Control of offspring sex ratio by injection of Toll-like Receptor 7/8 agonist Resiquimod into father mice

Sex control technologies hold great promise for enhancing animal production efficiency. X chromosome-linked toll-like receptor 7/8 (TLR7/8) genes are specifically expressed in mammalian X sperm but not Y sperm. In vitro treatment with the TLR7/8 ligand R848 can separate X and Y sperm by selectively reducing X sperm motility via inhibition of energy production. Fertilization using such treated sperm leads to sex-skewed embryos or offspring. However, whether R848 can be used in vivo to control offspring sex remains unclear. This study tested this hypothesis. We found that the X/Y sperm ratio in the upper layer of caudal epididymal sperm collected at 24 and 72 hours after intraperitoneal R848 injection was 24.09%/75.91% and 63.16%/36.84%, respectively. Mating superovulated females with males at these time points resulted in male- and female-biased litters. Using the upper layer of epididymal sperm collected at 24 and 72 hours after R848 injection for in vitro fertilization, the embryo sex ratio also skew toward male and female, respectively. Our findings demonstrate that intraperitoneal injection of R848 into male mice can dynamically modulate the proportion of mature X and Y sperm in the cauda epididymis, allowing offspring sex control through timed mating. This approach provides a simple and potentially practical method for effective sex ratio intervention in mammals.