Since freeze-dried spermatozoa can be stored at ambient or refrigerated temperature, the costs required for maintenance and shipping of spermatozoa can be reduced. To date, viable offspring in mice (Wakayama and Yanagimachi 1998 Nat. Biotech. 16, 639) and rabbits (Liu et al. 2004 Biol. Reprod. 70, 1776) have been produced by intracytoplasmic sperm injection (ICSI) using freeze-dried samples. The objectives of the present study were to examine whether freeze-dried rat spermatozoa can participate in full-term development by ICSI, and whether sonication prior to freeze-drying of the spermatozoa influences the offspring rate. Spermatozoa from cauda epididymides of Sprague-Dawley (SD) rats were collected in 10 mM TRIS/HCl buffer supplemented with 50 mM NaCl and 50 mM EGTA. A 2 × 3 factorial-designed experiment was conducted. The sperm suspensions were either sonicated for 10 s using a 10% power output from an ultrasonic cell disruptor or not sonicated. The sperm suspensions were then processed for freeze-thawing (100-μL sample in 1.0-mL cryotube was cooled in liquid nitrogen vapor, stored at -196°C for 48 h, and thawed in a 25°C water bath) and freeze-drying (100-μL sample in 1.5-mL polypropylene tube was frozen in liquid nitrogen for 20 s, lyophilized for 6 h by a freeze-drying apparatus, stored at 4°C for 48 h, and rehydrated with 100 μL ultra pure water), or were subjected to immediate use for ICSI. The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2–4 μm in diameter, as described previously (Hirabayashi et al. 2002 Transgenic Res. 11, 221). The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. The in vivo developmental potential of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa is shown in the table below. Viable rat offspring were produced in all six experimental groups, with the offspring rates at 2.5–35.0%. Sonication treatment of rat spermatozoa to induce membrane disruption and tail/midpiece dissociation from the heads was effective in increasing the offspring rate after ICSI. The positive effect of sperm sonication may be explained as facilitating decondensation of sperm heads by membrane disruption in the spontaneously activating rat oocytes. Thus, successful participation of freeze-dried rat spermatozoa into full-term development was demonstrated by applying the ICSI.
Table 1.
In vivo development of rat oocytes microinseminated with fresh, freeze-thawed, and freeze-dried spermatozoa
314 FULL-TERM DEVELOPMENT OF RAT OOCYTES MICROINSEMINATED WITH FREEZE-DRIED SPERMATOZOA
