Oviductal Fluid Proteome of Healthy and Repeat Breeder Jersey Cows (Bos taurus) Collected by Transcervical Catheter: A pilot study

Repeat breeder syndrome affects 10–24% of dairy cattle globally, yet its molecular basis remains poorly understood in part because the oviduct has never been accessible in a living animal without surgery. With custom built transcervical aspiration catheter (MLRIOFA; Design Registration No. 462145-001, Government of India) we collected oviductal fluid from conscious, standing Jersey cows, and used it to compare oviductal fluid proteomes of healthy (n =2) and repeat breeder (n = 2) animals sampled at confirmed standing natural heat. Tryptic digests were analyzed by LC-MS/MS and searched against the UniProt Bos taurus reference proteome at 1% false discovery rate. Across four samples, 105 nonredundant proteins were identified. Healthy animals yielded 77 and 76 proteins, respectively; repeat breeders yielded 55 and 25. That gap in proteome depth, nearly three-fold fewer identifications in the lower repeat breeder sample may itself reflect a sparser or more dilute luminal environment in sub fertile animals. Of the 105 proteins, 54 were shared between groups, 46 were exclusive to healthy animals, and only 5 to repeat breeders. Principal component analysis on log2-transformed spectral counts separated the two fertility phenotypes along PC1 (50.7% variance). Gene Ontology enrichment of the 46 healthy-exclusive proteins returned acutephase response, inflammatory signaling, cell-matrix adhesion, and hydrogen peroxide catabolism as the dominant biological processes. The most abundant healthy-associated proteins; alpha-2-macroglobulin (log2FC = 2.48), fibronectin, peroxiredoxin-2, clusterin, and annexin A1 collectively cover protease inhibition, oxidative stress defense, extracellular matrix integrity, and local immune regulation. The five-repeat breeder-exclusive proteins pointed in a different direction: retinoic acid metabolism, collagen fibril organization, and complement regulation, represented by ADH7, complement factor H, and collagen type III alpha 1 chain. The oviductal milieu of repeat breeder cows appears depleted in proteins that healthy animals use to support gametes, defend against oxidative damage, and maintain immune homeostasis. Whether these differences cause subfertility or reflect it remains to be determined with larger cohorts. The MLRI-OFA catheter makes those cohorts possible.