Annual Report

35. Cryopreservation and Transport of Mouse Spermatozoa at -79oC

Masanori Okamoto, Naomi Nakagata1, and Yutaka Toyoda2 (1Kumamoto Univ.; 2Obihiro Univ.)

Keywords: cryopreservation, in vitro fertilization, mouse spermatozoa, transport


Since the first reports of successful mouse sperm cryopreservation, mouse spermatozoa have also been successfully stored at -196oC, as have various other species. If frozen spermatozoa can survive cryopreservation at around -80oC for relatively long periods of time, storage and transport of mouse spermatozoa can be simplified, allowing for more widespread use in biomedical research. In the present study, two experiments were carried out, i.e., we examined the fertilizing ability of frozen spermatozoa which had been maintained at -79oC for relatively long periods of time and transported by packing in dry ice.

In experiment 1, spermatozoa obtained from adult Jcl:ICR male mice were frozen and stored in an ultra-low temperature freezer maintained at -79oC from 1 week to 8 months. ICR mouse oocytes were inseminated with frozen-thawed spermatozoa. In vitro fertilization rates of the frozen-thawed sperm after 1 week and 4 months of storage were high at 71 and 71%, respectively. These values did not differ significantly from the value (73%) of the control stored at -196oC. In contrast, the 8-month storage rate was significantly lower at 51%. In experiment 2, frozen spermatozoa obtained from adult Jcl:BDF1 male mice were transported in a Styrofoam box packed in dry ice over a 3-days period. After transport, ICR mouse oocytes were inseminated with frozen-thawed spermatozoa. Table 2. summarizes the results for sperm motility, in vitro fertilization and production of normal young with frozen-thawed mouse sperm after transport at -79 and -190oC. In vitro fertilization rate of frozen-thawed spermatozoa after transport at -79oC was high at 88%, which was not significantly different from that (84%) of the transported control at -190oC. After transferring two-cell embryos derived from frozen spermatozoa to the recipients, 37-62% of the embryos developed into offspring in both experiments.

These results indicate that mouse spermatozoa can survive cryopreservation for 4 months in an ultra-low temperature freezer and transport by packing in dry ice at -79oC. Liquid nitrogen containers and dry shippers are not always available in all laboratories, but ultra-low temperature (-80oC) freezers and dry ice are found in most major laboratories. The results obtained for the present method demonstrate the feasibility of sperm cryopreservation and transport at -79oC. This method is simple and convenient for preserving and transporting spermatozoa. For transport at -190oC, a dry shipper has to be used to transport frozen spermatozoa and it has to be returned to the consigner. In contrast, the containers holding frozen samples do not need to be returned in cases where dry ice is used. Thus, transport by packing in dry ice at -79oC is much easier than that by a dry shipper. The storage and transport of mouse spermatozoa can be simplified, thus allowing for the wider utilization of transgenic mice in biomedical research. We believe that the present method of transporting frozen spermatozoa will enable widespread exchange of transgenic mice between laboratories.

Publication:
Okamoto, M., Nakagata, N. and Toyoda, Y.: Exp. Anim., 50, 83-86, 2001.

Table 2. Sperm motility, in vitro fertilization and production of normal young with frozen-thawed mouse spermatozoa after transport by packing in dry ice and dry shipper

Cryopreserved BDF1 mouse spermatozoa were packed in straws and transported for 3 days. ICR mouse oocytes were inseminated with frozen-thawed spermatozoa. *Percentages of actively motile sperm were based on counts in the sperm suspension. # Based on data from two replicate experiments. a,Values with the same superscripts are not significantly different (P>0.05).


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