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The aim of this retrospective observational study was to evaluate the effect of estrous expression before the beginning of a synchronization protocol and at the moment of AI on pregnancy per AI (P/AI). A total of 2,842 lactating Holstein cows from 1 commercial herd were enrolled. In yr 1, cows underwent a Double-Ovsynch (DO) protocol for first AI (n = 1,536) and produced a total 305-d milk yield of 13,421 kg, whereas in yr 2, cows followed a presynch-ovsynch (PO) protocol (n = 1,306) and produced 13,868 kg over 305 d. Cows were classified as primiparous or multiparous and continuously monitored using ear-tag accelerometers. Estrus alerts were identified by the system, and intervals from the last alert before synchronizations were categorized as short (SI; 7–14 d) or long (LI; 14–30 d). The total number of alerts from calving to AI was also recorded, with cows categorized as 0, 1, or ≥2 alerts. Pregnancy was confirmed by ultrasonography on d 32 post AI. A logistic regression model assessed associations between estrous expression and P/AI, including estrus at AI, parity, number of alerts, and estrus interval classification. Cow was a random effect, and protocol was a fixed effect. Primiparous cows were more likely to express estrus at AI than multiparous cows (OR 1.79; 95% CI 1.52–2.10). Estrus at AI improved P/AI (OR 1.55; 95% CI 1.32–1.82). For cows showing estrus, P/AI was 65.4% ± 2.4% under DO and 51.1% ± 4.0% under PO. Without estrus, P/AI was 54.4% ± 1.8% and 44.5% ± 3.5% for DO and PO, respectively. Multiparous cows with ≥2 alerts had higher P/AI (OR 1.65; 95% CI 1.10–2.48) than those with 1 alert (OR 1.32; 95% CI 0.98–1.78) or none. No significant difference was found in primiparous cows. LI cows had lower P/AI (OR 0.62; 95% CI 0.51–0.76) than SI cows. Under DO, SI cows had higher P/AI (OR 1.79; 95% CI 1.47–2.17) than LI cows, and under PO, SI cows also showed greater P/AI (OR 1.67; 95% CI 1.30–2.14). In conclusion, cows with SI before synchronization and estrus at AI had improved P/ AI. Continuous monitoring can optimize protocol timing and reduce
blanket treatments through selective management.