Bos taurus bulls have been used widely for genetic improvements of Bos indicus cattle in developing countries. However, male fertility has not improved in sync with production performances in resultant crossbred cattle. The molecular basis of influence of genetic background (Bos taurus, Bos indicus, Bos taurus x Bos indicus) on bull fertility has not been understood well. In the present study, candidate genes involved in mitochondrial function (COX5A and COX3) and fertility and embryogenesis (YWHAZ, CHMP5 and GTSF1L) were studied in contrasting genetic backgrounds of bulls. For this purpose, semen from six bulls employed for artificial insemination (AI) purposes per genetic background was used. Since retrieval of RNA from spermatozoa is challenging, a modified protocol was worked out that yielded significant harvest of RNA for downstream applications. The relative expression of genes was studied by real-time PCR in taurine and crossbred in comparison to indicus bulls. The YWHAZ, CHMP5 and GTSF1L genes showed a significant higher expression in the spermatozoa of exotic and crossbred cattle (p <
0.05) vis-à-vis indigenous bulls. Since the bulls are of comparable fertility in terms of seminal attributes and genetic merit of their own order but varying in genetic background, the differences in gene expression may be set due to their biological differences that lead to benchmarking in relative reproductive success of taurine, indicine or crossbred bulls as male genetics.