Acipenser sinensis and Acipenser dabryanus are critically endangered species, so germplasm conservation via cryopreservation of sperm is necessary. Disaccharides can act as membrane-impermeable cryoprotectants, and enolase3 (ENO3) and plasma membrane Ca2+ ATPase isoform (PMCA2) are proteins associated with sperm quality. We considered seven characteristics of sperm quality in cultured brood stock from A. sinensis and A. dabryanus. We tested use of sucrose or trehalose alone and in combination at different concentrations for cryopreservation of A. dabryanus sperm. A low concentration of sucrose plus trehalose (S15 T15 ) was optimal. Mixing of the extender with sucrose, lactose, or trehalose alone or with pairwise mixtures revealed that a mixture of lactose and trehalose (L15 T15 ) gave the best results for both A. sinensis and A. dabryanus. Enolase3 and PMCA2 expression levels were measured in cryopreserved A. sinensis sperm via Western blotting. Relative ENO3 and PMCA2 expression levels were examined, and the relationship between disaccharide composition, sperm quality and protein expression was explored in A. sinensis. The results showed that relative ENO3 and PMCA2 expression levels were the highest at L15 T15 in cryopreserved A. sinensis sperm. There were significant positive correlations between ENO3 expression and percentage membrane integrity, and between PMCA2 expression and sperm motility parameters (percentage of motile sperm, curvilinear velocity, straight-line velocity and average path velocity; p < .05) in cryopreserved A. sinensis sperm. Our results indicate the optimal disaccharide combination and concentrations for cryopreservation of A. sinensis and A. dabryanus sperm and suggest that ENO3 and PMCA2 expression levels could serve as a valuable indicator of sperm quality in A. sinensis.

Institute of Hydrobiology Chinese Academy of Sciences Wuhan China University of Chinese Academy of Science Beijing China Key Laboratory of Freshwater Biodiversity Conservation Ministry of Agriculture of China Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China Sino Czech Joint Laboratory for Fish Conservation and Biotechnology Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China

Key Laboratory of Freshwater Biodiversity Conservation Ministry of Agriculture of China Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China Sino Czech Joint Laboratory for Fish Conservation and Biotechnology Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China

Key Laboratory of Freshwater Biodiversity Conservation Ministry of Agriculture of China Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China Sino Czech Joint Laboratory for Fish Conservation and Biotechnology Yangtze River Fisheries Research Institute Chinese Academy of Fishery Sciences Wuhan China University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Research Institute of Fish Culture and Hydrobiology Vodňany Czech Republic

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