Using an optimal method to separate low-motile and high-motile sperms in fresh bull semen treated with Resiquimod with the aim of separating sperms containing X from Y chromosomes

This study aimed to adjust and optimize the method of separating low-motile and high-motile sperms in fresh bovine semen treated with resiquimod, an agonist of Toll-like 7/8 receptors present in X-chromosome-containing sperms and causes a decrease in ATP and energy production. This method could separate X and Y sperms in fresh cow semen. Semen samples were obtained from five bulls in three replications, treated with 0.3 µM resiquimod, and subjected to swim up in two separation tools A and B (made of polyethylene and has an angle of 60 degrees for tool A and 80 degrees for tool B). There was no significant difference in the percentage of motile sperm in the upper layers of the groups (p>0.05); however, the percentage of motile sperm was higher in the lower layer of the control group (p<0.05). The ratio of the number of counted sperm in the upper layer of the control group was significantly more than the two treated groups (p<0.05) and was higher in tool B than in tool A (p<0.05). Equating the ratio of sperms counted in the upper layer to the total number of sperms counted in the upper and lower layers showed that the number of sperms in the upper layer of tool A is almost half of the control group. It is concluded that the use of 0.3 micromol of resiquimod and swim-up in instrument A can be effectively used to separate motile sperm from immobile sperm to separate sperm containing X chromosome from Y.