Simple SummaryReplacement heifer development is a critical component in beef production. The identification of the ideal uterine environment will greatly benefit reproductive efficiency. Additionally, the elucidation of how diet can influence this environment could alter management strategies. Therefore, our hypothesis was that different levels of protein supplementation would affect the uterine environment of beef heifers without inhibiting development or the ability to conceive. Supplemental protein did not greatly affect the uterine environment as it pertained to amino acid concentrations. However, uterine amino acid concentrations did change throughout development, and protein supplementation can influence uterine luminal fluid composition on d 14 post-insemination, which may affect conception rates.AbstractReplacement heifer development is one of the most critical components in beef production. The composition of the ideal uterine environment could maximize fertility and reproductive efficiency. Our hypothesis was that protein supplementation would affect the uterine environment of beef heifers without inhibiting development or reproduction. To test the effects of dietary supplementation on these outcomes, a randomized complete block design with repeated measures was implemented. Angus heifers (n= 60) were blocked by body weight (BW) and randomly assigned to one of three supplemental protein treatment groups (10% (CON), 20% (P20), and 40% (P40)). Mixed model ANOVAs were used to determine whether protein supplementation treatments, time, and the interaction or protein supplementation, semen exposure, and the interaction influenced uterine luminal fluid (ULF) and pregnancy outcomes. Amino acids (AAs) were impacted (p< 0.001), specifically, the essential AAs: Arg, Iso, Leu, Val, His, Lys, Met, Phe, Trp. Protein supplementation influenced multiple AAs post-insemination: Arg (p= 0.03), CC (p= 0.05), 1-MH (p= 0.001), and Orn (p= 0.03). In conclusion, protein supplementation did not affect the reproductive development via puberty attainment or the timing of conception even with alterations in growth. However, uterine AA concentrations did change throughout development and protein supplementation influenced ULF d 14 post-insemination, which may affect the conception rates.