Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica ) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO2, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN2 vapor. After thawing at 37℃ for 25 s, Isolate® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Chemicals
    Animals
    Epididymal sperm collection
    Sperm freezing and thawing
    Sperm motility
    Sperm viability
    Acrosome integrity
    Statistics
RESULTS
DISCUSSION
CONCLUSION
REFERENCES

• Saddah Ibrahim(Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University,Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology,)
• Nabeel Abdelbagi Hamad Talha(Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University,Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Sudan University of Science and Technology,)
• Jeongho Kim(Cheongju Zoo, Cheongju 28311, Korea)
• Yubeol Jeon(Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan 54596, Korea)
• Iljeoung Yu(Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Jeonbuk National University, Iksan 54596, Korea) Corresponding Author