ABSTRACT: Background: In food production animals, especially cattle, the diagnosis of gestation is an important issue because the timing of gestation has direct effects on the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection, or the accuracy, simplicity, or cost of the method, etc. A new method for detecting gestation, which involves assessing interferon-tau-related gene expression in peripheral blood leucocytes (PBL), was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods: PBL were collected on days 0 (just before artificial insemination), 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q)-PCR. PBL fractions were collected with flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solution. The expression levels of four interferon-tau-related genes, interferon-stimulated protein 15 kDa (ISG15), myxovirus-resistance (Mx) 1 and 2, and 2'-5'-oligoadenylate synthetase (OAS1), were then analysed in each fraction until day 28 of gestation using qPCR. Results: Miroarray analysis detected 72 and 28 genes significantly higher in whole PBL on days 14 and 21 of gestation compare to those on day 0, respectively. They involved interferon-tau responded genes. The expression levels of these genes increased with the progression of gestation. In the flow cytometry experiment, on day 14 all of the genes displayed significantly higher expression levels in the granulocyte fraction than the other fractions, and their expression levels gradually decreased until day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte fractions obtained with flow cytometry and density gradient separation. Conclusions: The expression profile of the four abovementioned genes could be a useful diagnostic biomarker of bovine gestation. Assessing the expression levels of ISG15, Mx1, Mx2 and OAS1 in a granulocyte fraction obtained with density gradient separation is a practical way of detecting gestation in cows within three weeks of insemination. Gene expression profiles were analyzed in the peripheral blood leucocytes at days 0 (n=5), 14 (n=3) and 21 (n=5) after artificial insemination in cow.
