Measurement of interferon-tau (IFN-τ) stimulated gene expression in blood leukocytes for pregnancy diagnosis within 18–20 d after insemination in dairy cattle

Abstract

The objective was to diagnose pregnancy within 18 d after insemination by measuring interferon-tau (IFN-τ)-stimulated genes (ISG) expression in circulatory leukocytes. Based on microarray results, three genes were selected [2′-5′ oligoadenylate synthetase 1(Oas1), myxovirus resistance gene 2 (Mx2), and interferon-stimulated gene 15 kDa protein (Isg15)] because they were known to be interferon-stimulated genes (ISG) and were also differentially expressed in the samples. The respective genes were assayed by using real time reverse transcriptase polymerase chain reaction (RTPCR). In the first experiment, RNA was isolated from leukocytes of pregnant ( n = 5) and non-pregnant ( n = 15) dairy cows on each of d 14, 16, 18, and 20 after insemination. The ISG expression (Mx2 and Isg15) was greater in pregnant compared with non-pregnant cows on d 18 and 20. In the second experiment, we attempted to define the shortest interval to pregnancy detection by measuring ISG on d 17 (Exp. 2A) or d 18 (Exp. 2B). For Exp. 2A, blood was collected from pregnant ( n = 16) and non-pregnant ( n = 16) cows on d 17 after insemination. On d 17, pregnant cows had greater Mx2 and Oas1 but Receiver Operator Characteristic (ROC) curves showed that the increase was too small to develop a suitable cut-off for a reliable pregnancy test. For Exp. 2B, blood samples were collected from pregnant ( n = 21) and non-pregnant ( n = 21) cows on d 18 after insemination. For both Mx2 and Oas1, primiparous pregnant cows had greater ISG expression than primiparous non-pregnant cows on d 18. For multiparous cows, however, pregnant and non-pregnant cows were similar for ISG expression on d 18. The possibility of correcting for pre-insemination ISG expression was examined in a final series of experiments. Blood samples were collected from cows ( n = 54) and heifers ( n = 24) during the luteal phase preceding insemination. A second sample was collected from the same cows [pregnant ( n = 13) and non-pregnant ( n = 41)] and heifers [pregnant ( n = 17) and non-pregnant ( n = 7)] 18 d after insemination. The ratio of the second sample (after insemination) to the first sample (before insemination) minimally increased the sensitivity of the ISG test for pregnancy detection. There was a large increase in ISG expression on d 18 of pregnancy in heifers (approximately 1 year of age) that could be measured for a reliable pregnancy test. The conclusion from the studies is that a reliable ISG-based method for pregnancy detection could be applied as early as d 18 but only in heifers. Older cows have a lesser response that reduces the sensitivity of the ISG test on or before d 18.