Early identification of bovine pregnancy status and embryonic mortality

Authors: BISHOP, JEANETTE - Colorado State University; GUZELOGLU, AYDIN - Colorado State University; SCHELLER, TOM - Colorado State University; DOCHEFF, JOSH - Blue Sky Dairy; GONZALEZ-BERRIOS, CAROLINA - Colorado State University; VAN CAMPEN, HANA - Colorado State University; NETT, TERRY - Colorado State University; Zezeski, Abigail; Geary, Thomas; THATCHER, WILLIAM - University Of Florida; HANSEN, THOMAS - Colorado State University

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/20/2025
Publication Date: 3/28/2025
Citation: Bishop, J.V., Guzeloglu, A., Scheller, T., Docheff, J., Gonzalez-Berrios, C.L., Van Campen, H., Nett, T.M., Zezeski, A.L., Geary, T.W., Thatcher, W.W., Hansen, T.R. 2025. Early identification of bovine pregnancy status and embryonic mortality. Biology of Reproduction. 112(5):981-995. https://doi.org/10.1093/biolre/ioaf066.
DOI: https://doi.org/10.1093/biolre/ioaf066

Interpretive Summary: Interferon-tau (IFNT) is produced only by the trophectoderm cells in the bovine conceptus as early as day 12. It was hypothesized that IFNT can be detected in peripheral blood, milk and/or cervical secretions and used to predict pregnancy status in lactating dairy and beef females. Recombinant bovine (rb)IFNT was generated, purified, and used to generate goat and rabbit anti-rbIFNT polyclonal antibodies. An enzyme-linked immunosorbent assay (ELISA, 5 h) for bIFNT was developed with no cross-reactivity with other type I or II IFNs. IFNT could not be detected reliably in serum, plasma, or milk samples, even at a limit of detection of 50-100 pg/ml on days 17-19 following artificial insemination (AI). However, when external os cervical swab samples were examined, IFNT was detected as early as day 15 and as late as day 25 of pregnancy. The most accurate days of IFNT detection were on days 16-19 after AI. Use of a custom bovine swab device reduced false negative rates to as low as 5% (94.5% Sensitivity) in dairy cows on day 17 and 0 to 3.4% (100 and96.6% Sensitivity) in beef cows on days 16 or 18, respectively. In summary, presence of IFNT in cervical swab fluid provides an excellent indication of pregnancy status in lactating dairy cows. Early identification of the open/non-pregnant allows for re-insemination earlier in lactation (days 21) compared to waiting until after ultrasound on day ~32-39. Also, the detection of IFNT on day 17, followed by loss of pregnancy by day 32 based on ultrasound provides a novel research tool for studying pregnancy loss caused by embryo mortality.

Technical Abstract: Interferon-tau (IFNT) is produced by the trophectoderm cells in the bovine conceptus as early as day 12 following fertilization. It was hypothesized that IFNT detection in blood, milk and/or cervical secretions could be used to diagnose pregnancy in lactating cows. Recombinant bovine (rb) IFNT was generated to produce goat and rabbit anti-rbIFNT polyclonal antibodies and an enzyme-linked immunosorbent assay (ELISA) for bIFNT was developed using these reagents. The IFNT ELISA did not cross-react with other type I or II IFNs and had a limit of detection of 50-100 pg/ml on days 17-19 post-AI. The IFNT ELISA detected IFNT in external os cervical swabs from days 15 to 25 post-AI, but did not detect IFNT in serum, plasma, or milk. The most accurate days of IFNT detection in cervical fluid were days 16-19 after AI. A custom bovine swab device used to collect cervical secretions reduced false negative rates to 5.5% (94.5% Sensitivity) in dairy cows on day 17 and 0 to 3.4% (100 and 96.6% Sensitivity) in beef cows on days 18 or 16, respectively. In summary, the detection of IFNT in cervical fluid by ELISA provides an accurate indication of pregnancy status in lactating dairy cows. Early identification of the open/non-pregnant cow allows re-insemination on day 21 compared to waiting until ultrasound on day ~32-39. Also, the detection of IFNT on day 17 followed by loss of pregnancy detected by ultrasound on day 32 provides a novel research tool for studying pregnancy loss caused by embryo mortality.