BackgroundThe Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.Methodology/Principal FindingsEmbryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.ConclusionThis study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3-3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.

The present study aimed to investigate the differentiation of chicken (Gallus gallus domesticus) primordial germ cells (PGCs) in duck (Anas domesticus) gonads. Chimeric ducks were produced by transferring chicken PGCs into duck embryos. Transfer of 200 and 400 PGCs resulted in the detection of a total number of 63.0 ± 54.3 and 116.8 ± 47.1 chicken PGCs in the gonads of 7-day-old duck embryos, respectively. The chimeric rate of ducks prior to hatching was 52.9% and 90.9%, respectively. Chicken germ cells were assessed in the gonad of chimeric ducks with chicken-specific DNA probes. Chicken spermatogonia were detected in the seminiferous tubules of duck testis. Chicken oogonia, primitive and primary follicles, and chicken-derived oocytes were also found in the ovaries of chimeric ducks, indicating that chicken PGCs are able to migrate, proliferate, and differentiate in duck ovaries and participate in the progression of duck ovarian folliculogenesis. Chicken DNA was detected using PCR from the semen of chimeric ducks. A total number of 1057 chicken eggs were laid by Barred Rock hens after they were inseminated with chimeric duck semen, of which four chicken offspring hatched and one chicken embryo did not hatch. Female chimeric ducks were inseminated with chicken semen; however, no fertile eggs were obtained. In conclusion, these results demonstrated that chicken PGCs could interact with duck germinal epithelium and complete spermatogenesis and eventually give rise to functional sperm. The PGC-mediated germline chimera technology may provide a novel system for conserving endangered avian species.

A simple one step centrifugation method was developed for purification of primordial germ cells (PGCs) of chick embryos. PGCs, constituting less than 0.1% of the total blood cells of stage 13-14 embryos that contained a microliter amount of blood, were concentrated at the interface of a 6.3% (w/v) and 14.4% (w/v) Ficoll bilayer by centrifugation at 800 x g for 30 min. the purity of these PGCs was 86%, which was 22 times that obtained previously.

Fluorescent reagent-labelled PGCs isolated from the blood of 2-day-old chick embryos were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, human IGF-1, bovine FGF-b, and murine LIF. In 7 experiments, the number of MCs increased by an average of 4.8 fold in 4 days. Intrinsic PGCs in the 5-day embryonic germinal ridge were observed loosely attached to the stroma cells, and they also increased 3.8 fold during culture for 4 days. These results indicate the possibility of applying this culture method to the production of transgenic chickens.

The effect of interspecific egg white on the development of chicken embryos was investigated in a surrogate eggshell culture system. Egg yolks were separated from fertile White Leghorn chicken eggs and cultured in different egg whites from turkey (group TK), guineafowl (group GF), and duck (group DK), and chicken (group CK) was used as control. The viability of chicken embryos in groups CK, TK, GF, and DK after 3 d culture in system II was 98.3, 90.2, 96.1, and 91.1%. The whole contents (egg yolk and surrogate egg white) were further transferred into an eggshell from a 1.5 times heavier chicken egg with air space (system III), and incubated for further 16 d, before moving them to a hatcher. No significant difference between the 4 groups was found in their viabilities, which ranged between 72.9 and 81.3%, until 14 d postincubation (P > 0.05). After 21 d, the viability decreased to 60.4, 57.4, 50.0, and 27.7% in groups CK, TK, GF, and DK. The viability in group DK was significantly lower than in the other groups (P < 0.05). Weight loss in system III was approximately 12% in all the 4 groups without significant difference (P > 0.05). Hatchability of the chicken embryo was 60.4, 55.3, 47.9, and 19.1% in groups CK, TK, GF, and DK, respectively, and that in group DK was significantly lower than in the other groups (P < 0.05). There was no difference between the other groups (P > 0.05). These results show that chicken embryos can develop to hatch in duck, guineafowl, and turkey egg whites. However, the hatchability decreases according to the phylogenetic distance. The present study will provide a tool for manipulation of avian embryos and eventual conservation of endangered wild birds.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.

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