Safeguarding genetic resources of curassow through cryopreservation and transplantation of post-mortem recovered testicular cells into recipient roosters

Cryopreservation and transplantation of testicular cells (TCs), especially spematogonial stem cells (SSCs), offers a new perspective for the genetic rescue of birds since traditional biobanking using semen and eggs are either inefficient or impractical. Here, we demonstrated that transplantation of TCs (which contained SSCs) from dead curassows were able to survive and colonize chicken testes, despite the phylogenetic distance between donors and recipients. Previously to transplants, TCs were collected, cryopreserved, thawed, and then labelled with PKH26. Subsequently, labelled TCs were transferred into gonads of sterilized recipient chickens, and monitored for their presence and development from 1 to 180 days after transplantation. The interval between collecting testes and processing them in the laboratory was crucial for TCs viability, but it did not affect the viability of undifferentiated spermatogonia within 24 hours post-mortem. Although positive correlation between testis weight and the total cells recovered was noticed, the number of undifferentiated spermatogonia comprised approximately 0.05% of the total TCs. Once the number of undifferentiated spermagonial stem cells was not sufficient, we decided to make transplants using crude TC fractions containing different types of germ and somatic cells. PKH26-positive cells were found in the cryosections of recipient testes until 42 days after transplantation, whereas the presence of the donor genomic DNA was confirmed until 120 days after transplantation. Taken together, our findings indicate that chicken testes can provide functional conditions for the survival and proliferation of germ cells rescued from individuals of another family of the Galliformes order. This approach represents a promising conservation tool for the recovery of testicular germ cells (including SSCs) from individuals of different ages (from embryos to adult males).