High-throughput sperm cryopreservation of eastern oyster Crassostrea virginica

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 (10–60 min) 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 60 min 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.

► High-throughput protocol was developed for sperm cryopreservation in eastern oysters.
► Two types of straws (0.5-ml French and CBS straws) were used as packaging containers.
► Effects of cryoprotectants, cooling rates, and thawing temperatures were evaluated.
► Effects of equilibration times and sperm concentrations were evaluated.
► The protocol was tested by automated filling, sealing, labeling, and freezing system.