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-NOTE-The long estrus period in mares associated with a very variable ovulation time remains a challenge for horse breeders who would like to breed just before ovulation [5]. The fact that the best time to inseminate is the one closest to follicular rupture stands unquestioned. It has been shown earlier that insemination of mares should be done from 3 days before to 12 hr after ovulation, otherwise the pregnancy rate is lowered or embryonic loss is increased [11]. But no accurate method for predicting the exact ovulation time in mares is yet well established.Endocrinological studies revealed cyclic changes in circulating and urinary estrogen conjugate (EC) concentrations at different stages of the estrous cycle. In both cases, estrogen levels registered a gradual increase during the follicular phase and then a decrease a few days before ovulation [6][7][8]. The EC in the urine is ten times higher than in the plasma due to the kidney's concentrating capacity and the resistance of EC metabolites to rapid degradation [3]. There are reports that estrogens excreted out through the urine can be an index of its secretion rate because they are not stored in the body [2]. Because estrone has been reported to be the principal EC component in urine [8], urine contains materials adequate for hormonal measurements and to estimate ovulation time on the basis of urinary changes in E1S concentrations.A total of 16 cycling draft mares of breeds Percheron and Breton between 4 to 16 years old were used in this study. The stages of the estrous cycle of each mare were first determined by routine rectal palpation, vaginal examination and ultrasonography. Urine samples were collected daily with a sterilized urinary catheter. The collection started on D-5 (n=6), D-4 (n=10) and D-3 (n=16) until the ovulation was confirmed by ultrasonography. Samples were centrifuged at 1,500 rpm for 5 min and stored at 30°C until assayed for E1S.Urinary E1S concentrations were determined in 2 hr with the EIA kit (Bri-Equine Oestrone Sulphate EIA: Bioresearch Ireland) which was originally designed for plasma E1S measurement. A preliminary experiment has been conducted to validate the plasma EIA kit for urinary E1S measurement. Urine samples from 2 mares in the last trimester of pregnancy (with high E1S concentrations) were diluted, and then assayed for E1S. The E1S standard curve ranged from 0.25 to 10 ng/ml urine. The intra and inter coefficients of variation were 5.7 and 7.7%, respectively. The recovery rates of E1S

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