Cryoprotectant Sperm Staining Media for Viable Sex Sorting
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Solution Overview
Problem
Existing sperm sorting technologies face challenges in accurately differentiating X-chromosome and Y-chromosome bearing sperm due to small DNA content differences, geometric variations, and staining processes that cause damage and reduce fertility, while cryopreservation with glycerol poses toxicity and osmotic shock risks.
Innovation Solution
A sperm staining media comprising a buffer, DNA selective dye, and cryoprotectant such as sugar alcohols or glycols is used, with cryoprotectant introduced at earlier stages to minimize damage, and a system for manipulating sperm samples includes a sheath fluid with cryoprotectant to enhance sperm survival.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Hoechst 33342 staining is used to differentiate X and Y chromosome bearing sperm, then DNA content differentiation is achieved, but sperm damage and reduced fertility occur due to elevated temperature and pH requirements
Solution Approach 1:
The patent applies preliminary action by adding cryoprotectant to the staining media before the staining process begins. This ensures that sperm are protected from damage during the subsequent elevated temperature and pH exposure required for Hoechst 33342 staining. The cryoprotectant is present from the start of the staining procedure, preventing cellular damage before it can occur.
Solution Approach 2:
The patent uses cryoprotectant as an intermediary substance between the harmful staining conditions (elevated temperature and pH) and the sperm cells. This intermediary agent absorbs or mitigates the harmful effects of the staining process, allowing the DNA differentiation to proceed while protecting the sperm from damage and maintaining fertility.
2Reliability
If glycerol is used as cryoprotectant for sperm storage, then freezing damage is reduced, but toxicity and osmotic shock occur
Solution Approach 1:
The patent applies parameter changes by using alternative cryoprotectants with different chemical properties than traditional glycerol. These alternatives (such as ethylene glycol, propylene glycol, or sugar-based cryoprotectants) have different toxicity profiles and osmotic characteristics, providing freezing protection while reducing or eliminating the toxic and osmotic shock effects associated with glycerol.
Solution Approach 2:
The patent employs cryoprotectants that can be easily removed or are less persistent in their toxic effects compared to glycerol. The alternative cryoprotectants allow for effective freezing protection during storage, but can be more readily eliminated before sperm use, reducing the impact of their potentially harmful effects.
3Measurement precision
If flow cytometry is used for sperm sorting, then sex determination accuracy is improved, but sperm viability decreases due to shear forces and pressure
Solution Approach 1:
The patent applies preliminary action by incorporating cryoprotectant into the staining media before sperm sorting. This pre-protection ensures that sperm are already shielded from damage before they undergo the shear forces and pressure of flow cytometry sorting, helping maintain viability while achieving accurate sex determination.
Solution Approach 2:
The cryoprotectant serves as an intermediary that protects sperm cells during the flow cytometry sorting process. It mitigates the harmful effects of shear forces and pressure exerted during sorting, allowing accurate X and Y chromosome differentiation while preserving sperm viability and fertility.
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 improves sperm viability and fertility by reducing damage during sorting and cryopreservation, maintaining sperm health through controlled osmotic conditions and gentle processing.
Implementation Method 1
a sperm population is generally stoichiometrically stained with a DNA selective fluorescent dye that binds to nuclear DNA
Implementation Method 2
Intracellular fluids, including water, are removed from the cell during this process to reduce the volume of intracellular fluids that freeze. Otherwise, intracellular fluids would crystallize and expand from their liquid volume.
Implementation Method 3
maintaining sperm health through controlled osmotic conditions and gentle processing
Data Source
AI summary
Embodiments of the present invention relate generally to processes, systems, and compositions useful in manipulating a ratio of viable X chromosome bearing sperm to viable Y chromosome bearing sperm in at least one sperm population and useful for preserving the resulting manipulated sperm population. In some embodiments a cryoprotectant may be incorporated into various medias used in manipulating the sperm sample, such as in a staining media, a sheath fluid, and a collection media.