Investigation of conceptus stimulated gene expression in buffalo peripheral blood mononuclear cells as potential diagnostic markers of early pregnancy.

Exploration of novel strategies for early pregnancy diagnosis is pivotal in enhancing the reproductive potential and monetary gains from dairy herds. In buffalo, the trophectoderm cells of the elongating conceptus secrete interferon-tau that stimulates the transcription of various genes in peripheral blood mononuclear cells (PBMC) during the peri-implantation period. We explored the differential expression of classical (ISG15) and novel (LGALS3BP and CD9) early pregnancy markers in PBMC of buffaloes during various stages of pregnancy. Natural heat was detected in buffaloes by assessing the vaginal fluid, and artificial insemination (AI) was done. Whole blood was collected from the jugular vein in EDTA-containing vacutainers for PBMC isolation before AI (0-day) and 20, 25 and 40 d post-AI. On day 40, transrectal ultrasonography examination was performed to confirm pregnancy. The inseminated non-pregnant animals served as control. Total RNA was extracted using the TRIzol method. The temporal abundance of ISG15, LGALS3BP and CD9 genes in PBMC was compared between pregnant and non-pregnant groups (n = 9 per group) using real time-qPCR. Results showed transcripts of ISG15 and LGALS3BP were more abundant at 20 d in the pregnant group compared to the 0 d and 20 d values of the non-pregnant group. However, due to variability in expression, threshold (Ct) cycle of RT-qPCR alone could not distinguish pregnant and non-pregnant animals. In conclusion, ISG15 and LGALS3BP transcripts abundance in PBMCs are potential candidate biomarkers for early prediction of buffalo pregnancy 20-days post-AI, but further work is required to allow the development of a reliable new methodology.