Approximately 3 mL of blood was collected from the lateral tail vein into plain vacutainer tubes. Samples were allowed to clot centrifuged at 3,000 rpm for 10 min and analysed within 2 h of collection to preserve biochemical integrity.

Serum samples were analysed for multiple biochemical and clinical parameters using standardized veterinary diagnostic platforms. Blood urea nitrogen (BUN), creatinine and uric acid were estimated by colorimetric dry chemistry slide methods, while electrolyte concentrations - sodium (Na⁺), potassium (K⁺) and chloride (Cl⁻) were determined using a potentiometric dry chemistry technique all performed on the FUJIFILM Dry-Chem NX700 analyser. SDMA concentrations were measured using a competitive immunoassay with a mouse monoclonal anti-SDMA antibody and cardiac biomarkers including NT-proBNP and cTnI were quantified using a fluorescent immunoassay these analyses were carried out on the BIONOTE Vcheck V200 system. Creatine kinase-MB (CK-MB) activity was also assessed by a colorimetric method.

The data obtained from both male and female tigers were subjected to statistical analysis using two-way analysis of variance (ANOVA). Statistical analyses were performed using the WASP (ICAR) statistical software to evaluate the correlation coefficients and other relevant parameters. This analysis was performed to compare the mean values of each biochemical and haematological parameter between sexes. Pearson’s correlation coefficients (r) were calculated for all paired biochemical parameters. Statistical significance of correlations was assessed using two-tailed tests, and p-values < 0.05 were considered statistically significant.

In the present study, mean BUN (15 ± 2 mg/dL) and creatinine (1.2 ± 0.1 mg/dL) concentrations produced a physiologically appropriate BUN:creatinine ratio of approximately 15:1, consistent with normal nitrogen metabolism and adequate hydration status in obligate carnivores. Biochemical profiling of conscious tigers further revealed uric acid (4.5 ± 0.6 mg/dL) and electrolyte concentrations within expected physiological ranges, including sodium (151 ± 2 mEq/L), potassium (4.6 ± 0.4 mEq/L) and chloride (111 ± 3 mEq/L). All parameters remained within the expected reference ranges for large felids with minimal inter-individual variation and corroborated with published reference ranges (13, 14). Mean SDMA concentrations (8.0 ± 0.5 μg/dL) demonstrated stability across age and sex indicating potential for establishing species-specific reference intervals.

Mean concentration of NT-proBNP (200 ± 25 pg/mL), cTnI (0.02 ± 0.01 ng/mL), and CK-MB (7.5 ± 1.0 U/L) in clinically normal tigers exhibited minimal inter-individual variability suggesting stable baseline cardiac biomarker profiles under the study conditions. These values align with reported ranges in other large felids, suggesting conserved cardiac biochemical baselines across the group (15).

The BUN–creatinine relationship reflects intact renal perfusion and protein metabolism with negligible extrarenal influence (16). The mean uric acid concentration aligns with typical mammalian purine metabolism in carnivores where uric acid is a minor excretory product compared to urea. In tigers, uric acid remains a sensitive but secondary indicator of renal tubular excretion efficiency (17). Values obtained in conscious tigers eliminate pharmacologic confounders and therefore represent true physiological baselines critical for identifying early CKD trends over time.

The narrow standard deviations observed across the cohort indicate physiological stability during sampling in a fully conscious state suggesting that restraint-related stress did not significantly alter electrolyte or uric acid homeostasis. The observed correlation between SDMA and creatinine (r = 0.80) supports their shared dependence on glomerular filtration kinetics; however, the tighter variance of SDMA values underscores its superior analytical precision and sensitivity to early nephron dysfunction (18).

The renal axis is central to metabolic and physiological homeostasis in Panthera tigris, integrating nitrogen turnover, osmotic regulation, and electrolyte balance (19). Within this system, the BUN–creatinine–SDMA triad functions as a sensitive, multifactorial indicator of renal performance, reflecting coordinated glomerular and tubular processes essential to metabolic clearance (20). The integration of SDMA substantially enhances renal diagnostic sensitivity. SDMA, a methylated arginine derivative released during protein turnover is almost exclusively eliminated by glomerular filtration and remains independent of muscle mass a limitation inherent to creatinine-based assessments (21, 22). This property is particularly relevant in tigers, where sexual dimorphism and muscle mass variability can obscure early renal decline when relying on creatinine alone (23).

SDMA therefore serves as an effective early biomarker for preclinical renal compromise, preceding detectable creatinine elevations (3, 20). Collectively, these findings emphasize the diagnostic value of the renal axis as an integrated indicator of physiological stability and renal reserve. The inclusion of SDMA strengthens early detection frameworks for chronic kidney disease (CKD) and subclinical nephropathy in Panthera tigris, facilitating earlier intervention improved hydration management and long-term conservation health monitoring. A diagnostic framework for interpreting SDMA and creatinine concentrations in Bengal tigers is presented in Table 1, enabling assessment of renal function and evidence-based clinical decision-making. Normal renal filtration is indicated by SDMA values <9–10 μg/dL with creatinine within reference limits. Mild SDMA elevation in the presence of normal creatinine reflects early nephron stress or subclinical renal dysfunction, warranting hydration optimization and dietary management. True renal insufficiency is characterized by concurrent elevations in SDMA (>10 μg/dL) and creatinine (>1.4 mg/dL), indicating compromised glomerular filtration and necessitating further diagnostic evaluation, including ultrasonography and targeted therapeutic intervention. Persistently elevated SDMA (>11 μg/dL) with stable creatinine is consistent with compensated chronic nephron loss, for which serial monitoring and dietary protein restriction are recommended.

The narrow variance across individuals indicates hemodynamic stability and the absence of acute or chronic myocardial strain. Correlational analysis among the three biomarkers (r = 0.42–0.51) delineates a strain–injury continuum where mild elevations in NT-proBNP may precede subtle cTnI increases signaling early compensatory adaptation rather than irreversible damage. The correlation matrix defines functional interdependence among renal, cardiac, and electrolyte parameters, outlining the biochemical architecture of homeostasis in Bengal tigers. These relationships identify how metabolic, renal and cardiovascular systems co-regulate under conscious physiology. Cardiac biomarkers provide quantitative insight into myocardial health and hemodynamic balance, offering a functional assessment of cardiac performance. In Panthera tigris, where clinical access is often limited and stress-induced cardiophysiological changes can confound interpretation these markers provide a highly informative index of cardiac performance. The NT-proBNP–cTnI–CK-MB triad provides a layered interpretive framework encompassing myocardial load, injury and recovery. NT-proBNP, a cleavage product of pro–B-type natriuretic peptide reflects ventricular wall stress and volume overload serving as a quantitative proxy for cardiac strain and diastolic dysfunction. In contrast, cTnI represents a high-specificity biomarker of myocardial cell membrane damage responding sensitively to ischemic or stress-induced cardiomyocyte injury (23). CK-MB though less specific provides complementary data on cardiomyocyte enzyme leakage and post-injury recovery kinetics, bridging the temporal gap between acute release and systemic clearance (24).

A reference-based interpretative framework for cardiac biomarkers in Bengal tigers is presented in Table 2, correlating biomarker concentrations with underlying cardiac physiology and associated clinical recommendations. NT-proBNP values of 150–250 pg./mL reflect normal ventricular preload and support routine cardiac surveillance, whereas concentrations >300 pg./mL indicate ventricular overload, warranting heightened anesthetic caution and fluid management optimization. cTnI concentrations <0.03 ng/mL signify myocardial stability, for which continued electrocardiographic monitoring is adequate. In contrast, cTnI values >0.06 ng/mL suggest myocardial injury and necessitate further diagnostic evaluation, particularly echocardiography. Collectively, these findings substantiate the clinical value of integrated cardiac biomarker panels in large felids, reinforcing their role in proactive cardiovascular monitoring, anesthetic risk stratification, and health optimization within conservation medicine programs.

The diagnostic flow integrates renal, cardiac, and electrolyte parameters into a tiered interpretation model for comprehensive tiger health evaluation. Each axis reflects a physiological checkpoint—from filtration to preload to ionic compensation.

This study establishes non-anesthetic baseline values for electrolyte (Na⁺, K⁺, Cl⁻) and uric acid parameters in conscious tigers, providing direct insights into hydration status, acid–base regulation and redox metabolism previously inferred from anesthetized or domestic carnivores. The strong Na⁺–Cl⁻ covariance (r = 0.92) confirms preserved tubular integrity, while the moderate association between uric acid and BUN (r = 0.58) reflects its integrated role in nitrogen metabolism and antioxidant defense. Together, these findings define a physiological metabolic framework linking renal filtration, osmotic control, and oxidative status under anesthesia-free conditions.

Integrated and physiologically coherent network of relationships between renal, metabolic, electrolyte, and cardiac biomarkers, reflecting real-world organ crosstalk rather than isolated laboratory values are recorded in Table 3. The strong positive correlations among BUN, creatinine, and SDMA (r = 0.75–0.82; p < 0.001) confirmed tightly coupled nitrogen handling and glomerular filtration, reinforcing their reliability in distinguishing true azotemia from transient biochemical fluctuations and in detecting early renal impairment independent of muscle mass. The moderate association between uric acid and BUN (r = 0.58; p < 0.001) suggested shared metabolic and oxidative pathways, which were accentuated during increased protein load or catabolic states. Electrolyte relationships further supported renal tubular homeostasis, with a very strong Na⁺–Cl⁻ correlation (r = 0.92; p < 0.001) indicating preserved tubular integrity, while the inverse Na⁺–K⁺ correlation (r = −0.36; p < 0.05) reflected hormonally regulated ionic balance mediated by aldosterone. Importantly, the moderate correlations linking SDMA with NT-proBNP, cTnI with NT-proBNP, and CK-MB with cTnI (r = 0.42–0.51) revealed a biologically plausible cardiorenal–cardiac continuum, in which early renal stress paralleled myocardial load and subtle injury, allowing differentiation between physiological adaptation and emerging pathology. All reported correlations were statistically significant (p < 0.05) reinforcing that the observed renal, electrolyte and cardiorenal associations represent true physiological relationships rather than random variation.

Table 4 presents an integrated, stepwise diagnostic flow model for comprehensive clinical assessment of tigers, linking key physiological axes to targeted clinical decision-making. The framework begins with renal axis evaluation (SDMA, creatinine, BUN) to assess filtration efficiency and nephron integrity, guiding hydration correction and renal-specific interventions. The second step addresses the cardiac axis by integrating NT-proBNP, cTnI, and CK-MB to evaluate ventricular load and myocardial injury, informing anesthetic risk stratification and the need for advanced cardiac imaging. Electrolyte assessment (sodium, potassium, chloride) follows to evaluate osmotic and acid–base balance, supporting fluid therapy decisions and endocrine monitoring. The final step emphasizes longitudinal biomarker trending- particularly SDMA and NT-proBNP to enable early detection of disease progression and support structured surveillance for proactive health management.

Integrating SDMA with conventional renal indices expands the diagnostic precision of kidney health monitoring, while NT-proBNP and cTnI provide parallel insights into myocardial strain and cardiomyocyte integrity. The moderate SDMA–NT-proBNP correlation (r = 0.46) establishes a functional cardiorenal axis - a biologically significant relationship never quantified in big cats. This relationship highlights the necessity of interpreting renal and cardiac systems as a unified physiological continuum.

Clinically, this study provides a tri-axial diagnostic model encompassing:

This model enables early detection of renal or cardiac compromise facilitates pre-anesthetic risk profiling and enhances welfare monitoring for geriatric, breeding and translocated tigers. Beyond veterinary application the findings bridge One Health physiology and conservation management.

In summary, this is the first report to define authentic anesthesia-free biochemical baselines for renal, cardiac, electrolyte and uric acid physiology in Bengal tigers. The study introduces both a novel methodology and a multidimensional diagnostic continuum that integrates filtration, myocardial performance and metabolic balance. Together, these contributions mark a significant advancement in big cat healthcare transforming it from periodic, anesthesia-dependent assessments to continuous, evidence-driven and welfare-positive health surveillance.