Development of serum-free cryomedia for slow-freezing of chicken primordial germ cells

The preservation of chicken genetic resources is important to both backup commercial breeds that support egg and meat production worldwide and conserve the genetic diversity of indigenous breeds, which is essential for managing breeding according to future demands. Because chicken embryos are attached to a large yolk, cryopreservation is technically impossible. In chickens, a unique developmental engineering approach based on the manipulation of primordial germ cells (PGCs), the embryonic precursor cells of gametes, has been developed. Cryobanking of PGCs is an innovative strategy for efficiently maintaining chicken genetic resources without breeding live birds. Cryomedia containing dimethyl sulfoxide or propylene glycol (PG) as permeable cryoprotectants, combined with serum as a non-permeable cryoprotectant, have been widely used for the cryopreservation of chicken PGCs. However, because the serum composition varies greatly, performance differences arise between lots. Consequently, there is a demand for serum-free cryomedia with specific known compositions. Here, we investigated the cryoprotective effects of ovalbumin and sericin, as alternatives to serum, on chicken PGCs. Two cryomedia were designed: PO comprising 7.5% PG and 5% ovalbumin and PS comprising 7.5% PG and 2% sericin. Following the culture of PGCs cryopreserved in these cryomedia, viability and cell doubling times recovered to levels comparable to those of an unfrozen control group at 6 and 2 d post-culture, respectively. When frozen-thawed PGCs were transplanted, their gonadal migration ability was significantly lower immediately after thawing, but recovered to levels comparable to those of the unfrozen control group after 4 d of culture. We successfully revived viable offspring from Hinai-dori, designated as a natural monument of Japan, from PGCs cryopreserved in these cryomedia. In conclusion, we developed two serum-free cryomedia that achieved > 60% recovery of viable PGCs after thawing while maintaining germline competency.