Proteomic Analysis of Dairy Cows with Persistent Subclinical Hypocalcemia

To delineate the serum proteomic profile of persistent subclinical hypocalcemia (pSCH) in periparturient dairy cows, elucidate its molecular pathogenesis, and provide a theoretical basis for early warning and precision prevention and control, we selected 12 Holstein dairy cows of similar age (2.92 ± 0.12 years), parity (1.56 ± 0.21), body condition score (BCS) (2.84 ± 0.04), milk yield (26.81 ± 0.22 kg/d), and day in milk (DIM) (6.60 ± 0.24 d) and no significant between-group differences as experimental animals. Based on serum calcium concentrations on postpartum days 1 to 4 and clinical presentation, the cows were divided into a healthy control group (serum calcium > 1.77 mmol/L on day 1 and >2.20 mmol/L on day 4 postpartum, n = 6) and a persistent subclinical hypocalcemia group (serum calcium ≤ 1.77 mmol/L on day 1 and ≤2.20 mmol/L on day 4 postpartum, n = 6). Serum samples were collected on postpartum days 1, 2, and 4 and analyzed using 4D-DIA quantitative proteomics. A total of 178 significantly differentially expressed proteins were identified (fold change > 1.2, p < 0.05), including 59 up-regulated and 119 down-regulated proteins. These differentially expressed proteins were mainly enriched in pathways involving endocrine and other factor-regulated calcium reabsorption, regulation of actin cytoskeleton, the tricarboxylic acid cycle (TCA cycle), and lipoic acid metabolism. The results indicate that the pathological state of pSCH is closely associated with abnormalities in calcium reabsorption regulation, actin cytoskeleton maintenance, and mitochondrial energy metabolism-related signaling pathways. The key proteins and pathways identified in this study provide a theoretical foundation for future in-depth research on the pathogenesis, prevention, and treatment of pSCH.