In order to realize the germplasm preservation of the important cultured fish in China, a systematical research on the cryopreservation of sperm, embryo and embryonic stem cell culture were done in this project, which established a technology system for preservation of fish germplasm at the cell, sperm and embryo levels.

1. A practical technology of high throughput fish sperm cryopreservation has been invented. The dilution ingredients suitable for different fish sperm cryopreservation have been developed and the survival rates, fertilization rates, and hatching rates were 65-90%, 80-94% and above 85% respectively. The results indicated this cryopreservation techonology has reached the application level in fishery production. 2. The sperm cryopreservation method forAcipenseridaehas been invented and the motility of the cryopreserved sperm has been increased to over 60%. 3. The sperm cryopreservation technology for 30 species of fishes including turbot has been developed and a sperm cryobank has also been established. These cryopreserved sperms have been applied industrially in fishery production. 4. The genetic structures have firstly been proven to have no difference of between fish fries produced by frozen sperm and fresh sperm using microsatellite marker method. 5. The fish embryo vitrification cryopreservation method has been invented, which has broken through the technical difficulty of cryopreservation in marine fish embryo. It is the first time to obtain freeze revived embryo and fry of marine fish in the world. 6. The programmed cryopreservation method forpagrosomus majorembryo was invented and the freeze revived embryo and fry ofpagrosomus majorwere obtained for the first time. 7. The isolation and culture method of marine fish embryonic stem cell was developed and the embryonic stem cells of sea perch andpagrosomus majorwere established. 8. The method of preparing embryonic stem cell transplantation chimera was established, which proved the developmental pluripotency of sea perch embryonic stem cell.

There are 90 papers published in this project, including 22 SCI papers. There are also 4 patents and one book obtained in this project. Since application of fish sperm cryopreservation method in China, more than 2 billion RMB, including direct and indirect economic benefit, have been generated. The project has brought our research of fish germplasm conservation to the international leading rank. Moreover, it has also promoted the development of fish cryobiology, construction of germplasm bank, and development of genetic breeding, which has generated remarkable social benefits.