture of 0.5 to 2 parts by weight of sodium pyruvate, 0.5 to 2 parts weight of gentamicin sulfate, 2 to 10 parts by weight of fetal bovine serum (FBS), and 100 parts by weight of a HEPES butter solution.

Assignees

• Mkbiotech Co Ltd
• Iac In Nat Univ Chungnam

Inventors

• Kim Min Kyu
• Nam Yoon Seok
• Park Kang Sun
• Ji Gook Bin
• Lee Ji Hye

Classifications

• A01N1/12

  Chemical aspects of preservation · CPC title

• A01N1/162 Primary

  Temperature processes, e.g. following predefined temperature changes over time · CPC title

• A01N1/147

  Carriers for immersion in cryogenic fluid for slow freezing or vitrification · CPC title

• A01N1/122

  Preservation or perfusion media · CPC title

• A01N1/125 Primary

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title