Interferon Tau (IFNT), the conceptus-derived pregnancy recognition signal in cattle, significantly modifies the transcriptome of the endometrium. However, the endometrium also responds to IFNT-independent conceptus-derived products. The aim of this study was to determine what proteins are produced by the bovine conceptus that may facilitate the pregnancy recognition process in cattle. We analysed by mass spectrometry the proteins present in conceptus-conditioned media (CCM) after 6 h culture of Day 16 bovine conceptuses (n = 8) in SILAC media (arginine- and lysine-depleted media supplemented with heavy isotopes) and the protein content of extracellular vesicles (EVs) isolated from uterine luminal fluid (ULF) of Day 16 pregnant (n = 7) and cyclic (n = 6) cross-bred heifers on day 16. In total, 11,122 proteins were identified in the CCM. Of these, 5.95% (662) had peptides with heavy labelled amino acids, i.e., de novo synthesised by the conceptuses. None of these proteins were detected in the EVs isolated from ULF. Pregnancy-associated glycoprotein 11, Trophoblast Kunitz domain protein 1 and DExD-Box Helicase 39A were de novo produced and present in the CCM from all conceptuses and in previously published CCM data following 6 and 24 h. A total of 463 proteins were present in the CCM from all the conceptuses in the present study, and after 6 and 24 h culture in a previous study, while expression of their transcripts was not detected in endometrium indicating that they are likely conceptus-derived. Of the proteins present in the EVs, 67 were uniquely identified in ULF from pregnant heifers; 35 of these had been previously reported in CCM from Day 16 conceptuses. This study has narrowed a set of conceptus-derived proteins that may be involved in EV-mediated IFNT-independent embryo–maternal communication during pregnancy recognition in cattle.

Current bovine pregnancy detection methods are not reliable until at least day 28 post artificial insemination (AI). The bovine estrous cycle is approximately 21 days; consequently, producers miss an opportunity to rebreed at the next estrous event. Therefore, commercial interest exists for the discovery of novel biomarkers of pregnancy which could reliably detect pregnancy status at or before day 21 of pregnancy. The objective of the present study was to use liquid chromatography tandem mass spectrometry (LC-MS/MS) to perform a global, label-free, proteomics study on (i) milk whey and (ii) extracellular vesicle (EV) enriched milk whey samples, from day 21 of pregnancy, compared with day 21 of the estrous cycle, in order to identify potential protein biomarkers of early pregnancy. The estrous cycles of 10 dairy cows were synchronized, they went through one (control) estrous cycle and these cows were artificially inseminated during the following estrus. These cows were confirmed pregnant by ultrasound scanning. Milk whey samples were collected on day 21 of the estrous cycle and on day 21 post AI. Milk whey samples and EV enriched milk whey samples were analyzed by LC-MS/MS and subsequent analyzes of the label-free quantitative data was performed in MaxQuant and Perseus. Four proteins (APOB, SPADH1, PLIN2 and LPO) were differentially expressed between the proteomes of milk whey from day 21 of pregnancy and day 21 of the estrous cycle (P < 0.05). Ten proteins (PIGR, PGD, QSOX1, MUC1, SRPRA, MD2, GAPDH, FOLR1, GPRC5B and HHIPL2) were differentially expressed between the proteomes of EV enriched milk whey from day 21 of pregnancy and day 21 of the estrous cycle (P < 0.05). These proteins are potential milk whey biomarkers of early pregnancy.

A robust electrochemical assay for sorbitol dehydrogenase (SORDH) activity in milk was developed using voltammetry and chronocoulometry at bare and polymer modified transducers.