Ming, we have a batch of experimental mice that need to be preserved. Could you explain sperm cryopreservation?

 

The principle of sperm cryopreservation is to lower or inhibit the metabolic activity of sperm through low-temperature treatment, thereby extending their preservation lifespan. After thawing, sperm retain a certain degree of motility and fertilization capacity. Let's delve into the details.

 

Mouse Sperm Cryopreservation
 

In the long-term preservation and breeding of genetically engineered mice, sperm cryopreservation offers significant advantages over traditional live colony maintenance. It substantially reduces housing costs, ensures genetic stability, and effectively prevents genetic drift and genetic contamination within the population.
 

Definition and Principle
 

Sperm cryopreservation, or sperm freezing, involves placing sperm in a cryoprotectant medium and subjecting it to ultralow temperatures. This process allows water molecules to maintain a disordered, vitrified state, forming glass-like ultra-micro crystals. This effectively prevents the formation of intracellular ice crystals and the resulting damage to cellular structures.

The underlying principle is the reduction or inhibition of sperm metabolism through low-temperature treatment, extending their storage life. Following thawing, sperm retain viability and fertilization capacity.

Compared to embryo cryopreservation, sperm cryopreservation requires fewer donor mice and is generally faster and simpler.
 

Historical Development
 

●1776:  Italian scientist Lazaro Spallanzani first discovered that partially frozen semen could regain some sperm motility upon thawing.

●1949:  Polge et al. discovered glycerol's cryoprotective properties, marking the true scientific entry of sperm cryopreservation technology.

●1990:  A breakthrough in mouse sperm cryopreservation was achieved. Takeshima, Nakagata, and Ogawa published a method demonstrating high success rates and reproducibility. This method used a cryoprotectant containing 3% skim milk and 18% raffinose in water.
 

Advantages and Selection Factors
 

1.Reduces long-term costs and saves animal housing space, while allowing preservation of a vast germplasm bank.

2.Prevents loss of valuable strains  in case of accidents or natural disasters.

3.Helps maintain strain stability  for transgenic lines susceptible to transgene instability.

4.Aids in controlling genetic drift  and prevents spontaneous loss of phenotypes.

While sperm cryopreservation is a cost-effective preservation measure, the decision is often based on the following factors:

1.The necessity to preserve a homozygous mutant genotype or a specific genetic background.

2.Comparison of post-thaw viability between sperm and embryos.

3.The ability to obtain sufficient numbers of embryos for freezing and breeding success.

4.A mouse strain's tolerance to sperm cryopreservation (Sztein et al., 2000a).

5.Differences in embryo viability based on genotype (Schmidt et al., 1985, 1987).
 

Operational Procedure
 

1.Filter a 1M DMSO solution. Place four 100µL droplets of 1M DMSO in a culture dish. One droplet is for washing embryos from the collection medium; the others are for holding the washed embryos.

2.Transfer a group of embryos from the collection medium into one droplet. Then, evenly distribute the washed embryos into the remaining three droplets. These embryos will eventually be transferred to cryotubes.

3.Use a 20µL pipette with a gel tip. Set the volume to 5µL and aspirate all embryos. Transfer them into a cryotube. Place the cryotube in a desktop cooler at 0°C for 5 minutes.
 

Notes:
 

a.The cryotube can be kept in the 0°C cooler for more than 5 minutes if needed (up to 20 minutes).

b.Before aspirating embryos, gently swirl the culture dish to concentrate the embryos in the center of the droplet. This facilitates aspirating all embryos within the 5µL of 1M DMSO solution.

4.Add 45µL of pre-equilibrated antifreeze solution (e.g., DAP213) from the 0°C cooler, and equilibrate for another 5 minutes.

5.Quickly secure the cryotube in a fixation clamp and immerse it directly into liquid nitrogen.

 

References:

[1] Zhang Caiqin, Zhao Ya, Tan Dengxu, et al. Research on Preparation, Preservation, and Breeding Techniques of Genetically Engineered Mice. Laboratory Animal Science, 2016, 33(04): 52-57.

[2] Luo Lin, Wu Meng, Lan Guocheng, et al. Study on Mouse Sperm Cryopreservation and In Vitro Fertilization with Frozen-Thawed Sperm. Proceedings of the Seventh China Animal Husbandry and Veterinary Science and Technology Forum, 2016: 329.

[3] Kumamoto University - Center for Animal Resources and Development (CARD) Online Resources

[4] https://mp.weixin.qq.com/s/ZwFMvYGeFABGTp52gNy85Q.

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