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Lactating dairy cows with a mature corpus luteum (CL) (diameter of ≥17 mm determined by ultrasonography) and having a follicle with a diameter of 10 mm (n = 49) were randomly assigned to three groups. The first group was treated with a single dose of exogenous prostaglandin (25 mg), while the second group was treated with 35 mg on day 0, and the third group served as control in order to evaluate the effect of rectal manipulation on the CL during ultrasonographic examination. Blood samples were collected daily for analysing progesterone (P4) concentrations. In group 1 the incidence of oestrus and artificial insemination (AI) in 10 days after treatment was 95% (19 of 20). The conception rate was 31.6%, and the average time to oestrus after treatment was 3.7 day. In group 2 the incidence of oestrus and A.I. was 84.2% (16/19). The conception rate was 31.2%, and the average time to oestrus after treatment was 2.8 day. In the untreated group only two cows (2/10) exhibited oestrus during the examined period and none of them became pregnant. There were no significant differences between the two treated groups in terms of reduction in the area of CL and P4 concentrations and of an increase in the area of the dominant follicles. At the same time, the decrease in the percentage changes relative to the area of CL and to the concentrations of P4 were statistically significant in both treated groups.