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The use of model organisms is important for basic and applied sciences. Several laboratory species of fishes are used to develop advanced technologies, such as the zebrafish (Danio rerio), the medaka (Oryzias latipes), and loach species (Misgurnus spp.). However, the application of these exotic species in the Neotropical region is limited due to differences in environmental conditions and phylogenetic distances. This situation emphasizes the establishment of a model organism specifically for the Neotropical region with the development of techniques that may be applicable to other Neotropical fish species. In this work, the previous research efforts are described in order to establish the yellowtail tetra Astyanax altiparanae as a model laboratory species for both laboratory and aquaculture purposes. Over the last decade, starting with artificial fertilization, the yellowtail tetra has become a laboratory organism for advanced biotechnology, such as germ cell transplantation, chromosome set manipulation, and other technologies, with applications in aquaculture and conservation of genetic resources. Nowadays, the yellowtail tetra is considered the most advanced fish with respect to fish biotechnology within the Neotropical region. The techniques developed for this species are being used in other related species, especially within the characins class.

Chicken embryo manipulation, especially germline manipulation, recently has progressed greatly by devising important experimental techniques, such as embryo culture and primordial germ cell (PGC) manipulation. Embryogenesis and embryonic development in chickens are affected by gravity at various developmental stages. Freshly collected PGC population seems to contain germline-competent PGCs in opposite-sex germline chimeric chickens, but these PGCs probably disappear from the population during in vitro culture. Long-term culture of chicken PGCs makes it possible to transfer foreign DNA into the germline and also contributes to preserving avian genetic resources. Propagation of endangered avian species could become possible via interspecies germline chimeras. Development of techniques producing somatic cell-derived offspring could also be useful to avian germline manipulation.

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.

Testicular gonocytes differentiate into spermatogonia and spermatogonial stem cells could be established by culturing spermatogonia in vitro. Spermatogonial stem cells differentiate exclusively into spermatozoa and thus they are very useful for genetic manipulation in chickens. The present study was carried out to examine the possibility of in vitro proliferation of testicular and ovarian gonocytes in chickens. Gonads were obtained from 19-day incubated chicken embryos and the dissociated gonadal cells were cultured in vitro. Then, non-adherent cells containing gonocytes were collected and cultured further. It was confirmed that a part of the testicular gonocytes proliferated in vitro, when analyzed by anti-CVH antibody. The proliferated testicular gonocytes occasionally formed cell colonies. The cultured testicular gonocytes were successfully implanted in the gonads by transferring them into the coelomic epithelium that corresponds to the future gonadal region of recipient embryos. However, no apparent proliferation was observed in the ovarian gonocytes in vitro. These results suggest that testicular goncytes can be cultured in vitro and are one of the candidate cells for genetic manipulation in chickens.

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated.In the present studies, sexually matured males and females of the type-I were mated each other. Form these mating, chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of muscular dystrophy, were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain.The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy.

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy.It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens.

This is the first successful report on producing the endangered domestic fowl, Kureko Dori (KD), which is a Kumamoto prefectural natural treasure, using germline chimeras transplanted from primordial germ cells (PGCs) in practice. The attempt was made to produce germline chimeras between KD fowl, as a model of rare and/or endangered poultry, and common White Leghorn (WL) for the proliferation of KD individuals. The PGCs of KD were collected from embryonic blood at stages 13 to 16 (H & H) and preserved in liquid nitrogen until injection. A certain number of frozen-thawed PGCs were picked up under a phase contrast microscope, and injected into the peripheral vein of WL embryos (common poultry) at stages 12 to 14. We obtained one male and 6 females as the putative germline chimera, they were raised until sexual maturity, and the progeny test was performed by mating them with KD of the opposite sex to analyze the efficiency of germline chimera. The average rate of offspring originating from transplanted PGCs of KD was only 1.4%, and the maximum was 5.3% from all 577. These KD offspring derived from the KD-WL germline chimeras also achieved fertility as normal. These results show that rare and/or endangered birds can proliferate through producing germline chimeras between the same species of bird.

The migratory ability of germ cells collected from gonads at different stages of embryonic development was investigated in the domestic chicken. In Experiment 1, primordial germ cells (PGCs) were collected from 2.5-day-old embryo. Also gonadal germ cells (GGCs) were collected from 6.5-, 10.5-, 14.5-, 18.5-, and 20.5-day-old embryos. PGCs and GGCs were labeled with the fluorescent dye PKH-26, and 20 fluorescently labeled PGCs or GGCs were injected into the vascular systems of 2-day-old recipient embryos. The total number of fluorescently labeled cells recovered from both the left and right gonads of the recipient embryos was counted 5 days after injection. The proportion of recipient embryos carrying fluorescently labeled cells was 100% when the donor GGCs were collected from 6.5-, and 10.5-day-old embryos. The proportion decreased when the donor GGCs were from embryos incubated for longer than 14.5 days. An inverse relationship was observed between the total number of fluorescently labeled cells in the recipient gonads and the age of the donor embryo. In Experiment 2, gonadal cell suspensions were prepared from the left gonads of newborn chicks and adult chickens. Gonadal cell suspensions were labeled with the fluorescent dye PKH-26 and injected into the vascular systems of 2-day-old recipient embryos. The proportions of recipient embryos with fluorescently labeled cells in their gonads were 100% (3/3), 100% (3/3), 100% (3/3), and 67% (2/3) when the injected gonadal cell suspension was from male newborn chicks, female newborn chicks, adult roosters, and adult hens, respectively. These results indicate the presence of migratory germ cells in the gonads of developing embryos until day 20.5 of incubation. Furthermore, these results also suggest the presence of migratory cells even in the sexually matured testes or ovaries.