FISHERIES RESEARCH INSTITUTE,MOA,TAIWAN

To study the cryopreservation of male gamete of white shrimp, Litopenaeus vannamei, various combinations of factors including proper extender, kind and concentration of three cryoprotectants, four equilibration times, five freezing rates and three thawing temperatures were examined. Practical cryopreservation of spermatophore in white shrimp was carried out. Due to the immobility of spermatozoa of white shrimp, the viability percentages of sperm before and after cryopreservation were assessed by staining the sperm cells with eosin-nigrosin. Best result was obtained in the subgroup in which shrimp spermatophores were treated with Ca-F saline (Calcium-free marine crustacean saline) at 25 oC, equilibrated in cryoprotectant of 5% DMSO for 30 min, frozen at rate of -2 oC/min until -80 oC, maintained for 2 min, and quenched in liquid nitrogen (LN2, -196 oC). The best thawing was found using water bath of 30 oC. The highest viability of sperm out of cryopreserved spermatophores based on this protocol was 34.4 ± 3.4%. Almost similar sperm viability (33.3 ± 3.9%) was observed in the subgroup cryopreserved at freezing rate of -1 oC/min. For long-term cryopreservation, equilibration in cryoprotectant of 5% DMSO for 30 min at ambient temperature of 25 oC resulted in 49.5 ± 8.3% viability in equilibrated sample, 44.3 ± 6.6% viability in one-day cryopreserved sample, and 33.0 to 37.0% in 10-, 20-, 30-, 40-, 50-, 60-,and 70-day cryopreserved samples. There was no significant difference among the last seven subgroups.This is the first report on the establishment of cryopreservation protocol for spermatophores of white shrimp based on the systematic combination of favorable factors. Further studies on the application could provide information for commercialization for both research and industrial use.