Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin–nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of −30, −80 or −80 °C and immediately stored in liquid nitrogen (cooling rates of −2, −4, −6, −8, −10, −12, −14 or −16 °C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 °C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of −2 °C/min between 25 and −80 °C before storing in liquid nitrogen. Optimal thawing was in a 30 °C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8 ± 2.9%) and cryopreserved spermatophores held for less than 60 days (87.3 ± 4.1%) did not differ (P > 0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P < 0.05) and varied from 27.3 ± 3.4 to 53.3 ± 4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6–72.2% and 63.6–64.1%, respectively) at rates comparable to fresh spermatophores (70.8–78.2% and 66.3–67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.