Conceptus signaling markers in immune cells enhance pregnancy prediction in beef cattle

In beef cattle, estrous synchronization aiming a second artificial insemination (AI) requires a reliable estimation of the pregnancy status 20 days (D20) after the first AI. The hypothesis is that the expression of interferon-stimulated genes (ISGs; ISG15, OAS1, RSAD2, and IFI44) and cytokines (IL1β and IL10) in mononuclear (PBMC) and polymorphonuclear (PMN) cells is regulated by interferon-τ (IFN-τ) and predicts the pregnancy status. PBMC and PMN were isolated from non-pregnant beef cows (N = 9), 10-12 days post-ovulation (D0), and stimulated with 100 ng/mL recombinant ovine (ro) IFN-τ or with pooled uterine flush (UF) from D18 pregnant cows. Both roIFNT and UF stimulated the expression of ISG15, RSAD2, and IFI44 in PBMC and PMN. Expression of IL1β was reduced by UF in both PBMC and PMN. On another experiment, PMN were isolated, and luteal blood perfusion was measured on D20 post-timed-AI in beef females. The accuracy of ISG expression and luteal blood perfusion to predict the pregnancy outcome was determined by ROC curve analysis. All gene combinations were tested, and the best association for increased accuracy (92.7%) and reduction of false-negative results (0.9%, 2/233) was obtained through the combination of the four ISGs (ISG15, OAS1, RSAD2, and IFI44). The criterion was that if the expression levels of at least one of the four genes were greater than the predefined cutoffs, the animal would be considered pregnant. In conclusion, the expression of ISGs and IL1β was upregulated by roIFNT and UF from pregnancy cows. The combined PMN expression of classical (ISG15 and OAS1) and unusual (RSAD2 and IFI44) ISGs provided the greatest predictive accuracy of the pregnancy status on D20 in females with active CL by Doppler and is a potential tool to be used in reproductive programs for beef cattle.