Loss of Myostatin Affects m6A Modification but Not Semen Characteristics in Bull Spermatozoa

N6-methyladenosine (m6A) modification is a key methylation modification involved in reproductive processes. Myostatin gene editing (MT) in cattle is known to enhance muscle mass and productivity. However, the changes in m6A modification in MT bull sperm remain poorly understood. In the MT and wild-type (WT) groups, we identified 25,542 and 22,253 m6A peaks, respectively, mainly concentrated in the coding sequence (CDS) and 3' untranslated region (UTR) of genes. The MT group showed an increase in gene transcription, but there was no significant difference in the overall m6A peaks pattern. There was also no significant difference in m6A motif and chromosome distribution between MT and WT groups. Most genes had less m6A modification sites. A total of 1120 m6A peaks were significantly different, corresponding to 1053 differentially m6A-methylated genes (DMMGs). These DMMGs are mainly associated with G protein-coupled receptor signaling pathways and the overall composition of the cell membrane. Furthermore, an MCL clustering analysis of 111 differentially m6A-methylated and expressed genes identified seven key genes (RHOA, DAAM1, EXOC4, GNA12, PRICKLE1, SCN1A, and STXBP5L), with the cytoskeleton and migration-related gene, RHOA, being the most important gene located at the center of the gene network. However, the analysis of sperm morphology and motility indicated no significant changes in semen volume, sperm count, sperm viability, plasma membrane integrity, acrosome membrane integrity, or mitochondrial membrane integrity. This study provides a map of m6A methylation in spermatozoa from MT and WT bulls, identifies key differential m6A genes that are affected by the myostatin gene but do not affect sperm morphology and viability in MT bulls, and provides a theoretical basis for the breeding quality of MT bulls.