A 2 x 3 factorial designed experiment was conducted in order to examine whether the freeze-dried rat spermatozoa can participate into full-term development following intracytoplasmic sperm injection (ICSI). A sperm suspension from cauda epididymides of Sprague-Dawley (SD) rats was prepared with or without ultrasonic treatment. The sonicated and non-sonicated sperm suspensions were processed for freeze-thawing (FT groups; 100-μl sample was cooled in LN2 vapor, stored for 1 day at -196°C, and thawed in a 25°C water bath) and freeze-drying (FD groups; 100-μl sample was frozen in LN2 for 20 sec, lyophilized for 6 h, stored at 4°C for 2 days, and re-hydrated with 100-μl ultrapure water.), or were subjected to immediate use for ICSI (fresh control groups). The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2-4 μm in diameter. The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. Viable rat offspring were produced from all six experimental groups. Ultrasonic treatment of rat spermatozoa was effective in increasing the offspring rate (23.3 vs 6.7% in fresh control groups, 35.0 vs 7.6% in FT groups, 9.2 vs 2.5% in FD groups). The acrosomal region appeared to be intact even after ultrasonic, FT and FD treatments as well as fresh controls, while the lateral dorsal region of the sperm membrane was more or less damaged in sonicated, FT and FD samples. Thus, the successful participation of freeze-dried spermatozoa into full-term development was demonstrated by applying the ICSI in the rat.
Hirabayashi M, Kato M, Ito J, Hochi S: Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads. Zygote 13: 79-85. 2005.
