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Updated: Jul 25, 2025
Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit
Published on: December 12, 2011
A novel, simplified method to prepare and preserve freeze-dried mouse sperm in plastic microtubes
Li Ly Yang1, Daiyu Ito1, Natsuki Ushigome1
1Faculty of Life and Environmental Science, University of Yamanashi, Yamanashi 400-8510, Japan.
Abstract:
Although freeze-drying sperm can save space, reduce maintenance costs, and facilitate the transportation of genetic samples, the current method requires breakable, custom-made, and expensive glass ampoules. In the present study, we developed a simple and economical method for collecting freeze-dried (FD) sperm using commercially available plastic microtubes. Mouse epididymal sperm suspensions were placed in 1.5 ml polypropylene tubes, frozen in liquid nitrogen, and dried in an acrylic freeze-drying chamber, after which they were closed under a vacuum. The drying duration did not differ between the microtube and glass ampoule methods (control); however, the sperm recovery rate was higher using the microtube method, and the physical damage to the sperm after rehydration was also reduced. Intracytoplasmic sperm injection (ICSI) using FD sperm stored in microtubes at -30°C yielded healthy offspring without reducing the success rate, even after 9 months of storage. Air infiltration into all microtubes stored at room temperature (RT) within 2 weeks of storage caused a drastic decrease in the fertilization rate of FD sperm; underwater storage did not prevent air infiltration. RT storage of FD sperm in microtubes for 1 week resulted in healthy offspring after ICSI (5-18%), but the addition of silica gel or CaCl2 did not improve the success rate. Our novel microtube method is currently the simplest and most effective method for treating FD sperm, contributing to the development of alternative low-cost approaches for preserving and transporting genetic resources.
Insights
Researchers developed a cost-effective method for freeze-drying (FD) sperm using plastic microtubes, improving sperm recovery and reducing damage. This technique preserves sperm viability for successful offspring production via intracytoplasmic sperm injection (ICSI) even after long-term storage.
Area of Science:
- Reproductive Biology
- Cryopreservation Technology
- Biotechnology
Background:
- Current sperm freeze-drying methods rely on expensive, fragile glass ampoules.
- There is a need for economical and robust methods for preserving genetic samples.
Purpose of the Study:
- To develop a simple, economical, and effective method for freeze-drying sperm using readily available plastic microtubes.
- To evaluate the viability and fertility of freeze-dried sperm preserved in microtubes.
Main Methods:
- Mouse epididymal sperm were freeze-dried in polypropylene microtubes after freezing in liquid nitrogen.
- Sperm recovery, physical damage, and fertilization rates after intracytoplasmic sperm injection (ICSI) were assessed.
- Offspring viability was evaluated after ICSI using sperm stored at -30°C and room temperature (RT).
Main Results:
- Freeze-drying sperm in plastic microtubes resulted in higher recovery rates and reduced physical damage compared to glass ampoules.
- Intracytoplasmic sperm injection (ICSI) with microtube-stored sperm yielded healthy offspring, with no success rate reduction after 9 months at -30°C.
- Room temperature storage led to decreased fertilization rates within 2 weeks due to air infiltration, unaffected by desiccants or underwater storage.
Conclusions:
- The novel microtube method offers a simple, cost-effective alternative for freeze-drying sperm.
- This approach facilitates the preservation and transportation of genetic resources, supporting low-cost biobanking strategies.
- Plastic microtubes provide a viable, robust alternative to glass ampoules for sperm cryopreservation.
