Early pregnancy detection in buffaloes targeting interferon-stimulated genes in duplex RT-qPCR

Early pregnancy diagnosis in buffaloes enhances reproductive efficiency and dairy production. This study aimed to develop and validate a duplex TaqMan-based RT-qPCR assay using two interferon-stimulated genes (ISGs), <i>ISG15</i> and <i>LGALS3BP</i>, for early pregnancy diagnosis in buffaloes. Whole blood samples were collected from artificially inseminated buffaloes before (day 0) and after (days 20, 25 and 40) artificial insemination (AI). On 40 days, post-insemination ultrasonography was performed to categorize animals as pregnant or non-pregnant. RNA from peripheral blood mononuclear cells (PBMCs) was isolated and converted into cDNA. A duplex TaqMan-based RT-qPCR assay was developed to predict pregnancy based on the expression of <i>ISG15</i> and <i>LGALS3BP</i> in PBMCs. Gene expression was evaluated in a test dataset at various time points. The assay's performance was validated using two additional datasets, under which blood samples were collected randomly at 20 ± 2 days to evaluate its utility for predicting pregnancy. Colour Doppler ultrasonography was utilized to confirm pregnancy at 40 days post-insemination for all the animals. Significantly different abundance of transcripts of <i>ISG15</i> and <i>LGALS3BP</i>, in pregnant buffaloes, was observed on day 20 post-insemination. A statistically significant fold change (p < 0.05) of <i>ISG15</i> and <i>LGALS3BP</i> transcripts was observed between pregnant and non-pregnant buffaloes. The receiver operating characteristic curve of validation datasets demonstrated the AUC = 0.95 and AUC = 0.90. The negative predictive value and positive predictive value range from 90% to 95% and 75% to 85%. In conclusion, the developed duplex RT-qPCR-based assay demonstrates high sensitivity and specificity in detecting early pregnancy in buffaloes.