Observation of the in vitro fertilization process in living oocytes using frozen–thawed sperm in rats

Previous studies have observed the fertilization process in rats using whole-mount preparation at different time-points after insemination. However, very few reports have described the various events during the fertilization process using an inverted microscope without whole-mount. Moreover, to the best of our knowledge, no reports have described the observation of changes in sperm motility associated with sperm penetration into oocytes. In this study, in vitro fertilization was performed using frozen–thawed sperm in various rat strains (SD, Wistar, LE, F344, and BN) and oocytes from the SD strain, and the process of sperm penetration into the oocytes and the subsequent development were observed. The sperm motility was assessed, and the correlation between the process of sperm penetration into the oocytes and sperm motility over time was examined. The motility of frozen sperm from the SD, Wistar, LE, and F344 increased at 2–3 h after thawing, at which time the sperm attached themselves to the zona pellucida. Sperm penetration into the zona pellucida occurred after 3–5 h, and pronuclei were formed in the cytoplasm of oocytes 5–9 h after insemination. The fertilities of frozen–thawed sperm from the SD, Wistar, LE, and F344 were 92.7%, 90.0%, 90.7%, and 68.7%, respectively. However, no increase in motility was observed after thawing of frozen sperm from the BN, and the fertility was only 21%. In addition, very few polyspermic oocytes were observed with use of frozen–thawed sperm of all strains. In summary, rats are suitable animals for the observation of sperm penetration into the oocytes, and we determined the timing of fertilization events in IVF using frozen–thawed rat sperm.