Cryopreservation Protocol Optimization for <i>Penaeus monodon</i> Sperm: Reagent Screening and Parameter Refinement
Dewei Kong,
Song Jiang,
Jianzhi Shi,
Qibin Yang,
Jianhua Huang,
Yundong Li,
Yangyang Ding,
Jieyi Wang,
Xinyu Qi,
Tianmi Liu,
Falin Zhou
Affiliations
Dewei Kong
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Song Jiang
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Jianzhi Shi
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Qibin Yang
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Jianhua Huang
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Yundong Li
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Yangyang Ding
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Jieyi Wang
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Xinyu Qi
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Tianmi Liu
Fisheries Technology Extension Center of Hainan Province, Haikou 570311, China
Falin Zhou
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of South China Sea Fishery Resources Exploitation and Utilization, Ministry of Agriculture and Rural Affairs, Guangzhou 510300, China
Penaeus monodon (black tiger shrimp) is one of the important shrimp species in aquaculture. Cryopreserving its sperm not only provides technical support for breeding but also effectively prevents the decline of genetic resources, promoting the sustainable development of its aquaculture industry. This study screened different types of diluents, cryoprotectants, and concentrations and explored equilibration time, cooling protocols, and thawing conditions, ultimately determining the optimal cryopreservation protocol for P. monodon sperm. The results showed that the optimal cryopreservation protocol involved using natural seawater as the diluent with 10% dimethyl sulfoxide (DMSO) as the cryoprotectant, in which the sperm suspension and cryoprotectant were mixed at a 1:1 (v/v) ratio and equilibrated at 4 °C for 30 min. Subsequently, cooling was performed using a programmable controlled-rate freezer: the temperature was reduced to −20 °C at −5 °C/min and held for 5 min; then cooled to −80 °C at −10 °C/min and held for 5 min; finally, the temperature was reduced to −180 °C at −20 °C/min. After cooling, the sperm samples were transferred to liquid nitrogen for long-term storage. The results demonstrated that thawing in a 37 °C water bath achieved the highest sperm motility compared to conditions at 27 °C, 32 °C, 42 °C, and 60 °C. After 15 days of liquid nitrogen storage, the sperm survival rate was 53.33 ± 9.18%. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed that the sperm structure was intact before freezing, with a rounded head, a distinct acrosomal spike anterior to the head, a concentrated nucleus in the head, dense chromatin, and a smooth cell membrane surface. However, after freezing and thawing, the acrosomal spikes of some sperm were fractured, and the membrane structure was damaged. Enzyme activity analysis showed that during liquid nitrogen storage from 0 to 15 days, the enzyme activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) in sperm gradually increased with significant differences observed compared to day 0 (p p > 0.05) but significantly increased on day 15 (p p > 0.05) but significantly increased from days 10 to 15 (p P. monodon sperm and will guide the optimization of cryoprotectant combinations and freezing protocols aimed at improving sperm survival rates.
