Abstract
Background:
Sperm separation is a reproductive biotechnology that separates X- and Y-chromosome-bearing sperm to predetermine offspring sex, offering major benefits for livestock breeding programs. The discovery of an easy, inexpensive, and effective method will be very useful as an alternative to flow cytometry. The swim-up method is a method that meets these criteria. Combining the swim-up method with the addition of a substance to separate X- and Y-chromosome-bearing sperm is very helpful in developing more efficient methods. The activation of TLR 7/8 using the ligand R848 is related to sperm morphometry, the proportion of X and Y chromosome-bearing sperm, and sexed sperm quality.

Aim:
This study aimed to determine the effect of activating TLR 7/8 using the ligand R848 on sperm morphometry, the proportion of X- and Y-chromosome-bearing sperm, and sexed sperm quality in buck.

Methods:
Fresh semen samples were collected from 3-year-old bucks using an artificial vagina. A completely random design was used with four treatment levels of ligand (R848) and seven replications. The level treatment of ligand (R848) consisted of T0 = 0 µM, T1 = 0,3 µM, T2 = 0,6 µM, and T3 = 0,9 µM. Morphometry, the proportion of X-Y chromosome-bearing sperm, immunostaining, and sexed sperm motility were the evaluated parameters.

Results:
Sperm length ranged from 7.02 to 9.99 µm and 8.54 µm on average. Sperm size ranges from 3.08 – 6.01 µm, with an average of 4.61 µm. The sperm head area ranges from 28.02 – 34.14 µm 2. The proportion of X and Y sperm was significantly different between the upper and bottom layers at T3 but not for other treatments. The use of ligand (R848) significantly (p<0.05) affected sperm motility at the bottom layer, but not in the upper layer. The results show that at the bottom layer, the total motility of T3 (79.56%) is significantly lower than that of T0 (89.78%), T1 (87.18%), and T2 (87.01%), whereas T0, T1, and T2 are not significantly different.

Conclusion:
Ligan (R848)-mediated activation of Toll-like receptors 7/8 selectively suppresses motility of X-chromosome-bearing sperm in buck, enabling effective enrichment of X-sperm via simple swim-up separation. Morphometric analysis, motility assessment, and immunofluorescence analysis confirmed that the bottom swim-up fraction was significantly enriched in functionally impaired X-sperm.

Key words: Buck; Ligand (R848); Morphometry; Sperm quality; Sperm sexing.