Article ID: 011045
The production of germline chimeric chickens by transfer of primordial germ cells (PGCs) is an effective technique for the preservation and regeneration of genetic resources in chickens. To date, the most widely used method to identify germline chimeric chickens is a testcross using differences in plumage color between the donor and recipient breeds. However, this method is time consuming and laborious. Therefore, simple high-precision molecular techniques for the identification of germline chimeric chickens need to be developed. In this study, we verified the practicability of using a Hinai-dori-specific microsatellite marker that was previously developed to identify germline chimeric chickens. A Hinai-dori-specific microsatellite marker ABR0633 can distinguish the Hinai-dori breed from White Leghorn (WL); thus, these two breeds were used as donor and recipient embryos, respectively, in this study. PGCs obtained from embryonic gonads of Hinai-dori were microinjected into either the subgerminal cavity or dorsal aorta of WL recipient embryos to produce germline chimeric chickens. A portion of both left and right testes was removed from the manipulated male chicks and was used for genotyping to verify the possibility of identifying germline chimeric chickens. Semen was then obtained from matured manipulated male chickens and was used for genotyping. Simultaneously, these chickens were crossed with Hinai-dori females by artificial insemination to produce offspring. No donor-derived Hinai-dori allele was detected in the testicular tissues from the manipulated male chicks. However, donor-derived Hinai-dori allele was detected in the semen from two manipulated male chickens (2/2) that were produced by microinjection of Hinai-dori PGCs into the dorsal aorta. In the progeny tests, Hinai-dori PGC-derived offspring were obtained from these two chickens. Moreover, only the donor-derived Hinai-dori allele was detected in the offspring that were judged as the Hinai-dori breed by plumage color. Therefore, the offspring were confirmed as the Hinai-dori breed both phenotypically and genotypically. We conclude that a Hinai-dori-specific microsatellite marker is suitable to identify germline chimeric chickens and the use of this method could reduce the time and labor needed for testcrosses.