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Possibility of Long-Term Preservation of Freeze-Dried Mouse Spermatozoa¹

Pharmacology & Pathology Research Center, Chugai Research Institute for Medical Science, Inc.,³ Gotemba, Shizuoka 412-8513, Japan Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd.,⁴ Tokyo 115-8543, Japan Research Unit for Functional Genomics,⁵ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan Department of Developmental and Medical Technology,⁶ Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan

Freeze-dried mouse spermatozoa are capable of participating in normal embryonic development after injection into oocytes. When the freeze-dried spermatozoa are used as a method for storage of genetic materials, however, it is essential to assure the relevance of long-term preservation over several decades or centuries. Thus, we applied the theory of accelerated degradation kinetics to freeze-dried mouse spermatozoa. Thermal denaturation kinetics were determined based on Arrhenius plots derived from transition-state theory analysis at three elevated temperatures: 30, 40, and 50°C. Accelerated degradation kinetics were calculated by extrapolation of Arrhenius plots. This theory also is being applied to the long-term stability of drugs. The estimated rate of development to the blastocyst stage at 3 and 6 mo and at 1, 10, and 100 yr of sperm storage at 4°C were 21.60%, 7.91%, 1.00%, 0%, and 0%, respectively. At –80°C, estimated development rates to the blastocyst stage that would be expected after 100 yr of storage did not decline significantly. In addition, after 3 or 6 mo of storage at 4 or –80°C, preimplantation development of the embryos derived from intracytoplasmic sperm injection (ICSI) was examined. The actual developmental rates to the blastocyst stage from ICSI by freeze-dried sperm stored for 3 mo at 4 and –80°C were 21% and 62%, respectively, and the rates for such sperm stored for 6 mo were 13% and 59%, respectively. These results indicate that the determination of accelerated degradation kinetics can be applied to the preservation of freeze-dried mouse spermatozoa. Furthermore, for long-term preservation, freeze-dried mouse spermatozoa appear to require being kept at lower than –80°C.

¹ Supported, in part, by the Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, and Science, Japan.

² Correspondence. FAX: 81 155 49 5643; hisuzuki{at}obihiro.ac.jp