Potential utility of RSAD2 transcript and protein in early detection of pregnancy in buffaloes.

This study investigates a novel early pregnancy marker in water buffaloes, focusing on RSAD2 mRNA expression, known to be upregulated by interferon-tau (IFNT) during pregnancy. While RSAD2 is primarily recognized for its antiviral effect, we hypothesized its role as a conceptus-induced component in regulating pregnancy in buffaloes. Given its differential expression compared to other IFNT-induced genes in cows, RSAD2 may serve as a biomarker for early pregnancy detection in buffaloes. RNA, cDNA, and plasma samples were obtained from archived samples collected before insemination (d0) and at d20, d25 and d40 after insemination. Twelve RNA samples, having optimal optical density and concentration, from six pregnant and six non-pregnant buffaloes were selected. The cDNA was analyzed to measure the abundance of RSAD2 mRNA using real-time quantitative PCR (RT-qPCR) and plasma for protein expression analysis using Western blot. The RT-qPCR analysis showed a transcript of RSAD2 increased significantly by 7-fold and 6-fold on d20 and d25, compared to both d0 and d40 in the pregnant group only. At d20, the sensitivity of RSAD2 was 100% and the specificity was 83.3%, and at d25-d both the sensitivity and specificity was 100%, indicating low incidences of misdiagnosing early pregnancy in buffaloes. In the non-pregnant group, RSAD2 expression remained low and did not change after insemination. Western blot analysis revealed an immunoreactive RSAD2 protein band. Densitometry analysis of the RSAD2-specific protein band, based on gray mean value, showed significantly increased expression of RSAD2 at d25 compared to d0 in the pregnant group. In conclusion, these results indicated that RSAD2 expressions at both the mRNA and protein levels show promising potential for detecting pregnancy at d25 post-insemination.