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Genetics / Genomics JoVE (Open Access) Citable · DOI

Mouse Sperm Cryopreservation and Recovery using the I·Cryo Kit

DOI: 10.3791/3713-v
What you'll learn
  • ✓ Prepare and load mouse sperm into cryopreservation straws using the I•Cryo kit
  • ✓ Execute controlled-rate freezing and long-term storage of mouse sperm
  • ✓ Recover cryopreserved sperm and perform in vitro fertilization (IVF)
  • ✓ Evaluate embryo development and breeding success from frozen-thawed sperm
Protocol

Here we demonstrate the newly developed I•Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later successfully recovered by IVF.

Difficulty
advanced
Total time
~2–3 hours per cryopreservation batch; recovery and IVF ~4–6 hours per experiment
Model organism
Mouse (multiple strains; 49 genetically modified lines demonstrated)
Biosafety
BSL-1

Steps

1
Prepare cryopreservation straws and supporting materials

Assemble and prepare cryopreservation straws, labels, and ancillary materials required for the I•Cryo kit protocol. Ensure all equipment and consumables are organized and ready for sperm loading.

▶ 00:49
2
Load and cryopreserve mouse sperm samples

Collect mouse sperm, load into prepared straws using the I•Cryo kit, and execute controlled-rate freezing protocol for long-term archival of genetic material.

▶ 01:42
3
Prepare female mice and oocytes for IVF

Prepare recipient female mice, collect and prepare oocytes, and set up in vitro culture conditions to receive recovered sperm for fertilization.

▶ 03:34
4
Thaw sperm and conduct in vitro fertilization

Thaw cryopreserved sperm using the I•Cryo kit protocol and perform IVF with prepared oocytes to assess fertilization and early embryo development.

▶ 04:33
5
Evaluate embryo development and breeding outcomes

Monitor two-cell embryo development and assess overall success rates of cryopreservation and IVF across multiple mouse strains and genetically modified lines.

▶ 06:05
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