Cryopreservation method of sperm of sea cucumber Apostichopus japonicus
ZHAO Tanjun, YIN Wenlu, ZOU Yang, LIU Li, SONG Jian, CHANG Yaqing, ZHAN Yaoyao*
Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs,Dalian Ocean University, Dalian 116023, China
Abstract: In this study, cryopreservation steps, cryopreservation solution, sperm to cryopreservation solution ratio and thawing temperature were investigated attempting to obtain an optimal cryopreservation technique for sperm of sea cucumber Apostichopusjaponicus. Twenty-seven cryopreservation solutions (Aj-1 to Aj-27) were made up containing final concentrations of cryopreservation agents [dimethyl sulfoxide (DMSO), glycerol (Gly) or 1,2 propylene glycol (1,2 -G)] of 5%, 10% and 15% and semen of the sea cucumber was cooled down in 3 methods including equilibrated for 5 min at 4 ℃, then to -80 ℃ for a night, and final liquid nitrogen storage; equilibrated for 5 min at 4 ℃, then equilibrated at -20 ℃, -80 ℃ for a night, and final liquid nitrogen storage; and three steps: for 15 min above surface of liquid nitrogen, for 5 min on surface of liquid nitrogen, and final liquid nitrogen storage to screen the optimal cryopreservation steps. Cryopreservation was carried out at different proportions of sperm and cryopreservation solution (volume fraction). After 48 h of cryopreservation, sperm thawing was conducted at 37 ℃ or 30 ℃ water bath, and the optimal cryopreservation solution, sperm to cryopreservation solution ratio and thawing temperature were screened based on the sperm motility, rapid movement time and life span. The results showed that the optimal cryopreservation of sperm of sea cucumber was observed under the conditions of mixture of the sperm with Aj-20 sperm cryopreservation solution (12 g/L glucose, 7 g/L sodium chloride, 0.7 g/L potassium chloride, 5 g/L trehalose, 0.1 g/L anhydrous calcium chloride and final concentration containing 10% DMSO prepared with pure water) in a ratio of 1∶2 (volume fraction), and then the mixture of sperm and cryopreservation solution were cryopreserved by using the three-step method. The cryopreserved sperm of sea cucumber were well recovered by shortly incubated in water bath at 30 ℃, with the maximal sperm viability (19.00±4.00)%, the longest rapid movement time (1 178.67 s±14.57 s), and the longest life span (2 817.33 s±93.76 s) in thawed sperm of sea cucumber from cryopreservation using the optimal cryopreservation technical condition, compared with other cryopreservation technical conditions. Further experiment demonstrated that the fertilization rate of sperm cryopreserved for 72, 120 and 408 h was more than 70% and the blastula hatching rate of fertilized eggs with sperm cryopreserved 72, 120 and 408 h reached more than 50%, respectively, indicating that the optimal technique screened in this study is suitable for long-term cryopreservation of sperm of sea cucumber.
2021, Vol. 36 
