We cryopreserved gonadal germ cells (GGCs) collected from-day-old chick embryos using the vitrification method. Gonadal germ cells were suspended in vitrification medium and plunged directly into liquid nitrogen. The recovery rates of GGCs after freezing via vitrification and slow-freezing methods were. . and. . , respectively. The survival rates of GGCs in the unfrozen control, vitrified and slow-frozen cells were. . ,. . and. . , respectively. The recovery and survival rates were significantly lower for vitrified GGCs than for slow-frozen GGCs (.). To evaluate the migratory ability of cryopreserved GGCs, frozen/thawed GGCs were labeled with PKH fluorescent dye, and GGCs were transferred into-day-old chick embryos. After incubating for three days, the embryonic gonads were collected and the number of PKH-labeled GGCs per chick was counted. These values for the control, vitrified and slow-frozen cells were. . ,. . and. . cells, respectively. No significant di erences were observed among treatments (.). These results demonstrate that it is possible to cryopreserve GGCs using vitrification, although the recovery and survival rates are lower than those observed using the slow-freezing method.

Abstract. The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 × g in the presence of 1.0 μg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate sntGGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts. Key words: Chicken, Cryopreservation, Gonadal germ cell, Karyoplast, Nuclear transfer, Serum starvation (J. Reprod. Dev. 54: [221][222][223][224] 2008) ince the birth of Dolly the sheep [1], the first mammal derived from the transfer of a somatic nucleus, somatic nuclear transfer technology has become key in reproductive and developmental biology. However, somatic nuclear transfer technology developed for mammals cannot be applied directly to avian species owing to major differences in the basic reproductive physiology between these two classes. An alternative strategy of producing somatic nuclear transferred offspring in avian species by using avian primordial germ cells (PGCs) or gonadal germ cells (GGCs) has been described by Tajima [2].PGCs are undifferentiated germ cells that subsequently differentiate into spermatogonia in the testis or into oogonia in the ovary. In avian, PGCs originate from the epiblast [3] and appear in the area pellucida [4]. After development of the vascular system, PGCs circulate along with embryonic blood cells and finally migrate into the embryonic gonads [5]. After migration, PGCs are called GGCs. Avian germline chimeras have been produced by transfer of cryopreserved PGCs [6] or GGCs [7] into recipient embryos. When a somatic nucleus is introduced into an enucleated PGC or GGC, which is then transferred into the bloodstream of a recipient embryo, the recipient theoretically produces sperm or eggs originating from the somatic nucleus.Somatic nuclear transferred PGCs (snt-PGCs) have been produced from PGCs and embryonic blood cells (EBCs) by electrofusion [8,9] and virus-stimulated fusion [9]. Ishiguro et al.[10] described a method for producing inter-genus somatic nuclear transferred GGCs (snt-GGCs) from chickens and quails. In all the reports mentioned here, EBCs were used as nuclear donors, but limited information is available as to whether cells other than EBCs can be used for producing snt-PGCs or snt-GGCs.Fibroblasts can be recovered readily from adult chickens and embryos, cultured in vitro and cryopreserved. These characteristics make them suitable as nuclear donors. In fact, the production of nuclear transferred of...