During the transition period, dairy buffaloes undergo profound metabolic, biochemical and immunological changes to support the onset of lactation and restore physiological homeostasis following parturition. In the present study, the occurrence of NEB was clearly evidenced by significant (P≤0.05) alterations in circulating energy metabolites, lipid fractions, liver enzymes, minerals, oxidative stress markers and inflammatory cytokines during the transition period.
Serum non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) are well-established indicators of adipose tissue mobilization and hepatic ketogenesis, respectively. In the present study, NEFA concentrations increased progressively from -21 days prepartum (294.35± 5.36 μmol/L) to peak levels at +7 days postpartum (412.80±5.14 μmol/L), followed by a gradual decline by +21 days postpartum. Similarly, BHBA concentrations rose from 36.82±1.22 nmol/mL at -21 days to a maximum at +7 days postpartum (58.95±1.76 nmol/mL) before decreasing thereafter (Fig 1 a,b). These trends reflect intensified lipolysis and increased hepatic uptake of fatty acids during early lactation, when glucose availability is prioritized for lactose synthesis (
Herdt, 2000;
Gross et al., 2011; Lisuzzo et al., 2024). Comparable patterns have been reported in dairy cows and buffaloes, wherein peak NEFA and BHBA concentrations occur within the first week postpartum, coinciding with maximal milk yield and lowest dry matter intake (
LeBlanc, 2010;
McArt et al., 2013). Similar metabolic alterations associated with negative energy balance have also been reported in postpartum buffaloes
(Sriranga et al., 2023). Persistently elevated NEFA and BHBA are known to impair immune cell function, hepatic metabolism and reproductive performance, thereby increasing the risk of metabolic disorders such as ketosis and fatty liver syndrome.
In the present study significant alterations in the lipid profile were observed during the whole transition period (Table 1). Serum total cholesterol, triglycerides, HDL-C, LDL-C and VLDL concentrations declined markedly around calving, reaching their lowest values at +7 days postpartum, followed by gradual recovery by +21 days. These changes are primarily attributed to reduced hepatic synthesis of lipoproteins, increased utilization of cholesterol for steroid hormone synthesis and impaired triglyceride export from the liver during NEB (
Grummer, 1995;
Bionaz et al., 2007). Hypocholesterolemia and hypotriglyceridemia during early lactation have been consistently reported in buffaloes and dairy cows and are considered indicators of compromised hepatic lipid metabolism (
Mondal and Paul, 2012;
Das et al., 2016). The reduced availability of circulating lipoproteins further exacerbates hepatic fat accumulation, contributing to oxidative stress and inflammation.
Activities of liver enzymes and concentrations of minerals and glucose also changed significantly during the transition period (Table 2). Serum calcium, phosphorus and magnesium concentrations declined around calving and early postpartum, reflecting increased mineral demand for colostrum secretion, milk synthesis and neuromuscular function (
Goff and Horst, 1997;
Goff, 2008). Subclinical hypocalcemia during early lactation has been associated with reduced feed intake, impaired immune function and increased susceptibility to postpartum disorders. Elevated activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) observed in the present study indicate increased hepatic workload, enhanced gluconeogenesis and intensified lipid metabolism during NEB
(Contreras et al., 2010; Puppel et al., 2015). Similar increases in hepatic enzyme activities during the transition period have been reported in buffaloes by
Kour et al., (2024), indicating enhanced hepatic metabolic stress around parturition. Serum glucose concentrations declined at calving and remained low until +14 days postpartum, confirming the persistence of NEB, with partial recovery by +21 days as feed intake improved and metabolic adaptation progressed
(Yadav et al., 2013).
Total protein and globulin concentrations declined at calving, likely due to hemodilution and the selective transfer of immunoglobulins into colostrum to support passive immunity in newborn calves.
Rathwa et al., (2023) similarly observed alterations in serum total protein and albumin concentrations during the transition period in Surti buffaloes, reflecting metabolic adaptation and hepatic functional changes. In contrast, albumin levels increased slightly, resulting in an elevated albumin-to-globulin ratio. These findings suggest a transient redistribution of protein fractions rather than impaired hepatic protein synthesis. Blood urea nitrogen (BUN) and creatinine concentrations peaked at calving, reflecting increased protein catabolism, enhanced amino acid oxidation for gluconeogenesis and transient renal adaptation to increased metabolic load (
Mondal and Paul, 2012;
Mishra et al., 2013; Mondal and Paul, 2012;
Mishra et al., 2013; Bademkiran et al., 2020). Similar increases in BUN around parturition have been linked to increased mobilization of body protein reserves during NEB (Table 3).
Markers of oxidative stress exhibited pronounced changes during the transition period, reflecting a clear imbalance between reactive oxygen species (ROS) generation and antioxidant defense mechanisms in dairy buffaloes. Malondialdehyde (MDA), a well-recognized marker of lipid peroxidation, increased significantly at calving and reached peak concentrations at +7 days postpartum (Fig 2a). This elevation in MDA indicates enhanced oxidative damage to cellular and mitochondrial membranes during early lactation, a period characterized by intensified metabolic activity and extensive mobilization of body fat reserves. The surge in circulating non-esterified fatty acids (NEFA) during negative energy balance (NEB) increases hepatic fatty acid uptake and mitochondrial β-oxidation, thereby promoting excessive electron leakage from the respiratory chain and subsequent ROS production. Concurrently, the activities of key antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase, declined markedly around calving and during the immediate postpartum period (Fig 2b,c,d). This transient suppression of antioxidant capacity may be attributed to overwhelming ROS production, depletion of endogenous antioxidant reserves and reduced availability of antioxidant precursors and micronutrient cofactors under conditions of NEB
(Bernabucci et al., 2005; Jain and Shakkarpude, 2024;
Sordillo and Aitken, 2009). Similar reductions in antioxidant enzyme activities during the periparturient period have been associated with increased susceptibility to metabolic stress and immune dysfunction. The temporal coincidence of peak MDA concentrations with elevated NEFA and β-hydroxybutyrate (BHBA) levels further supports the mechanistic link between excessive lipolysis, hepatic ketogenesis, mitochondrial overload and oxidative stress
(Contreras et al., 2010; Lisuzzo et al., 2024). The gradual restoration of antioxidant enzyme activities by +21 days postpartum suggests an adaptive upregulation of endogenous antioxidant defense systems as metabolic balance improves, feed intake increases and oxidative pressure is alleviated
(Yang et al., 2018; Sharma et al., 2017; Gessner et al., 2021). Altered antioxidant status during the transition period has been linked with metabolic stress and lactation-associated physiological adaptations in Murrah buffaloes
(Chaudhari et al., 2017). These findings highlight the importance of managing oxidative stress during the transition period to safeguard metabolic health and immune competence in dairy buffaloes.
Serum inflammatory cytokines exhibited significant modulation during the transition period, reflecting dynamic changes in immune competence and physiological adaptation. Pro-inflammatory cytokines, including TNF-α and IFN-γ, declined sharply at calving, reaching their lowest concentrations on the day of parturition (day 0), followed by a gradual increase during the early postpartum period (Fig 3a,b). This transient suppression of pro-inflammatory cytokines may represent a physiological adaptation to prevent excessive immune activation during parturition, which could otherwise damage maternal tissues. In contrast, IL-10, a key anti-inflammatory cytokine, increased at calving and gradually declined thereafter, suggesting a temporary shift toward an anti-inflammatory state that supports tissue repair and limits inflammatory damage associated with placental separation, uterine involution and tissue remodeling (Fig 3c). The observed cytokine profile indicates that parturition is accompanied by a tightly regulated immunosuppressive and anti-inflammatory phase, likely aimed at protecting the dam from excessive inflammatory responses while maintaining essential immune surveillance. The postpartum resurgence of pro-inflammatory cytokines reflects the re-establishment of immune competence and functional recovery of the maternal immune system, which is crucial for protecting against infections and supporting early lactation. Similar cytokine dynamics have been reported in periparturient dairy cows and buffaloes, highlighting their close association with metabolic stress, oxidative imbalance and overall transition period adaptation
(Contreras et al., 2010; Gomaa et al., 2021; Ciliberti et al., 2025). One limitation of the present study is the relatively small number of animals included (n = 12), which may restrict the broader applicability of the findings. Nevertheless, repeated observations on the same animals throughout the transition period and uniform management conditions helped minimize biological variation. Further investigations involving larger populations are warranted to confirm the present results.
