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Reviving rare chicken breeds using genetically engineered sterility in surrogate host birds

Significance In the fields of conservation biology and sustainable agriculture, the ability to cryopreserve and revive animal species is paramount to efforts to preserve genetic diversity. An innovative approach is to use sterile surrogate host animals for the transplantation of reproductive germ cells from rare/endangered animals. This technology has previously been utilized in mammals but is of particular importance for animals with lipid-filled zygotes/embryos, such as fish and birds, which render cryopreservation techniques inefficient. We demonstrate that the female chicken rendered sterile using genome editing technology can be used as a surrogate host for transplanted cryopreserved germ cells and only lay eggs of the transplanted rare chicken breed. Our results suggest a way to preserve the biodiversity of bird species. In macrolecithal species, cryopreservation of the oocyte and zygote is not possible due to the large size and quantity of lipid deposited within the egg. For birds, this signifies that cryopreserving and regenerating a species from frozen cellular material are currently technically unfeasible. Diploid primordial germ cells (PGCs) are a potential means to freeze down the entire genome and reconstitute an avian species from frozen material. Here, we examine the use of genetically engineered (GE) sterile female layer chicken as surrogate hosts for the transplantation of cryopreserved avian PGCs from rare heritage breeds of chicken. We first amplified PGC numbers in culture before cryopreservation and subsequent transplantation into host GE embryos. We found that all hatched offspring from the chimera GE hens were derived from the donor rare heritage breed broiler PGCs, and using cryopreserved semen, we were able to produce pure offspring. Measurement of the mutation rate of PGCs in culture revealed that 2.7 × 10−10 de novo single-nucleotide variants (SNVs) were generated per cell division, which is comparable with other stem cell lineages. We also found that endogenous avian leukosis virus (ALV) retroviral insertions were not mobilized during in vitro propagation. Taken together, these results show that mutation rates are no higher than normal stem cells, essential if we are to conserve avian breeds. Thus, GE sterile avian surrogate hosts provide a viable platform to conserve and regenerate avian species using cryopreserved PGCs.