Initiative on Avian Primordial Germ Cell Cryobanking in Thailand.

Abstract

Background: Biobanking the reproductive tissues or cells of animals preserves the genetic and reproductive ability of the species in long-term storage and promotes sharing of reproductive materials. In avian species, the primordial germ cell (PGC) is one of the most promising reproductive cells to be preserved in biobanks, due to self-renewal properties and direct access to the germ line mediated by PGC transfer. Methods: To conserve the genetic resource of local chicken breeds that are of conservation importance, we systematically isolated two types of pregonadal PGCs from chicken embryos-circulating and tissue PGCs. PGCs of individual embryos were separately isolated, cultured, and cryopreserved. Characteristics of cultured PGCs are described and evaluated. Results: The efficiency of PGC isolation from individual embryos was 98.9% (660/667). In most cases, both matching circulating and tissue PGC lines were isolated from the same embryo (68.2%, 450/660), whereas the remaining lines were from a single source, being either tissue (30.6%, 202/660) or circulating (1.2%, 8/660). Efficient PGC isolation and proliferation can be expected in cultures of circulating PGCs (68.7% and 64.3%, respectively) and tissue PGCs (97.8% and 80.7%, respectively). Following cryopreservation, recovered cells sustained PGC identities including expression of chicken vasa homolog and deleted in azoospermia-like proteins and migration ability to recipient embryonic gonads. Culture conditions equally supported proliferation of circulating and tissue PGCs from both sexes. Combining tissue PGC culture in the regimen prevented 30.3% loss of PGC cultures in the case where circulating PGC culture was ineffective. Cultured circulating and tissue PGCs were similar in morphology, but optimal culture characteristics were different. Conclusion: We applied the approach of PGC isolation from blood and tissue origins on a wide scale and demonstrated its efficiency for biobanking chicken PGCs. The workflow can be operated effectively almost year-round in a tropical climate. It was also described in ample and practical details, which are suitable for adoption or optimization in other conditions.