Abstract

Background and Aim: Conventional egg-yolk and milk-based extenders are widely used for semen cryopreservation but pose biosafety concerns and compositional variability that compromise standardization. Liposome technology offers a biosecure, uniform alternative. This study aimed to develop soy lecithin-derived nanoliposomes (NLs) using an ultrasonic-based process and to evaluate their efficacy as a Tris-based extender for Ongole-grade bull semen cryopreservation.

Materials and Methods: Soy lecithin NLs were prepared through probe ultrasonication (15–45 min) and ultracentrifugation, followed by physicochemical characterization using particle size analysis, scanning electron microscopy (SEM), and high-resolution transmission electron microscopy. Tris-NL (TNL) extenders were formulated at concentrations of 5–25 mg/mL and compared with a Tris–egg-yolk (TEY, 20%) control. Fresh semen from five Ongole-grade bulls was evaluated for motility, viability, and morphology before and after freezing. Kinematic parameters were assessed through computer-assisted semen analysis, plasma membrane integrity by hypoosmotic swelling test, acrosome integrity using fluorescein isothiocyanate-conjugated peanut agglutinin/propidium iodide staining, and DNA fragmentation by Halomax-sperm chromatin dispersion assay.

Results: Optimized sonication (45 min) produced stable NLs (mean diameter 76 nm, zeta potential −43.2 mV) with uniform spherical morphology. Among the tested formulations, TNL 5–10 mg/mL showed significantly higher (p < 0.05) post-equilibration motility (up to 98%), progressive motility, and kinematic parameters (velocity curved line, velocity average path, and velocity straight line) than TEY. Post-thaw evaluations demonstrated improved sperm viability (≈66%), reduced abnormalities (<7%), enhanced plasma-membrane and acrosomal integrity, and lower DNA fragmentation (~1.2%) in the 5–10 mg/mL groups. SEM confirmed smoother sperm surfaces with minimal cryo-damage compared with TEY.

Conclusion: Soy lecithin-derived NLs at 5–10 mg/mL serve as an effective and biosecure substitute for egg yolk in Tris extenders, enhancing motility, viability, and structural integrity of Ongole-grade bull spermatozoa. This locally developed, plant-based nanotechnology supports biosafety, import substitution, and sustainability of artificial insemination programs in Indonesia.

Keywords: computer-assisted semen analysis, cryopreservation, lecithin, nanoliposome, Ongole-grade bull, semen extender, sperm integrity.