Suboptimal responses to E2/P4-based timed AI protocols compromise pregnancy outcomes in fertile beef cattle: Roles of ovulation timing and semen viability.

Timed artificial insemination (TAI) protocols based on estradiol and progesterone (E2/P4) typically yield pregnancy rates (PR) near 50 % in beef cattle, falling short of the 65-70 % expected in fertile females. This study investigated physiological and procedural limitations that may compromise TAI success due to suboptimal synchrony between insemination and ovulation. Three experiments involving 840 suckled crossbred females evaluated follicular responses, semen type, and alternative reproductive strategies to enhance fertility. In Experiment I (n = 28), daily ultrasonography revealed that 46.4 % of females exhibited suboptimal ovarian responses, defined as no emergence/atresia of the new follicular wave, and ovulation delay or failure. In Experiment II (n = 348), cows with small follicles (<8.5 mm) at P4 removal had markedly higher PR when inseminated with cooled versus frozen semen (71.9 % vs 38.0 %), highlighting the relevance of sperm cell viability when ovulation is delayed. In Experiment III (n = 464), strategies combining TAI with estrus detection (ED), timed embryo transfer (TET), or both significantly increased PR compared to TAI alone (TAI: 53.6 %; TAI+TET: 67.9 %; ED-AI/TAI: 70.5 %; ED-AI/TAI+TET: 68.2 %), which was accompanied by increased twinning rates after TET, suggesting that many TAI failures were not due to uterine inadequacy or embryo mortality but to asynchrony in gamete encounter. Collectively, results suggest that most TAI failures stem from impaired synchrony between ovulation and sperm cell availability rather than uterine or embryonic limitations. Incorporating tools such as ultrasonography, semen selection, ED, or TET may enhance reproductive outcomes in commercial beef herds under E2/P4-based TAI protocols.