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ABSTRACT. Cooled canine semen solutions for storage were investigated with three stock solutions: egg yolk-citrate-glycine-glucose solution, egg yolk Tris-fructose citrate solution (EYT-FC), and egg yolk sodium citrate dihydrate solution (EYCD). For the control group, the second fraction of semen was examined. Nine male beagles and 37 female (47 experimental cases) beagles for artificial insem ination (AI) were used. The qualities of semen stored at 4°C deteriorated earlier in the control and EYCD groups. In the other two groups, sperm motility was 60% or higher after storage for 6 days and 20% or higher after storage for 12 days. On a comparison of these two groups, the sperm motility and viability were slightly higher in the EYT-FC group. A high conception rate was obtained by AI using semen stored at a low temperature for a maximum of two days in the control group and four days in the EYT-FC group. KEY WORDS: artificial insemination, canine, cooled semen.J. Vet. Med. Sci. 65(3): 307-312, 2003 For artificial insemination (AI) of dogs, fresh semen [3,12,18,20,21], cooled semen [6-9, 12, 15], and frozen semen [3,12,16,19] In studies of cooled canine semen, semen preparations with milk [6,8,9,13,15] and egg yolk [1,2,4,5,7,[11][12][13][14] as the extender have been investigated. In studies with egg yolk-citrate-glycine-glucose extender (EYC-GG) performed by Foote [4,5] and egg yolk Tris-glucose extender performed by Iguer-ouada and Verstegen [11], canine sperm could be stored at 4°C for a prolonged period, although these were in vitro studies.Some attempts at fertilization have succeeded when using cooled semen. Gill et al. [6] and reported a high conception rate for cooled semen within 24 hr. Successful fertilization with semen stored for a long period has been reported. Harrop [8] stored semen in sterilized milk for 4 days, and this is the first successful fertilization with cooled semen. Seager and Fletcher [15] also stored semen in sterilized milk for 1-4 days similarly to Harrop [8] and obtained a conception rate of 53.0% (8/15), but the semen volume and number of sperm used for AI and the frequency of collecting semen were not clarified.Therefore, although many in vitro experiments have been performed with cooled semen as mentioned above, semen preparation applicable to AI for several days has not been clarified.We therefore investigated extenders, storage methods, and AI methods with cooled semen in dogs. For the extender, EYC-GG with which Foote [4,5] succeeded in long-term storage of canine sperm, EYT-FC, which we used for frozen canine semen [19], and egg yolk sodium citrate dihydrate (EYCD), which has long been used for domestic animals, were investigated. MATERIALS AND METHODS Animals:Nine male dogs aged 2-5 years were repeatedly used. For AI, there were 29 female dogs aged 7 months-1.5 years at the initial estrus and two multiparous dogs. Because of repeated use, the number of cases was 47. Since many dogs were used for AI at the initial estrus, the conception results were obtained from 58 dogs...