Production of Live Offspring from Hamster Oocytes Injected with Freeze-Dried Spermatazoa Stored for One Year.

Abstract

Freeze-drying storage of mouse spermatozoa is now becoming a new preservation method for the maintenance of genetic resources. However, the freeze-drying method is not used widely as a preservation method of the mammalian spermatozoa. Successful production of live offspring from freeze-dried spermatozoa is limited in several animals, e.g. mice, rabbits and rats. Here, we report the birth of hamster offspring following intracytoplasmic injection with freeze-dried spermatozoa stored for one year. Firstly, cauda epididymal hamster spermatozoa were freeze-dried in 10 mM Tris-HCl buffer with or without 50 mM EGTA. The freeze-dried hamster sperm heads were injected into B6D2F1 mouse oocytes for the analysis of chromosomes at the first cleavage. The chromosome normality of hamster freeze-dried spermatozoa was examined after 1 day of sperm storage. The proportion of zygotes with normal sperm chromosomes were significantly higher in 50 mM EGTA solution compared with 0 mM EGTA (84%, 38/45 vs. 31%, 16/52, P < 0.05). Secondly, in vitro development of hamster oocytes injected with hamster spermatozoa freeze-dried in a solution with 50 mM EGTA and stored at 20 °C, +4 °C, or +25 °C for 1-day, 1-week, 1-month and 1-year were examined. Cleavage rates were no different among three storage temperatures, or storage periods (6888 %). Morula and blastocyst rate of hamster oocytes injected with freeze-dried spermatozoa stored at 20 °C, +4 °C, or +25 °C for 1-day, 1-week, 1-month, and 1-year were 36% (19/53), 38% (23/60), 44% (26/59) and 40% (21/53), 40% (24/60), 26% (10/39), 23% (8/35) and 16% (8/51), or 30% (2/40), 25% (11/44), 23% (9/40) and 4% (2/56), respectively. During 1-year of storage at 20 °C, the freeze-dried hamster spermatozoa maintained the developmental competence into morulae and blastocysts, but the storage at +4 °C or +25 °C for one year significantly decreased the developmental rates. Of 19 morulae and blastocysts transferred to three albino recipient females, two live golden offspring (11%) were obtained from freeze-dried spermatozoa after storage at 20 °C for one year. These results indicate that hamster spermatozoa freeze-dried in a solution with 50 mM EGTA are maintained the developmental competence at least for one year at 20°C. Further improvement for long-term preservation of hamster spermatozoa after freeze-drying has been necessary for producing more viable embryos.