Exploring the toxicological mechanisms of reduced fertility in dairy cows due to nonesterified fatty acids on the basis of network toxicology, transcriptomics and molecular docking.

High concentrations of nonesterified fatty acids (NEFAs) are normal metabolites of high-producing dairy cows in a state of negative energy balance (NEB), but they are thought to be strongly associated with reproductive disorders in dairy cows, which may contribute to reduced fertility in cows (RFC). There are few studies on the independent toxic effects of NEFA-mediated RFC. This study aimed to investigate the toxicological effects of NEFA-mediated RFC systematically via network toxicology, transcriptomics, and molecular docking techniques. A total of 403 potential targets of NEFA-mediated RFC toxicity were screened by comprehensively analyzing the GeneCards, OMIM, ChEMBL and Swiss Target Prediction databases. Further analysis via the GEO (GSE165476 dataset), STRING databases and Cytoscape software yielded eight hub targets, including MMP2, MAPK1, PRKACA and PRKCB. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these targets were involved in pathways related to metabolism, endocrine processes, cell death, and signal transduction, such as the AGE-RAGE signaling pathway in diabetic complications, the GnRH signaling pathway, and the MAPK signaling pathway. Molecular docking further confirmed the potential interactions between NEFAs and these hub targets. This study revealed that NEFAs may exacerbate the occurrence of RFC by interfering with endocrine regulation, inducing inflammatory responses, affecting angiogenesis and tissue remodeling, regulating apoptosis, and disrupting metabolic balance. The results of this study provide novel molecular insights into the mechanism of NEFA-mediated RFC toxicity and provide a scientific basis for emphasizing the importance of metabolite toxicity in dairy farming health management.