Investigations on the optimum timing of artificial insemination (AI) following automated activity monitoring (AAM) depending on different types of semen in heifers are limited and in part show controversial results. Therefore, the objective of this observational study was to determine the association between the timing of AI using different characteristics of estrus (i.e., the onset, peak, and end of estrus) and pregnancy per AI (P/AI) in Holstein heifers. Heifers were fitted with a neck-mounted AAM system and inseminated with frozen conventional and sexed semen. The pregnancy per AI (n = 4159) from 2858 heifers from six commercial dairy farms in Germany inseminated upon the alert of an AAM system was evaluated. Estrous intensity was classified based on peak activity into low (35 to 89 index value) and high (90 to 100 index value). We detected a quadratic association between the interval from the onset of estrus to AI and P/AI (p = 0.02). The greatest P/AI was observed for heifers inseminated from 9 to 32 h after the onset of estrus. The intervals from the peak of activity to AI and the end of estrus to AI were not associated with P/AI (p ≥ 0.05). Heifers inseminated with frozen conventional semen (50.1%) had a greater P/AI compared with heifers inseminated with frozen sexed semen (43.3%; p = 0.03). There were no interactions between the intervals from the onset, peak, or end of estrus to AI or the type of semen and the P/AI (p ≥ 0.05). The pregnancy per AI was not associated with estrous intensity (50.5% for low intensity vs. 53.0% for high intensity; p = 0.37). In conclusion, inseminating heifers between 9 and 32 h after the onset of estrus, as detected by the AAM, optimized the P/AI regardless of semen type.
