Full-term Development of Hamster Embryos Produced by Injecting Freeze-dried Spermatozoa into Oocytes

Abstract

Although injected freeze-dried hamster spermatozoa can develop into male pronuclei even after 12 months of storage at 4ºC, the developmental competence of hamster pronuclear oocytes is not well understood. Furthermore, production of live offspring from freeze-dried spermatozoa is limited in some animals, such as mice, rabbits and rats. Here, we report the birth of hamster offspring following intracytoplasmic injection with freeze-dried spermatozoa. The integrity of the sperm DNA after freeze-drying and rehydration is very important for the developmental competence of hamster embryos produced by injecting freeze-dried spermatozoa into oocytes. This study used the TUNEL method to examine DNA fragmentation and the chromosomal integrity of spermatozoa after freeze-drying using two freezing media: M2 medium, and a Tris-HCl buffered solution containing 50 mM EGTA (EGTA solution). The rate of DNA fragmentation when hamster spermatozoa freeze-dried in EGTA solution was used was significantly (P < 0.05) lower than that in M2 medium (4.3% vs. 41.4%), and the chromosomal integrity in EGTA solution was higher in EGTA solution than in M2 medium (81.1% vs. 41.0%). The percentage of morulae/blastocysts derived from hamster spermatozoa freeze-dried in EGTA solution was significantly (P < 0.05) higher than that derived from spermatozoa freeze-dried in M2 medium (62.2% vs. 12.5%). After transfer to foster mothers, 3 of 23 morulae/blastocysts developed into live offspring.