Y-Sperm Enrichment Through TLR 7/8 Validated Through Molecular and Biochemical Approaches in Sahiwal Bull.

Earlier studies reported the X-linked Toll-like receptors 7 and 8 (TLR7/8) in goats, dogs and mice spermatozoa. Conversely, Resiquimod R848, a TLR7/8-agonist, specifically suppresses the motility of X-spermatozoa. Basing above, this study focused on developing a simple immunological approach for producing Y-enriched Sahiwal bull semen using R848, validated through molecular and biochemical assays. Each ejaculate (N = 24; 8 ejaculate × 3 bulls) was divided into Group-I (Control, routine processing) and Group-II (Resiquimod-based sorting) before cryopreservation. Sorting efficiency was evaluated and validated by immunofluorescence for localization and qPCR to determine sperm sex-ratio by detecting X- and Y-bearing sperm through marker genes: PLP and SRY, respectively. The study showed that sex-sorting using Resiquimod at a 0.6 μM concentration yields a fraction rich in Y-cells, preserving the viability and motility at the fresh stage in sorted spermatozoa. The acrosome integrity (fresh stage), and plasma membrane- and DNA-integrity (frozen-thaw) did not differ significantly between G-I and G-II. Though the recovery rate was low, the technique achieves a high level of purity (> 78%) in sorted Y-cells. Standard curves for SRY and PLP genes confirmed a natural 1:1 ratio in the unsorted samples. In Y-enriched samples, Y-bearing sperm made up 78.18%, effectively shifting the sex-ratio towards male offspring. The immunofluorescence study confirmed the presence of TLR 7/8 receptors on the cranial head, acrosome and tail region in cattle sperm, revealing fewer than half of the sorted cells showing TLR7/8 expression on the tail, whereas it was nearly half in the unsorted sample. In conclusion, the study shows the effectiveness of Resiquimod-based sorting in harvesting motile, viable Y-cells without significantly affecting quality.