Simple SummaryThis study aimed to determine the levels of various hormones and metabolites in the blood of Holstein dairy cows during the crucial transition period, 7 days before parturition and a further 14 and 21 days postparturition, exploring the link between pregnancy probability after first artificial insemination and during the first 100 days in milk. The cows were split into two subgroups, those that were diagnosed as pregnant and those that did not conceive after further attempts at artificial insemination. The results showed that the levels of IGF-1 and progesterone exhibited significant differences among the two subgroups, which could have a diagnostic value in predicting pregnancy. These findings are relevant for dairy farm management, as they provide new information about the hormones and metabolites that affect the health and reproductive performance of dairy cows.AbstractThe purpose of this study was to determine the link between insulin-like growth factor 1 (IGF-1), progesterone (PROG), non-esterified fatty acids (NEFAs), β-hydroxybutyrate (BHB), and glucose (GLU) and pregnancy probability after the first artificial insemination (AI) and during the first 100 days in milk (DIM), during the critical transition period. We determined levels of serum IGF-1, PROG, NEFA, BHB, and GLU in Holstein dairy cows via ELISA, using blood samples collected 7 days before parturition (DAP) until 21 days postparturition (DPP). The group was split into cows diagnosed pregnant at 100 DIM (PREG) and those that did not conceive at 100 and 150 DIM (NPREG). Serum IGF-1 and PROG median levels at 7 DAP were significantly higher in PREG vs. NPREG (p =0.029), the only statistically significant differences across the subgroups. At 7 DAP, IGF-1 levels within the initial group showed a strong negative correlation with PROG (r =−0.693;p =0.006), while for the PREG subgroup, the IGF-1 levels exhibited a very strong positive correlation with GLU (r =0.860;p =0.011) and NEFA (r =0.872;p =0.013). IGF-1 and PROG levels detected at 7 DAP may be useful to predict pregnancy at 100 DIM. The positive correlation of NEFA and GLU levels during the transition period demonstrates that the initial group is not in NEB; thus, the NEFA level was not a decisive factor for reproduction success.