Study On Differences Of DNA Methylation And Nuclear Morphology Between Bull Sex-controlled X And Y Spermatozoa

There are differences between X and Y sperm,of which surface charge and DNA content have been found.While the differences of their DNA methylation and nuclear morphology are still unclear,and that is what we are looking at.The epigenetic and morphological differences between sex-controlled X and Y sperm in Holstein bulls were analysed,laying the foundation for elucidating the sex differences of mature sperm,revealing their genetic regulation mechanisms and improving the flow cytometry and cryopreservation techniques during the production of bull sex-controlled sperm.DNA methylation plays an important role in the regulation of gametes maturation and embryonic development.In order to clarify the DNA methylation status of mature sperm in Holstein bulls and identify the differentially methylated regions(DMR)and differentially methylated genes(DMG)in X and Y sperm,we sequenced and compared them in three Holstein bulls by Whole Genome Bisulfite Sequencing(WGBS),and the main results were obtained as follows:(1)The DNA of sex-controlled X and Y spermatozoa from 3 Holstein bulls was sequenced using Hi Seq-PE150 platform.A total of480 G of data volume was obtained,with an average of about 80 G per sample;(2)DNA methylation profiles of sex-controlled X and Y sperm in Holstein bulls were plotted at a single-base resolution;(3)there were 991 genes in regions with methylation levels higher in X sperm than in Y sperm,which were mainly enriched in intracellular ion transport,cell proliferation,apoptosis and migration,signal transmission and other pathways;(4)there were 1051 functional genes in regions with methylation levels lower in X sperm than in Y sperm,except for basic life activities such as ion transport and signal transmission,these genes were mainly enriched in ATP-binding,endoplasmic reticulum,and Golgi pathways,of which 14 genes were located in promoter regions.Sperm nuclear morphology is important for sperm quality.Fluorescent cell separation and sperm freezing are the main techniques for semen production,however,the effects of these two on sperm nuclear size and morphology are not well understood.In order to identify the difference of nuclear size and morphology between sex-controlled X and Y sperm of Holstein bulls and clarify the changes of nuclear size and morphology before and after freezing or flow separation,we analyzed the difference of nuclear size and morphology of sex-controlled X and Y sperm and studied the effects of flow separation and freezing on the nuclear size and morphology of bulls sperm.The main results obtained included:(1)the sperm images of 15 Holstein bulls were taken using a fluorescence microscope,and about 37,000 images were obtained by Nuclear Morphology Analysis with image J plugin;(2)there was no significant difference in the nuclear size and morphology of sex-controlled X and Y sperm in Holstein bulls;(3)flow cytometry and freezing,had a significant effect on the sperm nuclear size of Holstein bulls and a relatively small effect on the nuclear morphology;(4)the effect of freezing on the nuclear size of Y sperm was more obvious than that of X sperm in Holstein bull.The results of this study will provide a reference for improving or perfecting the production of bull sex-controlled sperm including fluorescent cell separation and cryopreservation,so as to promote the improvement of bull performance and economic benefits of dairy cows.