First successful of embryo development after artificial insemination using frozen sperm in Okinawa rail (Hypotaenidia okinawae)

Okinawa rail ( Hypotaenidia okinawae ) is listed as endangered species with a population of approximately 1,500 individuals. Reproductive technology is an effective ex situ conservation method to prevent species extinction by increasing population size. Sperm preservation is useful for artificial insemination and maintenance of genetic resources. In this study, ejaculated sperm were collected and frozen in a solution containing N-methylacetamide as a cryoprotectant. Thawed sperm were inseminated into the oviducts of the female. The fertilized egg was collected and placed in an incubator. One embryo in one egg developed normally until day 21 after egg-laying whereafter the embryo died. This embryo contained genetic information derived from the frozen sperm. This result shows that frozen Okinawa rail sperm could potentially fertilize oocytes via artificial insemination. This protocol also contributes to the maintenance of the populations of other endangered avian species.