Research Article | 12 May 2026

Enhancement of Y-bearing sperm enrichment in swamp buffalo using a TLR7/8 agonist (R848) combined with discontinuous Percoll density gradient centrifugation

Naela Wanda Yusria Dalimunthe1,2, Pakpoom Navanukraw3, Saksiri Sirisathien3, Sujira Thammawung4, Patchara Phuektes5, and Panisara Kunkitti3Show more
VETERINARY WORLD | Article No. 10 | pg no. 1943-1953 | Vol. 19, Issue 5 | DOI: 10.14202/vetworld.2026.1943-1953
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Abstract

          
Background and Aim: Sex sorting of sperm is a valuable reproductive biotechnology for improving herd productivity and genetic management in livestock. Recent advances have explored Toll-like receptor 7/8 (TLR7/8) activation using resiquimod (R848) as a biological approach to selectively modulate sperm function. However, its application in swamp buffalo remains limited. This study aimed to evaluate the effectiveness of R848 combined with discontinuous Percoll density gradient centrifugation (PDGC) for enriching Y-bearing sperm in frozen–thawed swamp buffalo semen and to determine the optimal concentration to maximize enrichment without compromising sperm quality. 
Materials and Methods: Frozen–thawed semen from five swamp buffalo bulls (Bubalus bubalis carabanensis) was incubated in tris-based medium containing 0, 0.03, 0.3, and 1 μM R848 at 37°C for 45 min. Following incubation, samples were subjected to PDGC (45%/90%) to isolate motile sperm populations. The pellet fraction was reprocessed and evaluated for sperm kinematics using computer-assisted sperm analysis. The relative abundance of Y-bearing sperm was quantified by SYBR-based quantitative polymerase chain reaction targeting the BRY4a gene, normalized against Glyceraldehyde-3-phosphate dehydrogenase. 
Results: Quantitative polymerase chain reaction analysis demonstrated that treatment with 0.03 μM R848 resulted in a significant increase in BRY4a expression (approximately 2.6-fold) compared to other groups (p < 0.05), indicating enhanced Y-chromosome enrichment. Sperm motility parameters showed no significant differences in total and progressive motility between treated groups (0, 0.03, and 0.3 μM) and post-thawed controls (p > 0.05). However, velocity-related parameters such as average path velocity and curvilinear velocity increased significantly, while linearity decreased, reflecting a hyperactivation-like motility pattern. In contrast, the highest concentration (1 μM) negatively affected overall sperm motility and kinematic performance. 
Conclusion: The combination of low-dose R848 (0.03 μM) and PDGC effectively enhances the relative proportion of Y-bearing sperm in frozen–thawed swamp buffalo semen without detrimental effects on motility. This approach represents a cost-effective and field-applicable alternative to conventional sperm sexing methods, with potential applications in buffalo breeding and conservation programs. Further validation using larger sample sizes and fertility trials is recommended. 
          
Keywords: buffalo breeding, density gradient centrifugation, frozen–thawed semen, R848, sperm motility, sperm sexing, swamp buffalo, Y-chromosome enrichment.