Cryopreservation and xenotransplantation of spotted sea bass (Lateolabrax maculatus) spermatogonia.

Cryopreservation of spermatogonia (SGs) is crucial for aquaculture, broodstock innovation, and the long-term conservation of aquatic genetic resources. This study developed an efficient cryopreservation protocol for spotted sea bass SGs and evaluated their post-thaw viability and transplantation potential. Initially, we characterized testicular tissue from 6-month-old male spotted sea bass, identifying various germ cell stages and strong Lmvasa expression in SGs. An optimized method for isolating and enriching SGs using Percoll density gradient centrifugation yielded a highly viable (99.6%) SG population, confirmed by morphology and germline markers (Lmvasa, Lmdnd, Lmnanos3). Subsequently, a cryopreservation protocol was optimized, with the optimal cryomedia comprising 1.3 M DMSO + 0.1 M Trehalose + 10% FBS + 35.2% extender in MEM, achieving a remarkable post-thaw SG viability of 91.83%. The transplantation potential of both fresh and 1-year cryopreserved SGs was evaluated by xenotransplantation into medaka larvae. Both fresh and 1-year cryopreserved SGs successfully colonized the genital ridges and formed germline chimeras, with 25% chimerism in fresh SGs and 20% in cryopreserved SGs, as confirmed by donor-specific Lmvasa 3'UTR PCR analysis. This study establishes a comprehensive system for SG isolation, cryopreservation, and functional assessment, providing a foundational tool for marine germplasm banking and surrogate broodstock technology.