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Protamine deficiency, thiols, and disulfide bonds were assessed and compared with previously published data for DNA fragmentation index (DFI) and high DNA stainability (HDS). In addition, in vitro embryo development and sperm DNA methylation were assessed using semen samples from 16 of these bulls. The percentages of DFI and HDS were negatively associated with NR56 and cleavage rate and positively associated with sperm protamine deficiency (<jats:italic>p<\/jats:italic>\u2009&lt;\u20090.05). Significant differences in cleavage and blastocyst rates were observed between bulls of high and low NR56. However, once fertilization occurred, further development into blastocysts was not associated with NR56. The differential methylation analysis showed that spermatozoa from bulls of low NR56 were hypermethylated compared to bulls of high NR56. Pathway analysis showed that genes annotated to differentially methylated cytosines could participate in different biological pathways and have important biological roles related to bull fertility. 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