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The objective of this study was to evaluate the association between GnRH administration at the supposed time of AI and circulating concentrations of LH in lactating Holstein cows, and to explore whether the intensity of estrous expression modulates this relationship. Additionally, we aimed to evaluate the effect of GnRH administration on the uterine response to produce 13,14-dihydro-15-keto-prostaglandin F2a (PGFM) on d 16 of the estrous cycle and explore the association of treatment on progesterone (P4) concentrations following ovulation. A total of 42 lactating cows were submitted to an Ovsynch synchronization protocol. Ovarian ultrasound (US) and blood samples (BS) were assessed at 7 and 14 d post-synchronization, and daily until spontaneous estrus event was detected. At the detection of spontaneous estrus, cows were assigned to receive either 100 mg of GnRH (treatment) or equivalent volume of 0.9% sodium chloride solution (control) at the supposed time of AI. Blood samples were collected immediately before treatment administration (0 h) and at 1 h intervals for the following 6 h to evaluate LH concentrations. Subsequent US exams were conducted at 24 h, 48 h, 7 d and 14 d to confirm ovulation and assess corpus luteum (CL) development. The CL and CL cavity diameter were used to calculate the total CL volume, and BS were collected to measure plasma P4 at 4, 7, 10 and 14 d post-estrus. A subset of cows (GnRH: n = 14; Control: n = 14) were subjected to an estradiol-oxytocin challenge 16 d post-estrus to assess PGFM release. Treatment influenced LH concentrations; however, this effect was dependent on the initial LH concentration. Cows were categorized by the median LH concentration at 0 h (0.81 ng/mL) into High- or Low-LH groups. In High-LH cows, GnRH-treated had greater LH concentrations at 1 h (GnRH: 4.6 ± 0.3 ng/mL; Control: 2.3 ± 0.3 ng/mL) and 2 h (GnRH: 3.0 ± 0.3 ng/mL; Control: 1.8 ± 0.3 ng/mL) compared with control cows. Estrous intensity did not modulate the LH response, but there was an interaction effect between treatment and intensity of estrus on CL volume and P4 concentrations post-estrus. This association was more evident at 14 d post-estrus, where control cows with lesser estrous intensity had smaller CL volume and lower P4 concentration than cows with greater estrous intensity and GnRH-treated cows with lesser intensity of estrus (GnRH-Greater: 5.0 ± 0.3 ng/mL; Control-Greater: 5.4 ± 0.3 ng/mL; GnRH-Lesser: 5.2 ± 0.3 ng/mL; Control-Lesser: 4.1 ± 0.3 ng/mL). GnRH-treated cows also tended to have greater PGFM concentrations compared with control (GnRH: 110.7 ± 9.2 pg/mL; Control: 86 ± 9.2 pg/mL). In conclusion, GnRH administration at the supposed time of AI increased circulating LH concentration, although it remains unclear whether this increase is sufficient to influence fertility outcomes. Moreover, estrous intensity appeared to modulate the effect of GnRH on CL development and P4 concentration post-estrus.