Chronological profiling of early pregnancy transcripts in Murrah buffaloes

Effective reproductive management in cattle, such as cows and buffaloes, requires early and accurate pregnancy detection. Early identification of pregnancy enables farmers to promptly identify non-pregnant animals for treatment and/or rebreeding, thereby reducing the calving interval. This study aimed to standardize the expression of the CCL8 and CXCL10 genes as markers for early pregnancy detection in Murrah buffaloes. Blood samples were collected on the 16^th day post-artificial insemination for gene expression analysis and on days zero, seven, 14, and 21 post-artificial insemination for progesterone concentration measurement. Buffaloes were categorized as pregnant (n = 6) or non-pregnant (n = 6) based on the resumption of estrus. Gene expression levels in peripheral blood leukocytes were analyzed using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) with SYBR green dye. Amplicons of CCL8, CXCL10, and GAPDH genes were measured 108, 117, and 158 bp, respectively. Results showed that CCL8 mRNA expression in pregnant buffaloes was 5.13 and 12.21 times higher compared to non-pregnant buffaloes, while CXCL10 mRNA expression was 4.19 and 22.17 times higher. These findings indicated significantly elevated CCL8 and CXCL10 mRNA expression levels in peripheral blood leukocytes of pregnant buffaloes on the 16^th day. Progesterone levels in the pregnant group were increased significantly from day zero to day 21, while no significant differences were observed between groups on days zero, seven and 14. Pregnancy was further confirmed via per-rectal examination on the 45^th day post-artificial insemination Therefore, CCL8 and CXCL10 gene expression profiling on the 16^th day could serve as reliable early pregnancy markers in Murrah buffaloes.