Effect of cryopreservation on DNA methylation in rainbow trout spermatozoa.
Sperm cryopreservation is a widely used biotechnology in fish. In the spermatozoa, DNA bears the genotype of the donor male, whose transmission to the progeny will allow the restoration of valuable genetic resources. Additionally, it is now known that fish spermatozoa epigenetic profile, particularly DNA methylation, is transmitted to the embryo. As a consequence, any alteration of DNA methylation in spermatozoa induces the risk of transmitting epigenetic alterations to the offspring leading to altering the progeny. Published data on cryopreservation impact on DNA methylation are limited and contradictory because they are species and cryoprotectant dependent. The aim of this study is to assess whether cryopreservation of rainbow trout spermatozoa can alter their DNA methylation profile. We also wanted to investigate whether the cryoprotectant molecules play a role in DNA methylation alterations. Sperm from rainbow trout mature males (n=12) was cryopreserved with dimethylsulfoxide (DMSO), methanol (MeOH) or glycerol and compared to fresh sperm (paired analysis). DNA methylation was studied by Reduced Representation Bisulfite Sequencing (RRBS). After adapter trimming and quality control of the raw data (Trimgalore https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), bisulfite treated sequencing reads were mapped to the rainbow trout genome (Swanson isolate: Oncorhynchus_mykiss.Omyk_1.0.dna.toplevel.fa.gz). Methylation call was achieved with Bismark (https://www.bioinformatics.babraham.ac.uk/projects/bismark/). The RRBS data of each sample covered 4 % of the reference genome, and comparison was achieved between fresh and cryopreserved samples for all CpG sites present in at least 6 males (0.7 to 1.5 x 106 CpGs analyzed with the paired design of DSS http://bioconductor.org/packages/release/bioc/src/contrib/DSS_2.34.0.tar.gz). We observed that sperm cryopreservation changed the DNA methylation level of very few CpG sites ( 335 to 564) and no differentially methylated regions were identified. Our project showed that RRBS allows the analysis of many biological replicates, whereas the amount of CpG available for paired comparison between fresh and cryopreserved samples remains low. Some CpG sites were affected by cryopreservation, but the biological significance of these observations must be investigated further.