Characteristics and in vitro fertilizing ability of giant panda ( Ailuropoda melanoleuca ) frozen‐thawed epididymal spermatozoa obtained 4 hours postmortem: A case report

When Chu‐Lin, a male giant panda (studbook #249), died at Madrid Zoo, his reproductive tract was removed 4 hr postmortem and the epididymal spermatozoa were collected. Extended sperm were kept at 5°C for 4 hr, loaded into straws, and frozen for 7 min in liquid nitrogen vapor before the straws were plunged into liquid nitrogen. Two straws were thawed and evaluated. Sperm motility was assessed in fresh, refrigerated, and thawed spermatozoa (75%, 60%, 35%, respectively). Sperm viability and acrosome status were estimated using a triple‐stain technique (TST). The results showed 33% live sperm with intact acrosomes after thawing. A hypoosmotic swelling (HOS) test demonstrated the retention of membrane integrity in 72% of thawed sperm. To evaluate the in vitro fertilizing ability of thawed sperm, a sperm penetration assay (SPA) was performed. The values obtained for the percentage of penetration and the penetration index were 62% and 1.78 sperm/oocyte, respectively. The results obtained demonstrate that epididymal sperm recovered from a giant panda postmortem can be successfully cryopreserved. The sperm fertilizing ability demonstrated in vitro after thawing may provide a final opportunity for this male to contribute to the currently small germplasm reserves of this endangered species, and to reproduce in the future through assisted reproductive technology. Zoo Biol 23:279–285, 2004. © 2004 Wiley‐Liss, Inc.