The present study aimed to contribute to the limited research on buffalo (Bubalus bubalis)
semen traits by incorporating genomic data. A total of 8465 ejaculates were collected. The genotyping
procedure was conducted using the Axiom® Buffalo Genotyping 90 K array designed by the
Affymetrix Expert Design Program. After conducting a quality assessment, we utilized 67,282 SNPs
genotyped in 192 animals. We identified several genomic loci explaining high genetic variance by
employing single-step genomic evaluation. The aforementioned regions were located on buffalo chromosomes
no. 3, 4, 6, 7, 14, 16, 20, 22, and the X-chromosome. The X-chromosome exhibited substantial
influence, accounting for 4.18, 4.59, 5.16, 5.19, and 4.31% of the genomic variance for ejaculate volume,
mass motility, livability, abnormality, and concentration, respectively. In the examined genomic
regions, we identified five novel candidate genes linked to male fertility and spermatogenesis, four
in the X-chromosome and one in chromosome no. 16. Additional extensive research with larger
sample sizes and datasets is imperative to validate these findings and evaluate their applicability for
genomic selection.