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 (TM) 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 degrees C/min from 5 to -80 degrees C and thawing at 30, 40 and 50 degrees C; 2) evaluate the effect of cooling rates of 10, 15, 20, 25 and 30 degrees C/min with 10% DMSO as cryoprotectant and thawing at 40 degrees C; 3) evaluate the effect of equilibration time (10-60 min) before freezing; 4) evaluate the effect of sperm concentrations from 1x10(8) to 1x10(9) 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 degrees C/min when thawed at 30 or 40 degrees C. Further evaluation of cooling rates of 10, 15, 20, 25 and 30 degrees C/min showed that 20 or 25 degrees 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 degrees C/min. Also, sperm concentrations ranging from 1x10(8) to 1x10(9) 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. (c) 2012 Elsevier B.V. All rights reserved.