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The objective of this observational study was to evaluate the association of estrous expression within 40 d in milk using a neck-mounted automated activity monitor (AAM; Heatime Pro; SCR Engineers Ltd., Netanya, Israel) with estrous characteristics of the first postpartum AI and reproductive performance in lactating Holstein cows. A total of 2,077 cows from 5 commercial dairy farms were included in the statistical analyses. Cows were classified according to the number of estrus events from d 7 until d 40 postpartum into 3 categories: 1) no estrus event (ANESTRUS); 2) one estrus event (ESTRUS1) and 3) 2 or more estrus events (ESTRUS2+). Generalized linear mixed models were used to analyze continuous or categorical data. Shared frailty models were used for time to event data. Overall, 52.7% (1,095/2,077) of cows had no estrus event detected by an AAM system from d 7 until d 40 postpartum. Herd-level prevalence of ANESTRUS ranged from 37.5% to 58.4%. Estrous expression from d 7 until d 40 postpartum affected estrous duration (P = 0.001) at first postpartum AI. Cows in ANESTRUS had the shortest duration (13.2 ± 0.33 h) compared with cows in ESTRUS1 (13.8 h ± 0.36) and ESTRUS2+ (14.8 h ± 0.41). Cows in ESTRUS2+ had a longer estrous duration at first postpartum AI compared with cows in ESTRUS1. For ANESTRUS, ESTRUS1, and ESTRUS2+ cows pregnancy per AI was 29.4%, 30.9%, and 37.8%, respectively. Estrous expression from d 7 until d 40 postpartum affected time to first AI and time to pregnancy. Compared with ANESTRUS cows, cows in ESTRUS1 (Hazard risk (HR) = 1.74; 95% CI 1.55 - 1.94; P = 0.001) and ESTRUS2+ (HR = 1.77; 95% CI 1.52 - 2.05; P = 0.001) had an increased hazard of being inseminated within 100 DIM. Median DIM to first AI were 70, 59 and 58 for cows in ANESTRUS, ESTRUS1, and ESTRUS2+, respectively. Compared with ANESTRUS cows, cows in ESTRUS1 (HR = 1.28; 95% CI 1.13 - 1.46; P = 0.001) and ESTRUS2+ (HR = 1.33; 95% CI 1.12 - 1.57; P = 0.001) had an increased hazard of becoming pregnant within 200 DIM. Median DIM to pregnancy were 127, 112, and 103 for ANESTRUS cows, ESTRUS1 and ESTRUS2+, respectively. In conclusion, cows with no estrous expression from 7 to 40 DIM had reduced estrous expression at first AI and inferior reproductive performance compared with cows that displayed estrous activity.