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The aim of this study was to investigate if a 2-step method of preventing suckling and cow-calf separation reduces the stress reaction in foster cows compared with a simultaneous separation method. Seven Swedish Holstein and 5 Swedish Red dairy cows were used as foster cows, each having a group of 4 calves. The foster cow-calf group was formed when calves were 1 wk old, and the calves were prevented from suckling at 10 wk of age. In 6 of the cow-calf groups, calves were prevented from suckling by simultaneous separation from the cow (control). In the other 6 groups, calves were fitted with a nose-flap, which prevented them from suckling while they were kept together with the cow for another 2 wk before they were separated (2-step). The behavior of the foster cows was observed at 4 observation periods, 0 to 2, 8.5 to 9.5, 24 to 26, and 72 to 74 h after the calves were prevented from suckling (2-step), after separation (2-step), and after calves were prevented from suckling by simultaneous separation (control). For both treatments, saliva cortisol was sampled once daily for 5 d at wk 10. This was repeated at wk 12 for the 2-step treatment. Heart rate was measured with the behavioral observations. Control foster cows vocalized more (P < 0.001) and walked more (P = 0.005) than the 2-step foster cows after prevention of suckling and after separation from the calves. When control cows were separated from their calves, they more frequently (P < 0.001) held their head out of the pen than was the case with 2-step cows when separated 2 wk after prevention of suckling. The variation in heart rate was larger in the control group compared with 2-step cows at 0 to 2 h after separation/prevention of suckling (P = 0.002). No effect of treatment was found on cortisol concentration. Our conclusion is that separating the 2 events "prevention of suckling" and "separation" reduces the stress experienced by the foster cow at weaning. [ABSTRACT FROM AUTHOR]