Integrative Analysis of MTHFR Gene Polymorphisms and Semen Quality Traits in Bulls: A Bioinformatics and Association Study

Background: This section clearly introduces the goal of the study and the genetic focus (MTHFR gene and SNPs in relation to bull semen). Methods: We conducted polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and Sanger sequencing of the MTHFR gene in bulls from four breeds: Sinan, Guanling, Australian Wagyu and Simmental cattle. Result: Four SNPs were identified: g.41883859T greater than C (Exon 8), g.41884250G greater than A/g.41884310T greater than C (Exon 9) and g.41886545T greater than C (Intron 12). These SNPs displayed moderate polymorphism and conformed to Hardy-Weinberg equilibrium. Furthermore, strong linkage disequilibrium was observed among the four loci. The dominant haplotype H1 and the diplotype H1H1 were identified. Associations revealed that the g.41883859T greater than C TT genotype increased ejaculate volume but reduced sperm density, while the g.41884250G greater than A AA genotype improved sperm density and motility. The g.41884310T greater than C TT genotype enhanced motility parameters. The H1H1 diplotype was associated with superior motility. Bioinformatics analysis suggested that the g.41883859T greater than C polymorphism altered the MTHFR mRNA structure and MTHFR interacted with enzymes involved in one-carbon metabolism.