Cryopreservation is a technique to maintain biological materials’ physiological and genetic stability at an ultralow temperature. For commercially important livestock or aquatic species, gamete and embryo cryopreservation could play a significant role in breeding programs and commercial production. For example, it could help overcome key problems such as asynchronous maturation and an unbalanced sex ratio. However, the physiochemical stresses imposed by cryopreservation can negatively affect gametes and embryos, leading to a poor survival rate. Recent studies on cryoinjury have demonstrated that the cryosensitivity of lipids is one of the key causes of cryodamage in mammalians, as lipid compositions in membranes of gametes and embryos are closely related to their cryoresistance. In addition, the cryotolerance of gametes and embryos in some mammalian species has been improved by lipid modification. However, studies on the role of lipids in the cryopreservation of gametes, embryos, and larvae are rare in fish and shellfish. Therefore, this review focuses on recent methodological advances to improve cryotolerance by lipid modification, including lipid application or manipulation in human and livestock sperm, oocytes, and embryos, and how these novel approaches could improve cryopreservation techniques in aquatic species, especially for oocytes and embryos.

Previous studies have shown that post-thaw sperm performance is affected by multiple stressors during cryopreservation, such as those induced by physical, chemical, mechanical and physiological changes. One of these is the balance disturbance between the antioxidant defense system and reactive oxygen species (ROS) production. This study investigated whether this disturbance could be alleviated by the addition of different antioxidants to cryoprotective solution [8% dimethyl sulfoxide (DMSO) in 1 µm filtered seawater] optimized for the sperm in dwarf surf clam Mulinia lateralis, the model bivalve species used in many different types of studies. Results showed that the addition of 20 μM coenzyme Q10 (Q10) to 8% DMSO achieved a D-stage larval rate similar to that of the fresh control at a sperm-to-egg ratio at least 50% less than the 8% DMSO treatment alone. The addition of other antioxidants (glycine, melatonin and polyvinylpyrrolidone) did not have any positive effects. The improvement in post-thaw sperm quality by Q10 could be due to its ability to significantly decrease ROS production and lipid peroxidation and significantly increase the motility, plasma membrane integrity, mitochondrial membrane potential, acrosome integrity, DNA integrity and activities of catalase and glutatione. In this study, 37 fatty acids (FAs) were quantified in dwarf surf clam sperm, with 21 FAs being significantly impacted by the cryopreservation with 8% DMSO. Thirteen of these 21 FAs were changed due to the addition of 20 μM Q10 to 8% DMSO, with approximately half of them being improved significantly toward the levels of fresh control, while the remaining half extended further from the trends shown with 8% DMSO treatment. However, no significant difference was found in the percentage of each FA category sum and the ratio of unsaturated/saturated FAs between the two treated groups. In conclusion, the antioxidant Q10 has shown the potential to further improve the sperm cryopreservation technique in bivalves.

Sperm cryopreservation technique has been published in many farmed bivalve species. One of the key factors preventing its application in aquaculture and/or cryobanking is the knowledge gap on the performance of resultant progeny at late developmental stages and subsequent generations. An effective strategy to overcome these challenges is to use a model species with a short generation interval, such as the dwarf surfclam Mulinia lateralis (three months). This study evaluated the parameters key to the development of a non-programmable sperm cryopreservation technique in this species, with a D-stage larval rate similar to control being achieved when the sperm were cryopreserved under the conditions (cryoprotectant agent: 8% dimethyl sulfoxide; equilibration period: 10 min; rack height: 4 cm; thawing temperature: 60°C and sperm to egg ratio: 1100:1) optimized. This technique is the most common method applied in bivalve and the results from this study were all within the ranges published for other bivalve species, indicating this species would be an ideal bivalve model species for addressing cryopreservation evaluation issues that need a long extended time to collect data and/or challenging field experiments.

The relative mortality rate of trochophore larvae exposed to cryopreservation is significantly higher at early larval developmental stages in blue mussels Mytilus galloprovincialis. However, little is known about cryodamage on the larval quality at cellular and molecular modifications in aquatic species, including M. galloprovincialis. This study aimed to investigate the effects of established cryopreservation technique on reactive oxygen species (ROS), total antioxidant capacity, apoptotic and antioxidant enzyme related gene expressions of trochophore larvae in M. galloprovincialis. The results showed that significantly lower D-larvae rate was produced from the cryopreserved trochophore larvae compared to fresh (p <.05). Nevertheless, there was no significant difference in the ROS production, total antioxidant capacity, and small molecular antioxidant capacity between fresh and cryopreserved trochophore larvae (p >.05). Similarly, the cryopreservation procedures had no adverse effects on the apoptotic and antioxidant enzyme related gene expression patterns between fresh and cryopreserved trochophore larvae (p >.05). These results indicate that the cryopreservation technique applied to M. galloprovincialis larvae in this study may not compromise the redox status of the post-thaw trochophore larvae.

High mortalities and highly variable results during the subsequent development of post-thaw larvae have been widely considered as key issues restricting the application of cryopreservation techniques to support genetic improvement programs and hatchery production in farmed marine bivalve species. To date, few studies have been undertaken to investigate the effects of cryodamage at the molecular level in bivalves. This study is the first to evaluate the effect of larval cryopreservation on the epigenetics of the resultant progenies of the Pacific oyster Crassostrea gigas. The results show that the level of DNA methylation was significantly (p < 0.05) higher and lower than that of the control when the trochophore larvae were revived and when they developed to D-stage larvae (day 1 post-fertilization), respectively, but the level returned to the control level from day 8 post-fertilization onwards. The expression of the epigenetic regulator genes DNMT3b, MeCP2, JmjCA, KDM2 and OSA changed significantly (p < 0.05) when the trochophore larvae were thawed, and then they reverted to the control levels at the D- and later larval developmental stages. However, the expression of other epigenetic regulator genes, namely, MBD2, DNMT1, CXXC1 and JmjD6, did not change at any post-thaw larval developmental stage. For the newly thawed trochophore larvae, the amount of methylated H3K4Me1 and H3K27Me1 significantly changed, and the expression of all Jumonji orthologs, except that of Jumonji5, significantly (p < 0.05) decreased. These epigenetic results agree with the data collected on larval performances (e.g., survival rate), suggesting that the effect period of the published cryopreservation technique on post-thaw larvae is short in C. gigas.