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ABSTRACT. Canine epididymides were excised and immediately stored at 4°C for 48 hr, and the qualities of caudal epididymal sperm after recovery and cryopreservation were evaluated. To confirm the fertility of the cryopreserved caudal epididymal sperm, artificial intrauterine insemination was performed. The sperm motility (61.0%) immediately after recovery from caudal epididymis stored at 4°C for 48 hr was significantly lower than those of sperm stored for 0 and 24 hr (88.6 and 80.7%, respectively), but there was no significant difference after freeze-thawing (0-, 24-, and 48-hr storage groups: 27.9, 24.3, and 28.3%, respectively). The incidence of abnormal sperm immediately after recovery was significantly higher in the 24-hr and 48-hr storage groups (19.3 and 27.7%, respectively) than in the 0-hr storage group (5.6%), and a significant difference was also observed after freeze-thawing. The incidence of immature sperm with cytoplasmic droplets was significantly higher in the 48-hr storage group (18.4%) than in the 0-hr storage group (4.7%), but there was no difference after freeze-thawing. By unilateral intrauterine insemination (2 × 10 8 sperm), 4 of 5 bitches (80%) conceived. The above findings demonstrated that sperm motility was good even enough the incidence of abnormal sperm was high in canine epididymal sperm that were recovered from the epididymis stored at 4°C for 48 hr and cryopreseved, and that artificial intrauterine insemination resulted in a high conception rate. KEY WORDS: canine, cryopreservation, epididymal sperm, intrauterine insemination, refrigeration.J. Vet. Med. Sci. 67(11): 1141-1147, 2005 Many wild animals, including those in the canine family such as the gray wolf (Canis palus) and American red wolf (Canis rufus), are on the verge of extinction [4]. At the deaths of these animals by unexpected accidents, recovery and cryopreservation of sperm from the epididymis are important for assisted reproductive techniques [1,16].Since it may take time to recover and cryopreserve sperm from the epididymis after the death of an animals, development of a technique to store the epididymides until recovery of sperm is necessary, and such techniques have been investigated in many animal species [2, 7, 9, 10, 13-15, 18, 21, 26-28]. For example, in dogs, when Yu and Leibo [27] stored canine epididymides at 4°C for 8 days after excision, sperm motility was significantly decreased after 5 hr, but sperm membrane integrity was maintained even after storage for 48 hr, and the zona pellucida-binding ability was maintained even after 192 hr. In sheep, it has been reported that sperm from recovery of the epididymis stored at 5°C showed better sperm quality after 24 and 48 hr than sperm recovered from epididymides stored at room temperature for the same period [13]. In mice, it has been reported that sperm motility decreased to between 10 and 15% within 24 hr when the epididymis was stored at room temperature after the animal died, but epididymis stored at 4°C maintained 30% of the sperm motility even after 10 da...