Optimization of ultra-low temperature cryopreservation and post-thaw reproductive performance of Ruditapes...

Cryopreservation serves as a vital technique for long-term germ preservation of aquatic species, and post-thaw sperm viability is essential to successful germplasm conservation. Here, we optimized the ultra-low temperature cryopreservation protocol for sperm of the Manila clam Ruditapes philippinarum. Based on preliminary viability assessment, sterile natural seawater was confirmed as the optimal diluent, and 0.2% (v/v) ammonia seawater was chosen for sperm activation. A 3×3 orthogonal experimental design was applied to screen the optimal combination of cryoprotectant type, concentration (10%, 15%, 20%) and cooling procedure for sperm cryopreservation of R. philippinarum. The optimized cryopreservation protocol was determined as follows. Sperm samples were collected via dissection of clams under low temperature. The obtained sperm were initially diluted with sterile natural seawater at a volume ratio of 1:1, and then blended with 10% dimethyl sulfoxide (DMSO) cryoprotectant at a volume ratio of 1:2. The diluted sperm were first equilibrated 15 cm above liquid nitrogen (LN₂) for 5 min, followed by another 10-min equilibration at 5 cm above LN₂, and finally preserved by complete immersion in LN₂. After 24 h of cryogenic storage, frozen sperm were thawed in a 38 °C water bath and activated with 0.2% ammonia seawater. The average fertilization rate reached 90.99 ± 1.14%, and the hatched larvae developed normally. This study lays a theoretical and technical foundation for germplasm conservation and selective breeding of high-quality Manila clam varieties.