Optimization of the TLR7/8 Activation-Based Sorting System for Goat Sperm

Background:Current research indicates that the immunological separation method based on differentially expressed proteins in X- and Y-chromosome-bearing sperm represents a novel approach for sex control. This method achieves X/Y sperm separation by directly activating these differential proteins. However, the technical limitations persist, and the inconsistent separation efficiency.Results:This study established a TLR7/8 activation-based sorting system for goat sperm and developed the corresponding technical protocol. Therefore, based on our laboratory's previous technique for sorting X- and Y-chromosome-bearing sperm using specific proteins, this experiment first investigated the interactions among sperm density, agonist R848 concentration, and incubation time. The results indicated that after binding of the agonist R848 to Toll-like receptors 7/8 on the tail of X-bearing sperm, the motility of X-bearing sperm was inhibited, causing them to accumulate in the lower layer of the sorting system. In contrast, the motility of Y-bearing sperm was unaffected, allowing them to swim to the upper layer. Regarding sperm acrosome integrity, no significant difference was found between the selected experimental groups and the control group, with values exceeding 50%. For sperm plasma membrane integrity, the selected experimental groups showed either a significant increase or no significant difference compared to the control group. SRY immunofluorescence staining revealed that the proportion of X-bearing sperm in the selected experimental groups was 88.29% ± 1.68%, 84.50% ± 0.18%, and 80.32% ± 1.94% in one set, and 80.95% ± 0.00%, 88.89% ± 0.00%, and 84.03% ± 2.38% in another. Subsequently, the effect of centrifugal force on the elution of R848 was studied. The results demonstrated that centrifugation could serve as an effective method for eluting the agonist R848. It was found that the optimal centrifugal force for sorted X-bearing sperm from dairy goats was 800 × g, while that for sorted Y-bearing sperm was 700 × g. Finally, the optimized sorting system was used for sperm separation. Via in vitro fertilization and subsequent embryo sex identification in goats, 45 embryos were analyzed. The results showed that in the X-sorting system experimental group, there were 21 male embryos (XY), accounting for 84.00%, and 4 female embryos (XX), accounting for 16.00%. In the Y-sorting system experimental group, there were 4 male embryos (XY), accounting for 20.00%, and 16 female embryos (XX), accounting for 80.00%.Conclusions:This study demonstrates that the interaction of goat sperm density, R848 concentration, and incubation time significantly impacts sperm sorting efficacy, while varying centrifugal forces enhances efficiency R848 elution. Specifically, the optimized system for sorting X-sperm-specific proteins in dairy goats uses a sperm density of 6×108 cells/mL, adds 1.4 μmol/L R848 to the sorting medium, undergoes two incubation sessions totaling 30 minutes, collects the lower sperm fraction, and performs elution via centrifugation at 800×g twice; conversely, the Y-sperm sorting system for dairy goats uses a sperm density of 6×108 cells/mL, adds 0.6 μmol/L R848 to the sorting medium, undergoes two incubation sessions totaling 45 minutes, collects the upper sperm fraction, and performs elution via centrifugation at 700×g twice.