Cryopreservation of Greenshell™ mussel (Perna canaliculus) sperm. I. Establishment of freezing protocol

Abstract

A series of eight experiments was conducted to determine the most suitable cryoprotectants (CPAs), dilution rates and methods of CPA addition as well as freezing method for the cryopreservation of Greenshell™ mussel (Perna canaliculus) sperm. The experiments utilized a fertilization assay to determine the treatments that gave the highest post-thaw fertility. The assays revealed that dimethyl sulphoxide (DMSO) gave the best results of the seven CPAs tested. A concentration of DMSO of approximately 12% [volume: volume] in Milli-Q water provided the best results. A dilution rate of 1:1 (sperm: CPA solution) was superior to 1:3 indicating that more concentrated sperm had higher post-thaw fertility. The time that sperm were exposed to the CPA solution prior to freezing had no effect over the range tested which gives some flexibility in the amount of time sperm can be exposed to CPA prior to freezing. The addition of the CPA in either one step or in 4 equal volume steps over two min had no significant effect on fertility and the two different freezing methods (programmable freezer or rack floating above liquid nitrogen) produced similar results. The post-thaw fertility of mussel sperm using the final protocol developed (12% DMSO plus 0.2M trehalose, sperm diluted 1:1, loaded into 0.5mL straws and cooled on a rack 3cm above liquid nitrogen followed by immersion in liquid nitrogen) was significantly reduced in comparison to fresh sperm. On average, approximately 363 x more sperm were required to achieve an equivalent level of fertilization as that of fresh sperm. The method is being used in mussel selective breeding, however, there is considerable between-animal variability. Further refinement to the method may help to improve post-thaw fertility.