Development of a programmable freezing technique on larval cryopreservation in Mytilus galloprovincialis

Abstract

This study investigated the factors important to the development of a programmable larval cryopreservation technique in Mytilus galloprovinvialis, including (1) larval developmental stages; (2) cryoprotectant agents (CPAs); (3) thawing temperatures; (4) sucrose concentrations to remove CPA after thawing and (5) straw volumes, using D-larval rate as the post-thaw survival indicator. The results showed that larvae at 25 h post-fertilization (PF) had the highest resistance to cryopreservation. A post-thaw D-larval rate higher than 80% was achieved when the larvae were cryopreserved with 10% ethylene glycol +7.5% Ficoll +0.2% polyvinylpyrrolidone, thawed at 28 °C and used 9% sucrose solution as the medium to remove CPA after thawing. No significant difference in post-thaw D-larval rates (P > 0.05) was found between larvae cryopreserved in 0.25 mL and 0.5 mL straws. The performance comparison experiment showed that although a significantly lower survival rate (P < 0.05) was found in cryopreserved larvae than that in fresh larvae at day 2 PF (D larvae), no significant difference (P > 0.05) was shown on relative mortality rate from day 8 PF (early umbonal larvae) to day 32 PF (spat) and on shell length at day 8 PF and day 32 PF between fresh and cryopreserved larvae. Therefore, the larval cryopreservation technique developed in this study would enhance the breeding program and all year-round hatchery production in this species.