Eui Kyeong, Korea National Park Service

The Asiatic black bear (Ursus thibetanus) is classified as Vulnerable on the IUCN Red List and is a primary focus of restoration efforts in South Korea. Establishing a stable sperm biobank is essential to preserve genetic diversity and mitigate spatiotemporal constraints in conservation breeding. While basic cryopreservation protocols utilizing 6% glycerol were established in the early 2000s, they often result in suboptimal post-thaw motility, posing a significant bottleneck for effective genetic resource management. To overcome these efficiency gaps, this study aimed to develop a highly optimized, high-efficiency protocol by evaluating the synergistic effects of various additives and advanced freezing techniques.
From 2020 to 2025, 268 semen samples were collected from 15 captive males via transrectal ultrasound-guided urethral catheterization. We conducted iterative optimization trials using an egg yolk–based extender (6% glycerol) to evaluate additives (Equex-STM, trehalose, HSP-70) and three freezing methods: vapor-phase, pellet/droplet, and programmable automated freezing. Post-thaw sperm quality was assessed using Computer-Assisted Sperm Analysis, viability, and morphology.
The highest post-thaw recovery was achieved through programmable automated freezing combined with 0.5% (v/v) Equex-STM. Under this optimized protocol (n=85), post-thaw motility was 48.37 ± 19.16% and viability was 67.30 ± 17.11%, nearly doubling the efficiency of conventional glycerol-only methods. Recovery rates related to fresh semen were 60.49 ± 22.78% for motility and 76.32 ± 25.44% for viability.
These findings provide a robust technological foundation for an advanced national sperm biobank, offering a vital tool for genetic management and sustainable restoration of endangered ursid populations.