Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.

The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

A 2×3 factorial designed experiment was conducted in order to examine whether freeze-dried rat spermatozoa can participate in 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; 100μl sample was cooled in liquid nitrogen 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 liquid nitrogen for 20 s, lyophilized for 6 h, stored at 4 °C for 2 days, and rehydrated with 100μl 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 2–4μm 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 in the fresh controls, while the lateral dorsal region of the sperm membrane was more or less damaged in the sonicated, FT and FD samples. Thus, the successful participation of freeze-dried spermatozoa in full-term development was demonstrated by applying ICSI in the rat.