ABSTRACT. The effects of the time period between canine epididymis removal and cooling on post-thaw caudal epididymal sperm quality were investigated. Sperm recovered from the epididymis stored for 6 hr at 4 or 20C exhibited similar motility. However, when the epididymis was stored for 12 hr or longer at 20C, sperm motility was significantly lower than that at 4C (p<0.01). The post-thawed qualities of sperm recovered from the caudal epididymides that had been stored at 20C for 0 or 6 hr and then at 4C for 24 hr after removal were not significantly different. Therefore, leaving the canine epididymis at 20C for up to 6 hr after its removal may have little effect on the post-thaw quality of recovered caudal epididymal sperm.KEY WORDS: canine, caudal epididymal sperm, frozen semen.J. Vet. Med. Sci. 71(6): 811-815, 2009 Many wild animals including canids are endangered. As artificial reproductive techniques to preserve these species, methods of gamete preservation have been investigated [1,5,7]. Gametes from male animals are generally collected by ejaculation. However, when animals die accidentally, sperm are recovered from the cauda epididymis, the sperm storage site [3,4,8].In a previous study [3], unilateral intrauterine insemination with sperm that was recovered from canine caudal epididymis in prostatic fluid (PF) and cryopreserved resulted in a high conception rate, 80.0%. We also showed that when caudal epididymal sperm were recovered after storage of the epididymis in sterile physiological saline at 4C for 48 hr, sperm motility was reduced, but intrauterine insemination with this post-thaw sperm again led to a high conception rate, 80.0% [4]. In these two experiments, the epididymis was kept at 4C immediately after removal, and the time period until the start of cooling after removal was not considered. However, when the bodies of male animals, such as wild animals, that have died accidentally are discovered, it is likely that several time have already passed under various temperature conditions, and so the epididymis can rarely be cooled immediately. This suggests that the time until cooling needs to be investigated.It has been reported that mouse caudal epididymal sperm shows a sperm motility of approximately 30% after 10 days of postmortem storage at 4C [6], but only shows a sperm motility of 10-15% after 24 hr of storage at room temperature [12]. Hishinuma et al. [2] reported that when the sika deer (Cervus nippon) epididymis is removed 4 hr postmortem and stored at 4C for 1-4 days, the caudal epididymal sperm motility is high, at approximately 50%, but, when removed 8-12 hr postmortem and stored at 4C for 1-4 days, the sperm motility is as low as 6.4%. These reports suggest that, in order to recover highly motile caudal epididymal sperm, it is important to store dead male animals immediately after death at 4C or to remove the epididymis as soon as possible and rapidly store it at 4C, thereby reducing the time of exposure to room temperature. However, in all the reported studies concerning...