Canine Oocyte Vitrification and Thawing Method
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Solution Overview
Problem
Canine oocyte vitrification and thawing techniques are inefficient due to the unique breeding physiology of dogs, where immature oocytes mature and ovulate within 2-3 days, making timely oocyte collection challenging, and existing in vitro maturation systems are insufficient for mature oocyte vitrification, limiting somatic cell cloning efficiency.
Innovation Solution
A method involving the collection of mature canine oocytes, followed by a vitrification process using specific solutions (mixed liquid, equilibrium solution, and vitrification solution) to prevent ice crystal formation and ultra-fast freezing, and a thawing process using tailored solutions to enhance oocyte viability, including denuding, equilibration, and storage in liquid nitrogen, followed by controlled thawing and recovery culture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional freezing methods are used for canine oocytes, then ice crystal formation occurs causing cell membrane damage, but this leads to low oocyte viability after thawing
Solution Approach 1:
The patent applies vitrification, a phase transition technique where the oocyte is transformed from a liquid state to a glass-like solid state through rapid cooling in the presence of cryoprotectants (ethylene glycol and dimethyl sulfoxide). This prevents ice crystal formation by transitioning water directly to a glassy state without passing through the ice phase, thereby preserving cell membrane integrity and oocyte viability
Solution Approach 2:
The patent uses cryoprotectants (ethylene glycol and dimethyl sulfoxide) as intermediary substances that mediate the freezing process. These cryoprotectants replace water molecules and prevent ice crystal formation by interfering with the freezing process, acting as a protective intermediary between the oocyte and the harmful effects of conventional freezing
2Productivity
If fresh oocytes are used for somatic cell cloning, then high cloning efficiency is achieved, but this requires timely oocyte collection within 2-3 days of ovulation
Solution Approach 1:
The patent applies preliminary action by collecting and vitrifying oocytes in advance before they are needed for cloning experiments. The vitrified oocytes can be stored long-term and thawed on demand, allowing experimental schedules to be planned independently of the estrous cycle timing, thus eliminating the time loss associated with waiting for optimal collection windows
Solution Approach 2:
The patent changes the physical state parameter of the oocyte from fresh to vitrified through controlled freezing processes. This parameter change allows the oocyte to be preserved in a stable frozen state and later recovered with maintained viability, decoupling the cloning efficiency from the timing of oocyte collection
3Quantity of substance
If in vitro maturation systems are used for canine oocytes, then oocyte availability increases, but the existing systems are insufficient for mature oocyte vitrification
Solution Approach 1:
The patent replaces the mechanical/chemical maturation system with a vitrification-based preservation system. Instead of relying on in vitro maturation systems to produce mature oocytes, the patent collects mature oocytes and preserves them through vitrification, substituting the maturation approach with a preservation approach that maintains oocyte quality and availability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly increases oocyte viability and nuclear transfer culture effects, allowing for high-quality frozen-thawed oocytes that can synchronize with experimental schedules, even when estrous cycles do not coincide, and demonstrates high efficiency in somatic cell cloning applications.
Implementation Method 1
a process of performing a vitrification process of the collected oocyte
Implementation Method 2
ultra-fast freezing, and a thawing process using tailored solutions to enhance oocyte viability
Data Source
AI summary
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.