TLR7/8 Signalling Affects X-Sperm Motility via the GSK3 α/β-Hexokinase Pathway for the Efficient Production of Sexed Dairy Goat Embryos

Abstract BackgroundGoat milk is most similar to human milk because of its abundant nutrients and ease of digestion. To derive more economic benefits, farmers need to obtain offspring with more does; however, the buck to doe sex ratio of offspring is approximately 50%. At present, artificial insemination after separation of X/Y sperm using flow cytometry is the primary means to control the sex of livestock offspring. However, flow cytometry has not been successful in separating X/Y sperm for sexing control in dairy goats.ResultsIn this study, a novel, simple goat sperm-sexing technology that activates the toll-like receptor 7/8 (TLR7/8), inhibiting X-sperm motility, was investigated. Results showed that TLR7/8 coding goat X-chromosome was expressed in approximately 50% of round spermatids in testis' cross-sections and sperm in the epididymis' cross-sections and ejaculate. Importantly, TLR7/8 was located at the tail of X-sperm. Upon TLR7/8 activation, phosphorylated forms of glycogen synthase kinase α/β (GSK3 α/β) and nuclear factor kappa-B (NFκB) were detected in the X-sperm, reducing mitochondrial activity, ATP levels, and sperm motility. High-motility Y-sperm segregated to the upper layer and low-motility X-sperm to the lower layer. Following in vitro fertilisation using the lower layer TLR7/8-activated sperm, 80.52 ± 6.75% of the embryos were XX females. TLR7/8-activated sperm was used for in vivo embryo production via the superovulatory response, and 88.89% of the embryos were XX females.ConclusionsOur study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3 α/β-hexokinase pathway, facilitating the efficient production of sexed dairy goat embryos.