Use of R848, a TLR 7/8 agonist to separate X and Y spermatozoa in buffalo: effects on cleavage rates, blastocyst production, and the ratio of male to female embryos.

The study aimed to separate X and Y buffalo sperm using R848, a TLR7/8 agonist. Semen from three Murrah buffalo bulls were collected and divided into aliquots for swim-up in modified human tubal fluid (mHTF) medium with varying concentrations of R848 (0, 0.03, 0.3, and 3 μM). After the treatments and swim-up, the uppermost and lowermost layers of semen were assessed using computer-assisted sperm analyser (CASA) and flow cytometry. TLR7/8 receptors were found present in approximately 50 % of sperm as determined by immunofluorescence assay. Sperm velocities (VCL, VAP, VSL) decreased with increasing R848 concentrations, whereas mitochondrial health (mitochondrial membrane potential and superoxide production) remained unaffected. Based on sperm velocity, sperm treated with 0.3 μM R848 was selected for in vitro fertilization. The cleavage and blastocyst production rates were normal across the groups, ranging from 69 % to 76 % and 21 %-26 %, respectively, indicating the treatment does not affect cleavage and blastocyst production rates. In this study, the efficiency of separating X and Y sperm was determined using embryo sexing. The blastocysts produced from the uppermost and lowermost layers of semen through IVF resulted in 75 % male and 75 % female, respectively. Thus, the study develops a simple new method for efficiently separating X and Y sperm in buffalo, enabling the selective production of male or female progeny.