Long-Term Preservation of Mouse Spermatozoa after Freeze-Drying and Freezing Without Cryoprotection1

Abstract

The widespread production of mice with transgenes, disrupted genes and mutant genes, has strained the resources available for maintaining these mouse lines as live populations, and dependable methods for gamete and embryo preservation in these lines are needed. 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 degrees C. Samples frozen without cryoprotection were maintained at -196 degrees C. After storage, spermatozoa were injected into the oocytes by ICSI. Zygotic chromosomes and fetal development at Day 15 of gestation were examined after 0, 1, 3, 6, 9, and 12 mo of sperm storage. When fresh spermatozoa were used for ICSI, 96% of resultant zygotes contained normal chromosomes, and 58% of two-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%, respectively; P > 0.05) and after 12 mo of sperm storage (mean of six endpoints examined: 87% and 52%, respectively; P > 0.05). Freeze-drying decreased the proportion of zygotes with normal karyoplates (75% vs. 96%; P < 0.001) and the proportion of embryos that developed into fetuses (35% vs. 58%; P < 0.001), but similar to freezing, there was no further deterioration during 12 mo of storage (mean of six endpoints examined: 68% and 34%, respectively; P > 0.05). Live offspring were obtained from both freeze-dried and frozen spermatozoa after storage for 1.5 yr. The results indicate that 1) the freeze-drying procedure itself causes some abnormalities in spermatozoa but freezing without cryoprotection does not and 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 sperm can then be stored at 4 degrees C and shipped at ambient temperatures. Both preservation methods are successful, but rapid freezing without cryoprotection is the preferred method for preservation of spermatozoa from mouse strains carrying unique genes and mutations.

Live rats resulting from injection of oocytes with spermatozoa freeze‐dried and stored for one year

“…2) was also reported in rabbit (Liu et al, 2004) regardless of significant reduction in full-term developmental potential of oocytes injected with freeze-dried spermatozoa. In mice, the process of freeze-drying induced chromosomal aberrations to some extent (Kusakabe et al, 2001;Ward et al, 2003;Kaneko and Nakagata, 2006). No chromosomal aberrations were observed in SD rat oocytes injected with fresh spermatozoa from which tails were removed by a piezo pulse, while chromosomal aberrations occurred in approximately one-fourth of oocytes injected with frozen/piezo-treated or fresh/sonicated sperm heads (unpublished data).…”

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

“…Intracytoplasmic injection of freeze-dried spermatozoa has been resulted in successful production of live offspring in mouse [1,[4][5][6]32], rat [3,7,33] and rabbit [2], and blastocyst-stage embryos in cattle [8,9] and pig [10]. However, long-term storage of FD spermatozoa at room temperature has not yet been achieved in any mammalian species.…”

A potential use of embryonic stem cell medium for the in vitro culture of preimplantation embryos

“…These days were chosen to provide information on the extent of fetal development as well as the frequency of embryonic loss after implantation, which can be detected by the presence of residual dark tissues, or implantation scar, in the uterus. Although we did not assess development to term in this study, normal fetuses at Day 14 of pregnancy rarely fail to develop to full term [32,33]. Furthermore, full-term birth occasionally results in accidental loss of pups due to cannibalization by the mother, which would impede quantitative comparison of the efficiency of fetal development between the KSOM and ESCM groups.…”