Upregulation of Urinary Alpha‐2‐<scp>HS</scp> Glycoprotein During Pregnancy of Murrah Buffaloes

Urine samples were systematically collected from inseminated Murrah buffaloes (Bubalus bubalis) on days 0, 7, 14, 21 and 28 (with day 0 representing the day of artificial insemination). Following confirmation of pregnancy via trans rectal palpation 45 days of insemination, the animals were categorised into pregnant and non-pregnant groups (n = 10 each). The urine samples on 0, 7, 14, 21 and 28 days of pregnant and one sample from non-pregnant preferably collected on 28th day was used for SDS-PAGE after diafiltration. We focused on urine samples obtained on 28th day of insemination as it could contain high fetuin-A levels associated with pregnancy. Each of the two urine samples from both the pregnant and non-pregnant groups underwent purification through wheat germ agglutinin (WGA) affinity separation. The resulting fraction/elute was subsequently employed for the determination and validation of fetuin-A through 1D SDS-PAGE, western blotting and MALDI-TOF analysis. The samples were used for measuring the concentration of fetuin-A by ELISA. Fetuin-A was significantly higher (1020.50 ± 19.75 mg/L) in pregnant Murrah buffalo urine than their non-pregnant counterparts (86.5 ± 6.33 mg/L), with a p < 0.05. On SDS-PAGE analysis, the WGA fractions revealed distinct protein bands, notably between ~54 to 70 kDa and slightly above ~91 kDa, in pregnant Murrah buffaloes compared with their non-pregnant counterparts. Following confirmation of fetuin-A at ~58 kDa through western blotting, the corresponding protein band was excised from the SDS gel and subjected to mass spectrometry for MALDI-TOF analysis. Peptide mass fingerprinting, coupled with protein BLAST, confirmed the upregulated protein as Alpha2-HS glycoprotein, also known as fetuin-A. These findings are crucial for elucidating the presence of fetuin-A in urine samples from pregnant Murrah buffaloes, offering valuable insights into their physiological status.