Sex-control technology have great economic value and is one of the hot topics in livestock research. To produce more milk, dairy farmers prefer female offspring. X/Y sperm separation is an effective method for offspring sex control. Currently, the major commercial production method for sperm separation is flow cytometry sorting in cattle. However, flow cytometry requires expensive equipment and long sorting times. So, a simple and inexpensive method for producing a higher number of dairy cows is required. In this study, R848 activates toll-like receptor 7/8 (TLR7/8), thereby separating X from Y sperm. The results showed TLR7/8 is expressed in the tail of X sperm. Immunofluorescence (IF) of testes, epididymis, and ejaculate shows that the number of TLR7/8 sperm cells is up to 50 %. Furthermore, TLR7/8 agonist (R848) affects mitochondrial function through the PI3K/GSK3α/β/hexokinase and PI3K/NFκB/hexokinase signalling pathways, inhibiting X sperm motility, while the motility of Y-sperm remains unchanged. The difference in sperm motility causes Y sperm (with high motility) to move to the upper layer and X-sperm (with low motility) to the lower layer allowing the separation of X and Y sperm. Based on this study, we reveal a simple and effective method for enriched X/Y sperms from cattle.
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