High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica

Abstract

Sperm cryopreservation is a valuable tool in germplasm preservation and breeding. Despite many studies, reliable and routine cryopreservation of oyster sperm remains a challenge. The goal of this study was to develop a reliable protocol for sperm cryopreservation of the eastern oyster Crassostrea virginica with high-throughput processing by using two types of 0.5-ml straws (French and CBS™ high security straws). The objectives were to: 1) evaluate the effect of 10% methanol, dimethyl sulfoxide (DMSO), and propylene glycol as cryoprotectants at cooling rates of 5, 20 and 40°C/min from 5 to −80°C and thawing at 30, 40 and 50°C; 2) evaluate the effect of cooling rates of 10, 15, 20, 25 and 30°C/min with 10% DMSO as cryoprotectant and thawing at 40°C; 3) evaluate the effect of equilibration time (10–60min) before freezing; 4) evaluate the effect of sperm concentrations from 1×108 to 1×109 for freezing, and 5) verify the established protocol by freezing sperm from 16 individual males. Among the three cryoprotectants, DMSO yielded the highest post-thaw motility at a cooling rate of 20°C/min when thawed at 30 or 40°C. Further evaluation of cooling rates of 10, 15, 20, 25 and 30°C/min showed that 20 or 25°C/min yielded the highest post-thaw motility (34±5%) and fertility (77±12%) for French straws and CBS straws (28±3% and 69±14%). Equilibration times of 10 to 60min did not cause significant differences in post-thaw motility when freezing with 10% DMSO at a cooling rate of 25°C/min. Also, sperm concentrations ranging from 1×108 to 1×109 at freezing did not cause significant differences in post-thaw motility. Sperm concentration after thawing was not different compared to that before freezing, and no agglutination was observed in the post-thaw samples. Finally, after thawing, sperm cryopreserved from 16 males with this protocol showed 58±24% fertility (from 18 to 86%) for French straws, and 54±21% fertility for CBS straws (from 18 to 95%). Overall, this study provided a reliable protocol for sperm cryopreservation in the eastern oyster with potential for high-throughput processing which can produce thousands of straws per day with homogenous and reliable quality.