As eastern oyster (Crassostrea virginica) aquaculture continues to expand, technological advancements are needed to support efficiency and sustainability. Access to high-quality gametes is one of the biggest restricting factors in aquaculture. Germplasm cryopreservation offers a solution as it enables continuous access to gametes by preserving genetic material. However, for oysters there is great individual variability in sperm quality and resistance to cryoinjury at sublethal temperatures. Bioinformatic assessment of sperm cell quality is an emerging tool that can be used to understand and optimize gamete quality for use in hatcheries. Residual mRNAs in conjunction with non-coding RNAs (such as miRNAs) can provide an understanding of molecular mechanisms and genetic regulation involved in sperm development and function. Our objectives were to investigate how miRNA and mRNA profiles relate to fresh and frozen-thawed sperm quality, based on percent motility and swimming velocity. We hypothesized that non-coding miRNAs play a role in the genetic regulation of mRNA transcription related to oyster sperm function.
Sperm were stripped from 20 oysters and evaluated using Computer Assisted Sperm Analysis software. Sperm were cryopreserved and reevaluated post cryo-storage. Males were ranked for quality and cryotolerance based on fresh and recovered post-thaw motility and velocity. An integrated analysis of miRNA and mRNA transcription was conducted for six oysters with low-quality vs. high-quality fresh sperm (n = 12 total) (Fig. 1A), and six oysters with low-cryotolerant vs. high-cryotolerant sperm (n = 12 total) (Fig. 1B). We found 119 differentially expressed genes (DEGs) from mRNA and miRNA families related to sperm quality (Fig. 1C), and 163 DEGs were identified as relating to cryotolerance (Fig. 1D). Functional annotation of DEGs revealed enrichment associated with the plasma membrane, channel activity, and oxidative stress, all of which impact a cells ability to fertilize. Interactions between miRNA regulation and mRNA transcription were also assessed, and 51 potential significant pairs were found. Our findings reveal RNAs that play critical roles in the regulation of oyster sperm quality and provide insight into mechanisms impacting sperm kinematics. Functional understanding of eastern oyster sperm lays the groundwork for precision breeding and more sustainable, profitable aquaculture.