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

ABSTRACT

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.