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Spermatozoa are the cells the most commonly used for cryopreservation of valuable genetic resources in aquaculture. It is known that fish spermatozoa transmit to the embryo not only their genetic but also their epigenetic profile, particularly DNA methylation. Therefore, any alteration of the DNA methylation profile in spermatozoa induces the risk of transmitting epigenetic alterations to the offspring. The aim of this study was to assess whether sperm cryopreservation can alter DNA methylation profile in rainbow trout spermatozoa. Its purpose is to know the risk of using cryopreserved sperm for the offspring. To trigger variable cellular response after cryopreservation, spermatozoa were cryopreserved with different cryoprotectants : dimethyl sulfoxide, methanol and glycerol. We confirmed that each of them provided a different level of cellular protection according to the cellular parameter (membrane and mitochondria quality, motility, fertilization ability). Following the analysis of DNA methylation by RRBS, we did not see an overall effect of cryopreservation. At the genome level, we were able to identify a small number (335 to 564) of differentially methylated cytosines (DMCs). Few are common between cryoprotectants, and no correlation was observed between the number of DMCs and cellular alterations after cryopreservation. We suggest that the DMCs indicate the presence of potentially sensitive regions to cryopreservation. In conclusion, sperm DNA methylation of rainbow trout would be only slightly sensitive to cryopreservation. Affected regions need to be confirmed before studying the impact on offspring.