Identification of four early pregnancy markers for cows by proteomics and metabolomics

Accurate and timely diagnosis of pregnancy in cows is crucial for optimizing reproductive management practices on cattle farms. Therefore, the present study aimed to identify molecular changes in the serum of pregnant and non-pregnant cows using the latest proteomics and metabolomics techniques, with the goal of discovering early pregnancy biomarkers in female cattle. Firstly, the serum samples of cows were collected and isolated on the 28-d post artificial insemination (AI). The pregnancy status of these cows was subsequently determined based on the echogenic appearances in the uterus via ultrasound examination on the 33-d post AI, and then the samples were accordingly classed as pregnant (n = 8) or non-pregnant (n = 22). For metabolomic analysis, samples from six cows in each group were selected, while for proteomic examination, samples from three cows in each group were chosen. Proteomic analysis identified 341 proteins, with KEGG pathway enrichment revealing their involvement in 20 signaling pathways, including immune and endocrine systems, signal transduction, and others. Among the proteins, eight proteins were found to be elevated and ten proteins were down-regulated in the serum of pregnant cows. The levels of the top differential proteins such as APOA1, HRG, CFH, A2M, and MYH1 between pregnant and non-pregnant cows were further measured by ELISA. The ELISA results revealed that the levels of APOA1, HGR, CFH, and MYH1 in the serum of pregnant cows were significantly higher than those in non-pregnant cows (<italic>p</italic> &lt; 0.05). Metabolome analysis demonstrated 20 up-regulated and 23 down-regulated metabolites in the serum of pregnant cows. However, none of these differential metabolites were deemed suitable as pregnancy markers. Overall, our findings show those four proteins hold potential as biomarkers for examining the early pregnancy status of cows.