Reproductive Technology

Long-Term Preservation of Mouse Spermatozoa after Freeze-Drying and Freezing Without Cryoprotection

Abstract
The production of mice with transgenes, disrupted, and mutant genes surpasses the resources available for maintaining them as live populations and demands establishing dependable methods for gamete and embryo preservation. Here we report the results of intracytoplasmic sperm injection (ICSI) with spermatozoa freeze-dried or frozen without a cryoprotectant after storage for periods up to 1.5 years. Freeze-dried samples were stored at 4°C. Samples frozen without cryoprotection were maintained at -196°C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at day 15 p.c. were examined after 0, 1, 3, 6, 9, and 12 months of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of 2-cell embryos transferred developed to normal viable fetuses. Similar results were obtained when spermatozoa were frozen without cryoprotection and then used for ICSI (87% and 45%; p>0.05), and after 12 months storage (mean of 6 endpoints examined: 87% and 52%; p>0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; p<0.001) and fetuses (35% vs. 58%; p<0.001), but similar to freezing, there was no further deterioration during 12 months of storage (mean of 6 endpoints examined: 68% and 34%; p>0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 years. The results indicate that (1) the freeze-drying procedure itself causes some abnormalities in spermatozoa, while freezing without cryoprotection does not; (2) long term storage of both frozen and freeze-dried spermatozoa is not deleterious to their genetic integrity. Freezing without cryoprotection is highly successful, simple and efficient, but like all routine sperm storage methods requires liquid nitrogen. Liquid nitrogen is also required for freeze-drying but with subsequent storage and shipment at 4°C and ambient temperatures respectively. In conclusion, both methods are successful but our present data show that rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

Key words: Embryo • Gamete Biology • Assisted Reproductive Technology • In vitro fertilization • Sperm

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