Sex selection technology is a pivotal innovation in animal husbandry and reproductive biotechnology. It significantly enhances livestock productivity, optimizes genetic resources, and scientific advancement. In dairy production systems, the preferential production of female calves not only increases milk yield but also mitigates economic losses associated with non-productive male offspring [1]. Conversely, beef production benefits from male-biased breeding through accelerated growth rates and superior meat quality [2], [3]. Currently, most methodologies center on pre-fertilization X/Y sperm separation for the purpose of sex ratio manipulation. However, traditional techniques such as centrifugation, electrophoresis, and immunological methods demonstrate subpar efficiency and reproducibility [4], [5], [6], [7], [8]. Although flow cytometry-based sperm sorting is regarded as the gold standard in the field of sperm sexing [9], [10], its widespread implementation is hampered by exorbitant instrumentation costs, high technical complexity, patent limitations, sperm injury during the sorting procedures, and the resulting loss of fertility [11], [12], [13], [14], [15], [16], [17], [18], [19].

Recent investigations into X sperm-specific biomarkers have unveiled novel opportunities for sperm separation. Toll-like receptors 7 and 8 (TLR7/8), initially recognized as regulators of the innate immune system [20], [21], exhibit distinct expression patterns in X sperm derived from murine and caprine sources. In murine models, TLR7 is localized to the mid-posterior region of the flagellum, while TLR8 is found in the sperm head and anterior flagellum [22]. In the case of Guanzhong dairy goats, research has indicated that TLR7/8 activation modulates X sperm motility through the regulation of the GSK3α/β-hexokinase pathway [23]. Although the expression of bovine TLR7/8 has yet to be comprehensively characterized, similarities in ATP-mediated motility control across species suggest the existence of conserved regulatory mechanisms [24]. Notably, studies on dendritic cells have demonstrated that TLR7/8 activation leads to the phosphorylation of GSK3α/β and NF-κB, thereby suppressing hexokinase activity and ATP synthesis-a pathway that holds potential for targeted manipulation of X sperm metabolism [25], [26].

The advent of high-potency TLR7/8 agonists has driven innovation in sex selection strategies. Compared to the traditional agonist R848 (TLR7/8 EC50: 0.075 μM and 0.48 μM), the pyridine [3,2-d] pyrimidine derivative (24e) has emerged as a more potent dual agonist, demonstrating TLR7/8 activation (EC50: 0.024 μM and 0.01 μM) and enhanced induction of NF-κB [27]. Despite its pharmacological superiority, the mechanism of 24e in bovine remains unclear, and its application in bovine sex selection has yet to be investigated. This study aims to address these knowledge gaps by precisely characterizing TLR7 and TLR8 expression in bovine sperm, developing 24e-mediated sex-sorting protocols, and elucidating the downstream signaling impacts on sperm motility and metabolic function. TLR7 and TLR8 are key receptors for energy metabolism in mammalian sperm. In this context, sex selection is achieved by inducing energy metabolism differences between X and Y sperm to enable sperm stratification. The results of this study not only establish a novel approach for preparing bovine sex-controlled semen but also provide mechanistic insights relevant to mammalian reproductive biotechnology.