Exploring the sources of variation in the serum metabolic profile of Italian Mediterranean buffaloes

This cross-sectional study investigated the main sources of variation in key blood metabolites related to energy, protein, and hepatic-muscular profiles in lactating Italian Mediterranean buffaloes, focusing on the effects of parity, DIM, season, and ECM. Blood samples were collected from 855 clinically healthy lactating buffaloes for serum analysis. Metabolites were grouped into 3 functional profiles: energy profile, including glucose, cholesterol (CHOL), triglycerides (TRI), nonesterified fatty acids (NEFA), and BHB; protein profile, including total protein, albumin (ALB), globulin (GLO), urea, creatinine (CREA), and albumin-to-globulin ratio (A/G); and hepatic-muscular profile, including alanine aminotransferase (GPT), aspartate aminotransferase (GOT), alkaline phosphatase, lactate dehydrogenase (LDH), γ-glutamyl transferase (GGT), creatine kinase (CK), total bilirubin, high-density lipoproteins (HDL), and low-density lipoproteins (LDL). Mixed linear models were used to assess metabolites variation, with parity, DIM, season, and ECM classes as fixed effects and herd sampling day as a random effect. Overall, most metabolites showed limited variability and remained within physiological ranges, confirming the metabolic stability of the species. Parity significantly affected several indicators. Cholesterol exhibited a nonlinear pattern, peaking in third-parity animals (126 mg/dL). Total protein and GLO increased progressively with parity (from 6.69 to 6.95 g/dL and from 3.44 to 3.80 g/dL, respectively), whereas ALB and A/G were lowest in animals with parity ≥5 (3.16 g/dL and 0.82, respectively). Several hepatic-muscular biomarkers, including GOT, LDH, CK, and HDL, generally decreased with increasing parity. Days in milk were significantly associated with metabolic variation. Nonesterified fatty acids and BHB peaked during early lactation (≤75 DIM) and decreased thereafter (0.39-0.25 mmol/L, 0.40-0.35 mmol/L, respectively), reflecting progressive restoration of energy balance. Cholesterol increased after 50 DIM, whereas TRI reached the lowest concentrations between 51 and 75 DIM before increasing in late lactation. Total protein, ALB, and GLO were lowest at 116 to 140 DIM and subsequently increased, whereas CREA and urea rose progressively in late lactation. Aspartate aminotransferase peaked within the first 50 DIM (136 U/L) and decreased thereafter, whereas GPT and LDL increased after 75 and 50 DIM, respectively. Season significantly influenced only CREA, which was slightly higher during the cold season than the hot season (1.65 vs. 1.61 mg/dL). However, significant DIM × season interactions were observed for CHOL, NEFA, and GGT, whereas parity × season interactions affected urea and LDL, indicating context-dependent metabolic modulation. Increasing ECM was associated with coordinated metabolic upregulation, including increases in CHOL (from 118 to 126 mg/dL), BHB (0.37 to 0.40 mmol/L), GOT (120 to 126 U/L), GGT (27.5 to 30.2 U/L), and CK (185 to 210 U/L). In conclusion, buffaloes in the present study exhibited a stable and well-regulated metabolic profile, with most variation reflecting physiological adaptations to parity, DIM, and productive performance. These findings provide valuable information for clinical interpretation and herd management, although further studies under different environmental conditions are needed to confirm broader applicability.