The world population of buffaloes (Bubalus bubalis) is estimated at 205 million, reared across more than 66 countries; this represents a 24.89% increase from the year 2000 to 2022 (1).

World buffalo milk production is estimated to have reached 143 million tons in 2022, marking a 49% increase from 2011 (1). The largest herds of buffalo are found in developing countries such as India (54.53%), Pakistan (21.29%), China (13.10%), and Nepal (2.50%) (1). These herds are predominantly managed through family-owned and extensive farming systems. Buffalo’s fresh milk represents more than 15% of the total global production of whole fresh milk in 2022 (Table 1), and this percentage increased to 34% within Asia (Table 1). In Europe, 89% of buffalo are bred in Italy, where the buffalo population has grown from 12,000 heads in 1947 to 435,972 in 2023 (3). Italy boasts 2,381 farms, with over half (50.48%) located in the Campania region. This concentration has positioned Italy as the seventh largest producer of fresh buffalo milk in the world, significantly benefitting economically from the production of Mozzarella di Bufala Campana Dop cheese, which generated 893 million euros in 2023 (4). Another important aspect is related to the intensification of the Italian breeding system, with 39% of the animals raised on farms with more than 500 heads (3). The greater interest in buffalo products, such as milk and meat, has increased the transition from farmyard activity to commercial holdings and large herds, which is more similar to the dairy cattle industry (5). However, the use of techniques that have not always been developed specifically for buffalo species can increase the incidence of various conditional diseases (6). Late gestation and early lactation, which is also known as the transition period, represent the time of the highest incidence of metabolic and reproductive diseases (7).

In particular, the imbalance between energy intake and energy demand resulted in a para-physiological negative energy balance (NEB) in dairy ruminants (8, 9). Body resources, such as muscle, adipose, and bone tissues, are then mobilized in the form of amino acids, non-esterified fatty acids (NEFAs), and calcium to support energy requirements for the growth of the fetus and the onset of lactation (7). These metabolic adaptations are also accompanied by alterations in inflammatory responses that modify immune function and may generate oxidative stress in animals (10). Therefore, the mobilization of body resources can increase the risk of several interrelated diseases, although a certain degree of immune activation and inflammation is usually found in periparturient animals. These diseases have a negative impact on the growth, health, lactation, and reproductive performance of the animals (7, 11, 12).

The body condition score (BCS) has been proposed to estimate the energy balance and body reserves instead of variations in live weight (6). However, NEFA appears to be more sensitive to energy balance (11). A NEFA concentration above 0.29 mEq/L in the prepartum period or 0.57 mEq/L in the postpartum period has been identified as a critical threshold for an increased risk of metabolic diseases in dairy cows (13). Along with NEFA, β-hydroxybutyrate (BHB), one of the main ketone bodies produced by fatty acid oxidation, is also a common indicator of NEB during the transition period, with a threshold of 1.0 mmol/L in the postpartum period of cows (9, 14). However, a specific threshold for NEFA and BHB in buffaloes has not yet been established, and the dairy cows’ cut-offs of these parameters are frequently applied in this species (15).

The hypothesis is that greater lipomobilization could cause more severe alterations in the biochemical profile of buffaloes. To date, there is very limited information relating to metabolic profile curves in the buffalo species during the transition period in intensive farming, and in particular, given its tropical origin, it could present qualitative and quantitative variations that do not overlap with what was found in dairy cattle. Therefore, this study aimed to evaluate the biochemical profile of Mediterranean buffaloes with different levels of lipomobilization from the prepartum period to the postpartum period.

In accordance with the principles of the 3Rs (replacement, reduction, and refinement), the choice to conduct the experiment on a single farm was influenced by the desire to minimize the number of animals used, while ensuring robust results, an objective made possible by the homogeneity of the experimental conditions. The farm under study allowed us to have only one exposure scenario, especially in terms of the type of feed and rationing, which allowed for the execution of accurate metabolic profiles on an appropriate number of subjects. To date, studies on the metabolic aspects of the buffalo species have been conducted either using minimal numbers of animals or constructing curves through single sampling on multiple subjects, reconstructing possible profiles through interpolations. In this study, we preferred to construct real profile curves with a considerable expenditure of energy and funds considering a total number of 1,064 samples and 36,176 analyses.

The negative energy balance naturally occurs from late gestation to early lactation in ruminants, although buffaloes seem to show a different trend than dairy cows (19, 20). One of the metabolic adaptations is adipose tissue lipolysis, resulting in an increase in the NEFA (21, 22). The release of the NEFA is also related to the formation of ketone bodies, particularly BHB, which can negatively influence animal health by causing metabolic disorders (5). Typically, the NEFA peak is followed by the BHB peak, but large variations in the timing of the peak can occur (23). This study aimed to evaluate the biochemical profile from the prepartum to postpartum periods in Mediterranean buffaloes with mild (NEFA-I; NEFA≤0.29 mEq/L), medium (NEFA-II; 0.29 < NEFA<0.57 mEq/L), and severe (NEFA-III; NEFA≥0.57 mEq/L) lipomobilization at 7 weeks before calving (T-7).

The pattern of NEFA and BHB concentrations suggested that the NEFA-III group exhibited more energy deficiency than other groups during the dry-off period, resulting in increased lipid mobilization, although the same TMR was used for all animals. In addition, the NEFA-II group showed lipolytic activity indicative of energy deficiency, although less evident than the NEFA-III group. As shown in the studies of Fiore et al. (19) and Deka et al. (21) on dairy buffaloes, all groups showed a peak in NEFA and BHB near calving, probably due to the smaller abdominal space resulting in reduced voluntary dry matter intake and increased energy requirements for calving and the onset of lactation (21, 24). However, the NEFA-III group continued to show the highest NEFA concentration during the postpartum period. Furthermore, BHB showed the highest values in the same group at 1 and 5 weeks after calving (T + 1 and T + 5). This finding is in line with the trend of BHB in dairy cows. In fact, the highest percentage of hyperketonemia cases occurs before the 10th day of lactation, with a second increase occurring between the 1st and the 2nd month of lactation (25). Buffaloes generally show a peak in milk production between 20 and 30 days postpartum, with a consequent increase in energy demand (19). However, an increase in BHB may not only be related to an impairment of Krebs cycle function but also to an energy deficiency (26). Therefore, the NEFA and BHB results of the NEFA-III group suggested the persistence of energy deficits during the postpartum period. In addition, the higher BHB levels at 1 and 5 weeks postpartum could indicate both increased metabolic stress from greater lipomobilization and a potential Krebs cycle impairment.

The lower GLU concentration in the NEFA-II and NEFA-III groups at calving probably causes higher lipomobilization and the formation of BHB. Furthermore, the association of this result with a possible impaired Krebs cycle function could have caused the significant BHB peak in the NEFA-II group. Moreover, GLU showed the highest concentration in the NEFA-III group at 1 and 2 weeks after calving (T + 1 and T + 2) despite the highest lipomobilization during the same period. This result could support the hypothesis that GLU concentrations are poorly modified parameters during the initial conditions of metabolomic alterations (26).

The total cholesterol concentration decreased during the prepartum period of the ruminants and then increased subsequently after calving, as shown in this study (19, 27). The decrease in CHO may be associated with steroidogenesis for the production of estrogens, progesterone, and glucocorticoids during the last period of pregnancy (19, 28). Furthermore, this decrease may represent a limiting factor for lipoprotein synthesis, particularly for very low-density lipoprotein (VLDL) composed of cholesterol esters (29, 30). Cholesterol esters are derived from the activity of lecithin cholesterol acyltransferase (LCAT), whose functionality is reduced before and shortly after calving. The reduced levels of CHO potentially associated with reduced activity of LCAT may predispose animals to fatty liver disease (30–32). The NEFA-II group showed the lowest concentration during the first 3 weeks of the study (T-7, T-6, and T-5) and in the last 3 weeks (T + 4, T + 5, and T + 6), further predisposing this group to fatty liver disease.

The production of free radicals as reactive oxygen species (ROS) is particularly increased during the transition period in animals due to increased metabolic demands and lipid peroxidation. The excess of radicals over antioxidants induces oxidative stress that increases the susceptibility of animals in the transition period to various production diseases (20, 33). The NEFA-III group in this study started with the lowest concentration of antioxidants (BAP), which increased during the prepartum period, which is in agreement with the lowest concentration of radicals (D.ROMS). Uric acid is one of the antioxidants considered in the BAP test (33). Its concentration was the highest at the beginning of the study in the group NEFA-III, in contrast to the BAP levels, and remained elevated during the postpartum period. The increase in uric acid in dairy cows is considered a response to oxidative stress resulting from inflammation (34). In contrast, the NEFA-II group showed lower concentrations of antioxidants, uric acid included, during the prepartum period, with the highest concentration of radicals. These results suggest a higher risk of oxidative stress for the NEFA-II group.

Considering the above results and the absence of differences in both the BCS and the diet of the animals, it can be assumed that the NEFA-III group was able to manage lipomobilization without exacerbating oxidative stress during both the prepartum and postpartum periods. On the other hand, the NEFA-II group was not fully able to manage lipomobilization, resulting in an increased risk of oxidative stress during the prepartum period. These conditions may suggest a possible genetic and/or metabolic deficiency of the NEFA-II and NEFA-III groups that could increase the risk of metabolic diseases.

Inflammatory events may occur around parturition due to differentiation and proliferation of the mammary gland, the interaction between the uterus and placenta, and physical effort during calving (29, 35). Serum proteins are divided into ALB, α, β, and γ–GL (36). A decrease in TP levels during the dry-off period of buffaloes, as observed in this study, up to the onset of lactation has previously been shown, probably due to hormone synthesis, fetal growth, especially for muscle masses, and gluconeogenesis (20, 37). On the contrary, ALB is a negative acute-phase protein whose concentration should remain constant during pregnancy in healthy cows (38). The NEFA-II group showed the lowest levels of TP and ALB during the prepartum period, suggesting an inflammatory status probably related to the highest values of radicals during the same period. An inflammatory condition should also be characterized by a higher concentration of GL, especially in α–GL, as it is more composed of positive acute phase proteins (Hp, ceruloplasmin, α1-acid glycoprotein, and α1-antitrypsin) (35, 36). In our study, α–GL and Hp showed no differences among the groups during the prepartum period.

A slight increase in Hp can be evidenced, especially during the last weeks of pregnancy, as demonstrated in this study, coinciding with the mammary gland development (35). Inflammatory events may also be inferred by an increase in β-GL levels, as found in ketotic cows (39–41). Furthermore, a decrease in γ–GL is observed in dairy cows during the last month of pregnancy until the first days of lactation due to the transfer of immunoglobulins from the blood into the colostrum (39, 42, 43). The buffaloes in this study showed an increase in β-GL and a decrease in γ–GL at or around the time of calving across all groups.

Lysozyme (LYS) is an antibacterial enzyme produced by neutrophils and macrophages and is present in all body fluids. Along with the complement system (COM), LYS plays an important role in the innate defense against pathogens. Typically, LYS concentrations are usually higher in buffaloes than in cows (17–20 vs. 1–3 μg/mL) (44, 45). A lower LYS level is indicative of inflammation and is associated with a decrease in COM levels (35). In our study, the NEFA-III group showed lower levels of both LYS and COM during the prepartum period. After calving, COM levels remained lower in this group compared to others, while LYS levels increased. Furthermore, LYS concentrations across all groups were generally lower at all time points compared to previously reported values for the buffaloes (44). Similarly, COM levels during the first three weeks (T-7, T-6, and T-5) of the study in the NEFA-III group were lower compared to previous findings (45). These results suggest that all groups experienced an inflammatory state close to calving. However, the NEFA-III group may be more susceptible to inflammation, considering their lower levels of γ–GL, LYS, and COM and higher levels of NEFA (35).

Iron is an important component of proteins involved in oxygen transport and energy production (46). Its level usually ranges between 130 and 250 μg/dL in ruminants (47), although according to Rocha et al. (48), its concentration can range between 93 and 287 μg/dL in buffaloes. Our animals were within the normal range, whereas an increase was observed in all the groups at T-1. Increased iron levels may be related to causing liver disease (47). NEFA values suggest that lipomobilization is close to calving across all groups, which could have a negative impact on liver health. Interestingly, the NEFA-III group showed the highest values among groups at 1 week after calving. In contrast, the lower Fe levels evidenced during the postpartum period compared to the prepartum period may be due to inflammatory events, which are normal during the peripartum period (47). Regarding K and Mg, all groups showed an increase in the concentration of both minerals at the time of calving. Considering that K is predominantly intracellular, the increase may suggest an intensification of metabolically active body tissue and muscle, probably due to calving itself (49). Furthermore, the NEFA-III group showed the highest values during the first 3 weeks of the study. On the contrary, the increase in Mg could be related to an enhanced requirement for milk production with a consequent increase in bone mobilization (50).

The results of the present study suggest that further refinements in buffalo-specific nutrition and metabolism are needed. Indeed, animals with severe lipomobilization did not show signs of oxidative stress, unlike those with medium lipomobilization. In recent years, it has been hypothesized that certain nutritional requirements, particularly relative to amino acids, may be related to inflammatory, immune, and antioxidant system responses, as well as to the proper functioning of metabolism itself (9, 15, 51). Therefore, insights with more accurate analytical techniques, such as metabolomics, could be useful for identifying and assessing potential nutritional deficiencies and/or increased requirements in these animals. This, in turn, could impact their metabolism, inflammatory, immune, and antioxidant responses. Furthermore, greater lipomobilization could adversely affect and/or be associated with a reduced ability to adequately regulate anti-inflammatory and immune responses. Therefore, animals with high lipomobilization might be more susceptible to infectious diseases (metritis, mastitis, and pneumonia), even in the absence of immunosuppression related to pathological levels of BHB.

In summary, this study evaluates the biochemical profile of Mediterranean buffaloes with varying levels of lipomobilization during the transition period, from the prepartum to postpartum periods, and describes the trends for the three examined NEFA thresholds. Buffaloes with higher NEFA levels (>0.29 mEq/L; NEFA-II and NEFA-III) 7 weeks before calving should be monitored more closely to mitigate the risk of metabolic diseases. Furthermore, the medium (NEFA-II) and severe (NEFA-III) lipomobilization groups could be associated with differences in the animals’ ability to manage their metabolic status. Specifically, severe lipomobilization was most associated with a greater energy deficit in both prepartum and postpartum periods but without oxidative stress. On the contrary, medium lipomobilization was associated with a less severe energy deficit, accompanied by an inflammatory status and oxidative stress during the prepartum period. These conditions suggest different response mechanisms to metabolic stressors, depending on the ability of buffaloes to maintain homeostasis. Therefore, further studies are needed to investigate the distinctive characteristics of this species in greater depth.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal studies were approved by Il protocollo sperimentale dello studio è stato autorizzato dal Ministero della Salute (autorizzazione n. 758/2017-PR). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

AL: Data curation, Writing – original draft, Methodology, Writing – review & editing. EM: Data curation, Methodology, Writing – original draft, Writing – review & editing. GC: Writing – original draft, Data curation, Validation, Writing – review & editing. AM: Investigation, Writing – original draft, Writing – review & editing. BM: Conceptualization, Writing – original draft, Writing – review & editing. JS: Conceptualization, Writing – original draft, Writing – review & editing. GV: Data curation, Writing – original draft, Writing – review & editing. ID: Writing – original draft, Writing – review & editing. LS: Formal Analysis, Writing – original draft, Writing – review & editing. EC: Methodology, Writing – original draft, Writing – review & editing. PR: Data curation, Writing – original draft, Writing – review & editing. BC: Formal Analysis, Writing – original draft, Writing – review & editing. EF: Data curation, Writing – original draft, Writing – review & editing. DV: Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.