Optimization of glycerol and egg yolk concentrations for cryopreservation of Saanen buck sperm under tropical conditions in the Mekong Delta, Vietnam.

Background and Aim: Sperm cryopreservation is a cornerstone technology for genetic resource conservation and artificial insemination. However, tropical climatic conditions often compromise post-thaw sperm quality due to enhanced oxidative and thermal stress. This study aimed to optimize glycerol and egg yolk concentrations in a Tris-citrate-glucose (TCG) extender to improve post-thaw sperm quality of Saanen bucks raised in the Mekong Delta, Vietnam. Materials and Methods: The experiment was conducted in two sequential phases. In Phase 1, five glycerol concentrations (4%, 6%, 8%, 10%, and 12%) were tested to identify the optimal permeating cryoprotectant level. In Phase 2, four egg yolk concentrations (5%, 10%, 15%, and 20%) were assessed in combination with the optimal glycerol concentration. Semen from four healthy Saanen bucks was collected using an artificial vagina, diluted in the respective extenders, equilibrated, and frozen in liquid nitrogen. Post-thaw evaluations included overall and progressive motility (phase-contrast microscopy), viability (eosin-nigrosin staining), membrane integrity (hypo-osmotic swelling test), acrosome integrity (Giemsa staining), and DNA fragmentation (sperm chromatin dispersion test). Data were analyzed using one-way analysis of variance with Tukey's post hoc test at p < 0.05. Results: Glycerol concentration had a significant effect on post-thaw sperm quality (eta squared = 0.93-0.97), with 10% providing the best balance between cryoprotection and cytotoxicity. Incorporating 15% egg yolk with 10% glycerol significantly improved sperm viability (65.5%), membrane integrity (72.3%), and reduced DNA fragmentation (12.4%) compared with other treatments (p < 0.05). Higher glycerol or egg yolk concentrations adversely affected motility due to increased osmotic stress and viscosity. Conclusion: The combination of 10% glycerol and 15% egg yolk in TCG extender provides optimal cryoprotection for Saanen buck semen under tropical conditions. The resulting post-thaw sperm exhibited high motility, viability, and DNA integrity. This protocol can serve as a region-specific standard for buck semen cryobanking and artificial insemination in tropical climates, supporting genetic improvement and conservation initiatives in Vietnam and other developing regions.