The provenance of xenogenic offspring from guinea fowl males was determined using the protocol illustrated in .
Xenogeneic offspring from guinea fowl.
Two male chicken (Gallus domesticus) PGC cell lines were derived from single embryos heterozygous for the dominant naked neck gene (Nana) 
. The cell lines were transfected 
, and clones expressing green fluorescent protein (GFP) were selected (). Two clones were expanded, one from each parental cell line (169.4; 143–151 days in culture and 167.2; 138 days in culture) and used to inject 141 guinea fowl (Numida meleagris) embryos at the equivalent of Stage 13–15 
Colonization of xenogeneic testis (Left panel) GFP positive chicken primordial germ cells in culture on irradiated BRL feeder cells.
Following injection of the in-vitro produced chicken PGCs into guinea fowl embryos after 60 h of incubation, colonization of the guinea fowl gonad by the GFP-expressing chicken PGCs was examined histologically in embryos sacrificed at various times during incubation and in the peri-hatching period. All 12 embryos examined revealed that GFP-expressing PGCs were incorporated into the gonad ().
A total of 51 embryos hatched and a cohort of 14 male putative interspecific germline guinea fowl chimeras was reared to sexual maturity. The 37 embryos not retained for germline testing comprised females, males whose testis were examined histologically for evidence of incorporation of GFP-expressing germ cells and keets that died before sexual maturity. The sex ratio was unaffected by the introduction of male germ cells.
Semen was collected from the 14 chimeras reared to sexual maturity and analyzed for GFP expression by flow cytometry (). Semen from 5 of the putative interspecific germline guinea fowl males contained between 26% and 78% GFP-expressing sperm suggesting that the in vitro produced chicken PGCs had extensively colonized seminiferous tubules in the guinea fowl testis and were actively undergoing spermatogenesis. These five males were mated to female chickens. Their eggs were collected, incubated and either hatched or opened during development (E7) to evaluate the presence or absence of the naked neck phenotype and GFP expression (). Because both the naked neck and GFP loci were heterozygous for the expressed alleles, the rate of germline transmission of the injected chicken PGCs is twice the rate of GFP or naked neck transmission. As shown in , approximately 50% of the offspring express either GFP or naked neck, indicating that most of the offspring were the product of the union of chicken eggs with a chicken sperm.
The provenance of the GFP offspring from the in vitro-produced chicken PGCs was confirmed by sequencing the site of insertion of the GFP locus in both the cell line and the GFP-expressing chicks sired by the interspecific chimeric males. Integration site-specific PCR assays were developed for typing the offspring. In each case, the insertion site of the GFP locus in the genome of the offspring was identical to that of the PGC line inserted into the germline of the interspecific sire, confirming the functionality of sperm produced in a xenogenic testis and derived from in vitro cultured PGCs. Examples of this analysis are shown in .
Interspecific hybridization of birds is frequent and well documented 
. Accordingly, the production of hybrids was estimated from each of the five matings of chimeras to chickens by the absence of a comb and hatching after 22 days of incubation as opposed to 21 days for chickens. Of the 192 offspring hatched from the interspecific chimeric guinea fowl males, only 6 were interspecific hybrids.
The strategy of using interspecific chimeras to propagate endangered species of birds requires that the offspring are reproductively normal. Accordingly, offspring from the interspecific chimeras were reared to sexual maturity, mated to wild-type chickens and their reproductive performance was recorded. The average fertility and hatching rate of the 5 hens tested was 71% and 94%, respectively and of the 4 roosters tested was 78% and 87% respectively, indicating that the offspring is reproductively normal.