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Abstract:In order to establish a system to facilitate the systematic collection, preservation, and provision of laboratory rats (Rattus norvegicus) and their derivates, the National BioResource Project-Rat (NBRP-Rat) was launched in July 2002. By the end of 2008, more than 500 rat strains had been collected and preserved as live animals, embryos, or sperm. These rat resources are supplied to biomedical scientists in Japan as well as in other countries. This review article introduces NBRP-Rat and highlights the phenome project, recombinant inbred strains, BAC clone libraries, and the ENU-mutant archive, named the Kyoto University Rat Mutant Archive (KURMA). The future direction of rat resources are also discussed.

We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.