The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r = 0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.