297 BIRTH OF LIVE RATS THROUGH IN VITRO FERTILIZATION USING CRYOPRESERVED SPERMATOZOA: SPERM FERTILITY IMPROVED BY FREEZING AT – 150°C

Abstract

We previously reported that damages to spermatozoa by cold shock can be avoided by cooling slowly at 0.5°C/min to 5.0°C (Seita et al. 2005 Reprod. Fertil. Dev. 17, 277-278). The objective of the present study was to develop an in vitro fertilization (IVF) system with frozen-thawed rat spermatozoa for more efficient reproduction of live offspring. We examined the effect of freezing temperatures (cooling 5.0°C to pre-plunging) on post-thaw sperm motility, plasma membrane integrity, and fertility in vitro. Epididymal spermatozoa of Wistar rats were collected in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM (Nova Animal Sales, Inc., Scituate, MA, USA) at room temperature. Samples were loaded into 0.25-mL straws and cooled to 5.0°C at 0.5°C/min in a programmable freezer. Next, the samples were exposed to liquid nitrogen (LN) vapor at various freezing temperatures (-120°C, -150°C or -180°C) above the LN level for 15 min and then plunged into LN. Straws were thawed in a 37°C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 × 10⁶ sperm/mL in a droplet of 200 ¼L of R1ECM and then pre-incubated for 5 h. Ovulated oocytes were introduced into the droplet and co-cultured for 10 h. The oocytes were denuded, fixed, and/or examined for two pronuclei (2PN) formation microscopically. The denuded oocytes, which were fertilized with spermatozoa frozen at -150°C and were microscopically confirmed to have 2PN formation, were transferred to pseudo-pregnant recipient females. IVF was also performed by the same method using fresh spermatozoa as the control. Differences in the sperm motility and plasma membrane integrity were analyzed by ANOVA, and the IVF data were analyzed by chi-square test. At 2 h after thawing the motility of spermatozoa frozen at -150°C was significantly higher than that of spermatozoa frozen at -180°C (19.8% and 11.1%; P < 0.05), although the sperm plasma membrane integrity was not significantly different among different freezing temperatures, -120°C, -150°C, and -180°C (18.2%, 23.5%, and 17.9%; P > 0.05). The percentage of oocytes with 2PN was not significantly different between the -150°C frozen and the control (fresh spermatozoa) groups [59% (131/221) and 62% (155/251); P > 0.05], although that of frozen spermatozoa at -120°C and -180°C [20% (38/188) and 23% (35/153)] were significantly lower than that of frozen spermatozoa at -150°C (P < 0.05). A total of 168 putative fertilized zygotes with 2PN were transferred to eleven recipients, and 87 live young were born. In conclusion, our results indicated that post-thaw motility of cryopreserved rat spermatozoa was improved by using a suitable cooling protocol, and the IVF system used in the present study would effectively produce offspring from the cryopreserved epididymal rat spermatozoa. To our knowledge, this procedure is the first successful production of live offspring from cryopreserved rat spermatozoa through in vitro fertilization.