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Flow cytometry is a common method for sex control in dairy cows and can effectively sort X\/Y sperm, but its equipment is expensive. Recent studies have shown a new inexpensive potential method that utilizing R848 (a TLR7\/8 agonist) to effectively stratify mammalian X\/Y sperm by activating X sperm specific proteins. However, the molecular mechanism has not been fully elucidated. In this study, R848 altered X sperm motion trail and stratified X\/Y sperm by activating TLR7\/8, without significant negative effect on sperm quality. Besides, we identified a total of 60 differential metabolites between the semen of control and R848\u2010treated group (17 upregulated, 43 downregulated) by LC\u2013MS and GC\u2013MS. The differential metabolites mainly clustered in glycolysis, TCA cycle and CoA biosynthesis pathways, which are related to sperm energy metabolism. Next, we verified and found that the protein expression levels of LKB1, AMPK, CPT1, and phosphorylated ACC in the R848\u2010treated group were significantly reduced compared to control group. Conversely, the phosphorylation levels of PI3K, NF\u03baB, GSK3\u03b1\/\u03b2, and I\u03baB\u03b1 significantly increased, while the activities of glucose metabolism\u2010related enzymes such as hexokinase (HK), phosphofructokinase\u20101 (PFK1), pyruvate kinase (PK), and pyruvate dehydrogenase (PDH) were significantly decreased in R848\u2010treated group. These results suggest that R848 modulates glycolysis and fatty acid \u03b2\u2010oxidation of X sperm via the LKB1\/AMPK signaling pathway, therefore diminishing ATP level and motion performance of X sperm, achieving the separation from Y sperm of dairy cows.<\/jats:p>","DOI":"10.1096\/fj.202500923r","type":"journal-article","created":{"date-parts":[[2025,7,2]],"date-time":"2025-07-02T10:54:26Z","timestamp":1751453666000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["TLR7\/8 Agonist R848 Regulates X Sperm Energy Metabolism in Dairy Cows via Inhibiting Glycolysis and Fatty Acid \u03b2\u2010Oxidation by LKB1\/AMPK Pathway"],"prefix":"10.1096","volume":"39","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2362-3011","authenticated-orcid":false,"given":"Xing","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology Northwest A&amp;F University  Yangling Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-7208-944X","authenticated-orcid":false,"given":"Songmao","family":"Guo","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology Northwest A&amp;F University  Yangling Shaanxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-4059-2469","authenticated-orcid":false,"given":"Zhangtao","family":"Hu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology Northwest A&amp;F University  Yangling Shaanxi 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