Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm

A large number of female goats are needed for the dairy goat industry; therefore, the development of a method to ensure the birth of more females than males in a single pregnancy will lead to economic benefits. Increasing the number of X-sperm would be an effective way to increase the proportion of female offspring. In this study, goat semen was incubated at pH 7.4 in alkaline diluent combined with resiquimod (R848) and the number of X-sperm was enriched by the swim-up method. The percentage of X-sperm was determined using the double TaqMan qPCR method. Sperm total motility, progressive motility, average path velocity, straight-line velocity, and curvilinear velocity were measured using a computer-aided sperm analysis system, and the functional parameters of the sperm plasma membrane, the acrosome, mitochondrial activity, ATP content, and reactive oxygen species levels were also measured. Lastly, the ratio of female embryos was determined by in vitro fertilization, and the number of female kids and the pregnancy rate of does was assessed by artificial insemination. The results showed that dilution of semen in an alkaline buffer containing R848 could enrich the number of X-sperm to 85.57% ± 3.27%. The progressive motility, average path velocity, straight-line velocity, curvilinear velocity, mitochondrial activity, and ATP level of the collected X-sperm-enriched semen were significantly reduced, but its total motility, plasma membrane, and acrosome were not affected. The in vitro fertilization experiments showed that the rate of female embryo production using X-sperm-rich seminal fluid could reach 83.25% (174/209), which was significantly higher than the proportion of female embryos in the control group, 47.71% ± 1.80% (104/218). As determined by artificial insemination, the number of female kids in the test group increased by 62.79% (243/387), which was significantly higher than that in the control group (47.65%, 193/405). There was no significant difference in pregnancy rate between the test group and the control group (71.71% vs. 78.48%). Therefore, this study demonstrated that use of a pH 7.4 diluent containing R848 is a simple and effective method of X-sperm enrichment for dairy goat production. Its application would allow does to produce more female offspring for herd expansion and milk production.