Fertilization by short-term stored sperm alters DNA methylation patterns at single-base resolution in common carp (Cyprinus carpio) embryos

Sperm after short-term storage in vitro is widely used for artificial fertilization in aquaculture. It has been shown that short-term storage affects sperm motility characteristics, resulting in diminished fertility. However, the detrimental effects of short-term sperm storage on embryos development have remained unexplored in single-base methylome resolution. The main aim of the present study was to investigate DNA methylation in the offspring of common carp (Cyprinus carpio) derived from short-term stored sperm. Sperm were stored in artificial seminal plasma on ice (0-2 degrees C) for 0, 3 and 6 days in vitro, fertilization was performed using oocytes from a single female, and embryos were collected at the mid-blastula stage. In the DNA methylation study, DNA from both sperm and embryos was extracted and analysed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Concurrently, DNA methylation levels of embryos in single base were evaluated through whole genome bisulfite sequencing (WGBS). Sperm storage showed negative effects on sperm motility, viability, and DNA integrity, but had no effect on global DNA methylation of spermatozoa and resulting embryos. Results from the WGBS showed that methylation of 3313 differentially methylated regions (DMRs)-target genes was affected in the embryos fertilized with the 6-day-stored sperm, and the identified DMRs were mainly involved in cell adhesion, calcium, mitogen-activated protein kinase and adrenergic signalling, melanogenesis, metabolism and RNA transport. Such results suggest that prolongation of storage time may have certain impacts on embryonic development. These initial results provide valuable information for future consideration of the DNA methylome in embryos generated from short-term stored sperm, which are used for genetic management of broodstock in aquaculture.