TMT-based quantitative proteomic analysis of serum from domestic sheep in early pregnancy

Context Timely pregnancy diagnosis is critical for reducing non-production days and improving the reproductive efficiency of sheep flocks.Aims The aim of this study was to investigate the serum proteomic profile of ewes in different gestational stages and explore the potential of differentially expressed proteins as biomarkers for early gestation.Methods Serum samples were collected from two groups: pregnant sheep (n = 4) and non-pregnant sheep (n = 4) on Day 14 after mating. The differentially expressed proteins were detected using tandem mass-tag (TMT) labelling with liquid chromatography tandem-mass spectrometry. The differentially expressed proteins were confirmed using enzyme-linked immunosorbent assay (ELISA). Furthermore, six selected proteins were evaluated in ewes (n = 35) on Day 14 from mating by using ELISA to assess their potential as biomarkers for early pregnancy. Receiver-operating characteristic (ROC) curves were used to analyse the diagnostic accuracy of these proteins.Key results Twenty proteins, of a total of 555 proteins detected, showed differential expression between pregnant and non-pregnant sheep. From the ROC-curve data, CHI3L1 (AUC = 0.992), PSMB4 (AUC = 0.976) and LGALS3BP (AUC = 0.844) had high predictive value in the diagnosis of early pregnancy.Conclusions In this study, CHI3L1, PSMB4 and LGALS3BP were found to be differentially expressed proteins in the serum of pregnant sheep. Therefore, these three proteins carry a high probability as candidate biomarkers for early gestational embryo attachment in ewes.Implications Early pregnancy diagnosis enables timely identification of non-pregnant ewes, resulting in optimised resource allocation and enhanced economic benefits for farmers by reducing production costs. The present study enables to advance the diagnosis of pregnancy in ewes to the first oestrous period after breeding. The differentially expressed protein in serum was found to be a highly specific and sensitive biomarker for early pregnancy diagnosis in ewes. Its application in production can reduce the number of days of mating loss in ewes, reduce production costs and improve economic efficiency.