Temporal analysis of circulating nucleic acid in early days of pregnancy in buffalo

Improving reproductive efficiency in livestock relies mainly on the ability to detect pregnancy quickly and accurately. Recently, circulating nucleic acids (CNAs) have been exploited for prenatal diagnosis in humans and animals. In the current investigation, serum samples were collected from pregnant animals ( n = 30) and non-pregnant animals ( n = 20) on 0th, 6th, 12th, and 18th day post artificial insemination. Total DNA was isolated from these serum samples. Two CNA tags (Bov-B and ART2A) derived from repetitive sections of the bovine genome were amplified using DNA extracted from serum samples. The expression analysis of these CNAs was done using real-time polymerase chain reaction assay, and copy number of each tag was calculated in pregnant and non-pregnant animals. The average number of copies of Art2A increased approximately threefold ( P < 0.01) from day zero of pregnancy (7,000 copies) to the day 18 of pregnancy (> 21,000). Similarly, BovB levels in the pregnant group increased significantly (approximately 2.9-fold) from day zero (93,900 copies) till day 18 (> 2, 72,310 copies) ( P < 0.01). There was no significant change observed on the 6th and 12th day of pregnancy and on the 18th day in the non-pregnant animals. In conclusion, based on these findings, the defined cut-off value can distinguish between pregnant and non-pregnant animals with a sensitivity of nearly 80% and specificity of nearly 70%. It is possible to employ these two CNA tags as biomarkers for early detection of pregnancy in buffaloes.