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An efficient procedure for cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-µL straws in Eagle’s minimum essential medium (MEM) with various concentrations of dimethyl-sulfoxide (DMSO; 0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% bovine serum albumin). Post-thaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish GFP-nos1 3’UTR mRNA and biotin dextran prior to cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3’UTR mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25°C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20°C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave lower hatching rates (~3%) and generated a low percentage of germ-line chimeras (~1.1%). In contrast, incubation of cryopreserved sample for 16 h followed by transplantation of the GFP-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes. |
