Early pregnancy diagnosis in dairy cows using circulating nucleic acids

Early and accurate pregnancy diagnosis in dairy cattle is a prerequisite for successful herd management. However, most of the currently available methods allow an early diagnosis only approximately 30 days after insemination. Recently, circulating nucleic acids (CNAs) have been used successfully as biomarkers in prenatal diagnosis at different gestational stages in human and animals. Here we show that CNAs can also be used as markers for the detection of early pregnancy in cattle. Serum samples were collected from multiparous pregnant (N = 24) and nonpregnant (N = 16) dairy cows at different days after insemination (Days 0, 20, and 40). Isolated serum DNA was preprocessed using a modified serial analysis of gene expression technique, which generated concatemerized short sequence tags for downstream next generation sequencing. Bioinformatic analysis identified sequence tags specific for pregnant dairy cows at Day 20 after insemination. The identified CNA-tags originated from repetitive regions of the bovine genome. Tag sequences that showed increased hit counts per animal were used to design quantitative real-time polymerase chain reaction assays. These quantitative polymerase chain reaction assays were applied to CNA samples from matched pregnant (N = 12) and nonpregnant cows (N = 16) at different times after insemination (Day 0, 20, and 40). At Day 20 after insemination the quantities of the interspersed repeats Art2A and BovB were increased in the pregnant cows compared with the nonpregnant control cows (P < 0.05). The best performing CNA biomarker BovB yielded an area under the receiver operating characteristic curve of 0.76. At a defined cutoff value, the pregnant and the control groups can be distinguished with a sensitivity of 83% and specificity of 75%. These results suggest that CNAs can be used as biomarkers for the detection of early pregnancy in cattle.