Early pregnancy diagnosis in cows using corpus luteum blood flow analysis based on colour Doppler ultrasonography and mRNA analysis

Background: Reproductive efficiency is paramount in the dairy industry, where early pregnancy detection of dairy cows will allow to detect the non-pregnant animals early, thus enabling to re-synchronize them and getting them pregnant leading to decrease in calving interval, which, in turn, is critical for maximizing productivity and economic gain. The objective of this study was to evaluate the colour Doppler ultrasonography (CDUS) and peripheral blood leukocytes (PBLs)-based pregnancy-associated biomarker mRNAs expression for the earliest detection of pregnancy status in the dairy cows at post insemination. Intensively managed animals were ovulation synchronized and subjected to timed artificial insemination (TAI). On day 20, corpus luteum blood flow (CLBF) was evaluated using CDUS in 30 cows. The percentage of the incoming blood flow (as an area) of the corpus luteum (CL) was determined using an image analysis software. On day 35, the same operator performed a final pregnancy diagnosis using transrectal ultrasonography to confirm the pregnancy. Blood samples were collected on day 20 and 28 after TAI for biomarkers analysis. The mRNA expression levels of ISG15, MX1, MX2, and PAG9 genes in PBLs were determined by quantitative polymerase chain reaction (qPCR).

Results: The identified CLBF cutoff point resulted 100% sensitivity and negative predictive value (NPV) in determining non-pregnant status on day 20 in the cows. Overall, MX2 and ISG15 mRNAs showed the most significant (P < 0.05) expression levels in pregnant animals on day 20 and 28 compared to non-pregnant animals. Among them, MX2 showed the highest expression levels on both days, ascertaining it as the better candidate biomarker for the earliest identification of pregnancy.

Conclusions: The CDUS-based CLBF analysis on day 20 after TAI can be potentially used for the early identification of non-pregnancy status in dairy cows and MX2 could be a potential mRNA candidate for the identification of pregnancy in cows. Further studies should be conducted in large scale to validate these findings due to the small sample number used in the current study.