Abstract

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0°C at 0.5°C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150°C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0°C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 × 10⁶ sperm/mL into a 200-¼L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 ± 6%, 24 ± 5%, and 18 ± 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.