FAM216A and CCDC166 are not essential for male fertility

Context: Bull fertility is crucial for livestock reproductive efficiency, yet studies on bull sperm and its molecular mechanisms remain limited. Direct research in cattle is challenging, while gene-knockout mouse models provide an effective alternative for investigating gene functions. Aims: This study aimed to investigate the role of two testis-enriched genes, FAM216A and CCDC166, in male fertility. Methods: We evaluated the conservation of FAM216A and CCDC166 in mammals through phylogenetic analysis and sequence alignment. Their expression patterns were investigated using published single-cell transcriptome data and RT-PCR. The role of these genes in reproductive function was assessed through knockout mouse models, histochemical staining, and fertility analysis. Key Results: FAM216A and CCDC166 are highly conserved across mammalian species and abundantly expressed in testis tissues. Knocking out these two genes does not affect male fertility in mice. Conclusions: Knockout of FAM216A and CCDC166 did not impair male fertility in mice, suggesting functional redundancy. Under the experimental conditions applied, they do not appear to be key factors for male fertility, and similar outcomes may be expected in bulls. Implications: Although FAM216A and CCDC166 are not essential for male fertility in mice, this study provides critical guidance for future research. It facilitates more targeted investigations of other candidate genes and avoids unnecessary studies, while also laying a foundation for exploring their functions in other physiological processes and diseases.