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The objective of this observational, retrospective study was to determine the association between the timing of artificial insemination (AI) and pregnancy per AI (P/AI) in nulliparous Holstein heifers inseminated either with sexed or conventional semen considering different characteristics of an estrus event (i.e., onset, peak, and end) using an automated activity monitoring system. A total of 4,265 AI services from 2,919 heifers based on the alert of Heatime (SCR Engineers Ltd., Netanya, Israel) were evaluated from 6 commercial dairy farms in Germany. The mean (± standard deviation) duration of an estrus event was 16.0 ± 4.8 h. Overall P/AI was 49.1%. Heifers inseminated with conventional semen (51.4%; 933/1,644) had similar P/AI compared to heifers inseminated with sexed semen (46.9%; 1,277/2,621; P = 0.14). Heifers at a younger age at AI were more likely to get pregnant (P = 0.01). The interval from onset of estrus to AI was associated (P = 0.01) with P/AI, with the greatest P/AI for heifers inseminated within 9 to 32 h after the onset of the activity alert. Whereas the interval from peak activity to AI was not associated (P = 0.58) with P/AI, there tended (P = 0.08) to be an association between the interval from end of estrus to AI and P/AI. There were no interactions between the intervals from onset, peak or end of estrus to AI and type of semen on P/AI. In conclusion, inseminating heifers within 9 to 32 h after onset of estrus increased P/AI irrespective of type of semen.