PARENT SESSION
REPRODUCTIVE TECHNOLOGIES

Wednesday, August 4, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(629) THE POSSIBILITY OF THE LONG-TERM PRESERVATION OF FREEZE-DRIED MOUSE SPERMATOZOA.

Kawase, Yosuke¹, Araya, Hiroshi², Kamada, Nobuo¹, Jishage, Kou-ichi¹, Suzuki, Hiroshi^{3, 4}, ¹ Pharmacology & Pathology Research Center, Gotemba, Japan² Clinical Pharmacology department, Gotemba, Japan³ National Research Center for Protozoan Diseases, Obihiro, Japan⁴ Department of Developmental and Medical Technology, Bunkyo-ku, Japan

ABSTRACT- Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injected into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials from animals, however, it is essential to assure the relevance of long-term preservation for several decades or centuries. Thus, we applied the determination of accelerated degradation kinetics for freeze-dried mouse spermatozoa. The denaturation kinetics as a function of temperature has been determined based on the Arrhenius plots derived from transition state theory analysis at 3 elevated temperatures, i. e., 30, 40, and 50°C. This theory is being used for the long-term stability of drugs. The procedures of ICSI and freeze-drying of sperm were similar to those described by Kimura and Yanagimachi (1995) and Kaneko et al. (2003), respectively. The linearity was best met when the reciprocals of the percentage of the development to the 2-cell or blastocyst stage were plotted against time. The extrapolation of the Arrehenius plots leads to the estimation of each temperature. In addition, at 3 months after storage at 4 or -80°C, the post-implantation development of the embryos derived from ICSI was examined. The estimated percentages of development to the 2-cell stage at 3 months, 1, 10, and 100 years after sperm storage at 4°C were 94.50, 84.56, 22.42, and 0.00%, and those to the blastocyst stage were 21.60, 1.00, 0.00, and 0.00%, respectively. At -80°C, estimated development rates to the 2-cell stage were 98.00% at 3 months-100 years after storage, and those to the blastocyst were either 59.00% at 3 months, 1, and 10 years after storage, and 58.99% at 100 years after storage, respectively. Actually, developmental rates to 2-cell stage of embryos from ICSI by freeze-dried sperm stored for 3 months at 4 and -80°C, were 92.46% (319/345) and 98.58% (348/353). Development to blastocyst at 3months after storage at 4°C and at -80°C was 21.16% (73/345) and 62.04% (219/353). These results indicate that it is possible to apply the determination of accelerated degradation kinetics to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to be required to be kept at less than -80°C.

KEY WORDS: ICSI, Arrhenius plots, freeze-dried mouse spermatozoa, determination of accelerated degradation kinetics