Haematological and serum biochemical reference intervals for Indian spitz dogs in tropical conditions

Introduction

Every veterinary textbook carries a table of "normal" canine haematological values, yet those values were typically generated from a narrow base Beagles and Labrador Retrievers maintained in temperate research colonies [1] . When a clinician in Manila or Bangkok runs a complete blood count on an Indian spitz and compares the numbers against those textbook ranges, there is a real chance that perfectly healthy dogs will be flagged as abnormal simply because their breed and their environment differ from the reference population [2] . Breed effects on blood parameters are well documented. Greyhounds, for example, carry higher packed cell volumes and lower white-cell counts than other breeds, differences large enough to cause diagnostic confusion if breed-specific ranges are not used [3] . Sighthounds as a group show elevated creatinine levels that do not indicate renal disease [4] . For Indian spitz dogs a popular companion breed across South and Southeast Asia no dedicated referenceinterval dataset exists in the peer-reviewed literature. Climate compounds the breed effect. Chronic heat exposure triggers physiological adaptations that alter baseline haematology: expanded plasma volume for thermoregulation, shifts in Leucocyte trafficking, and changes in protein metabolism [5] . Dogs living year-round in Tropical conditions may therefore display haematological profiles that deviate from temperate norms even when they are in excellent health [6] . The American Society for Veterinary Clinical Pathology (ASVCP) has published guidelines for reference-interval determination that recommend breed-, age-, and sex-specific partitioning when sample sizes permit, along with standardized outlierdetection and confidence-interval methods [7] . Applying these guidelines to a well-characterized cohort of Indian spitz dogs living under tropical conditions would fill a diagnostic gap that currently forces clinicians to use approximations. Manila's climate mean annual temperature 27.5 °C, relative humidity above 75% for most of the year provides a representative tropical environment for this purpose [8] . The Manila College of Veterinary Sciences maintains a clinical caseload that includes a large number of Indian spitz dogs, making recruitment of a suitably sized, health-screened cohort feasible [9,10] . This research aimed to (a) establish reference intervals for 20 haematological and 12 serum biochemical parameters in clinically healthy Indian spitz dogs living in tropical Manila, (b) test for age-and sex-related differences, and (c) compare the derived intervals with published general canine ranges to quantify the extent of breed-climate deviation [11,12] .

Ethical Considerations

The research was approved by the Manila College of Veterinary Sciences Institutional Animal Care and Use Committee (Protocol MCVS-IACUC-2022-063, approved 8 December 2022). All dogs were client-owned or kennelowned; written owner consent was obtained before blood collection. Dogs received a complimentary health certificate and parasite screening report as compensation for participation. Blood collection followed standard veterinary practice and caused no more than momentary discomfort equivalent to a routine clinical blood draw.

Material and methods

Material

Eighty-four Indian spitz dogs (42 males, 42 females) were enrolled between February and July 2023 from households and breeding kennels within the Manila metropolitan area. Inclusion criteria: age ≥ 6 months, body condition score 4-6 on a 9-point scale, no clinical illness within the preceding 30 days, current on rabies and core vaccinations, negative faecal flotation for gastrointestinal parasites, and negative rapid antigen test for heartworm (Dirofilaria immitis). Exclusion criteria: pregnancy, lactation, recent surgery, concurrent medication (including antiparasitics within 14 days), and any abnormality on physical examination (heart murmur, lymphadenopathy, fever, dehydration). Dogs were stratified into four age groups: juvenile (6-12 months, n = 18), young Adult (1-3 years, n = 24), mature (3-6 years, n = 26), and senior (> 6 years, n = 16). Methods Blood samples were collected between 07:00 and 09:00 after an overnight fast (≥ 8 hours) to minimize postprandial lipemia. Four millilitres were drawn from the cephalic vein: 2 mL into EDTA tubes for haematology and 2 mL into plain tubes for serum biochemistry. EDTA samples were analysed within 2 hours of collection on a calibrated automated veterinary haematology analyser (20 parameters including CBC, five-part differential, reticulocyte count, and red-cell indices). Serum was separated by centrifugation (3,000 rpm, 10 minutes), aliquoted, and processed on a bench-top chemistry analyser for 12 parameters: total protein, albumin, globulin, ALT, AST, ALP, BUN, creatinine, glucose, total bilirubin, cholesterol, and triglycerides. Outlier detection followed a two-step procedure: the Dixon Q-test for extreme values and Horn's algorithm for robust identification of moderate outliers, both recommended by the ASVCP [7] . Reference intervals were calculated as the central 95% (2.5th to 97.5th percentiles) using the non-parametric method for datasets with n ≥ 40, with 90% confidence intervals around each limit generated by bootstrapping (2,000 iterations). Age and sex effects were tested by two-way ANOVA with Bonferroni correction for multiple comparisons (significance threshold p<0.05). Analyses were conducted in R v4.3.1 [13,14] .

Limitations

The sample was drawn entirely from the Manila metropolitan area, so the results may not extend to Indian spitz dogs in cooler highland regions or rural settings with different nutritional and parasitic exposures. The sample size (n = 84) was adequate for overall reference-interval calculation but limited the power of age-and sex-stratified subgroup analyses, particularly in the senior group (n = 16). Diurnal and seasonal variations in haematological parameters were minimized by standardizing collection time and confining recruitment to a six-month window, but a full 12-month sampling protocol would strengthen the seasonal component. Finally, the research did not measure haemoglobin electrophoresis or iron studies, which would have been informative for characterizing the lower albumin values observed.

Results

After outlier removal (six values across four parameters), 84 dogs contributed to the final reference-interval calculations. The overall haematological profile showed a mean RBC count of 7.04±0.68 × 10¹²/L, haemoglobin of 14.3±1.4 g/dL, and packed cell volume of 43.1±4.2%. The leucocyte profile was characterised by a neutrophil-dominant differential (72.4±5.1%) with a relatively low lymphocyte proportion (18.7±4.3%).

Comprehensive interpretation

The reference intervals generated here broadly overlap with published general canine ranges but show important breedclimate nuances. The neutrophil-dominant leucocyte profile likely reflects a chronic mild stress leucogram induced by sustained tropical heat, a physiological shift rather than a pathological one. The marginally lower albumin may similarly represent heat-related plasma volume expansion. Age effects followed expected mammalian patterns higher leucocyte counts and lower erythrocyte counts in juveniles and support the ASVCP recommendation to use agepartitioned intervals in practice. The absence of clinically significant sex differences after Bonferroni correction simplifies clinical application, as a single set of breed-climate intervals (partitioned only by age) can be used for both sexes.

Discussion

The haematological profile documented here for Indian spitz dogs in tropical Manila is the first breed-specific dataset of its kind. Compared with the widely cited Beagle-derived reference ranges in standard textbooks [1] , the most notable differences were the higher neutrophil percentage and the lower lymphocyte proportion. Kiral and colleagues [5] reported similar leucocyte shifts in dogs exposed to ambient temperatures above 30 °C for extended periods, attributing them to cortisol-mediated demargination of neutrophils and redistribution of lymphocytes to tissues. Red-cell parameters, by contrast, showed only minor deviations from published norms. RBC count, haemoglobin, and PCV all fell comfortably within textbook ranges, suggesting that the erythropoietic response in Indian spitz dogs is not markedly different from that of temperate breeds or that any heat-related plasma expansion is offset by adequate erythropoietic drive [3,6] . The age-related trends higher WBC in juveniles, lower RBC are consistent with the well-established phenomenon of physiological leucocytosis in young growing dogs, where bone marrow granulocyte output is elevated to support rapid immune system maturation [4] . The clinical implication is straightforward: a clinician seeing a six-month-old Indian spitz with a WBC of 14 × 10⁹/L should not reflexively suspect infection if the differential is unremarkable. Serum albumin merits particular attention. The mean value of 3.04 g/dL, though within the accepted canine range, sits at the lower end and may create diagnostic ambiguity when a mild drop occurs during illness. Using the breed-climate-specific lower limit of 2.44 g/dL rather than the textbook 2.6 g/dL would prevent false hypoalbuminaemia calls in healthy animals [7,15,16] .

Conclusion

The main contribution of this research is a set of rigorously derived, ASVCP-compliant reference intervals for 20 haematological and 12 serum biochemical parameters in clinically healthy Indian spitz dogs living under tropical conditions in Manila, Philippines. These intervals fill a gap that has forced veterinary practitioners to rely on Beaglebased norms for a breed whose physiology and environment differ meaningfully. The supporting evidence shows that while most red-cell and biochemistry values overlap with general canine ranges, the leucocyte differential and serum albumin deviate enough to cause diagnostic misclassification if breed-climate context is ignored. The neutrophil-dominant, lymphocyte-low differential in healthy Indian spitz dogs is a physiological adaptation to chronic heat exposure, not a sign of disease; using temperate-breed norms for this parameter would risk unnecessary workups in healthy animals. For the broader field of veterinary clinical pathology, the findings reinforce the ASVCP's call for breed-and population-specific reference intervals rather than one-sizefits-all tables. As companion-animal populations in tropical countries grow and diversify, the diagnostic infrastructure should keep pace by generating local reference data for the breeds and climates that practitioners actually encounter. Open questions include whether seasonal variation within the tropics (wet vs dry season) produces clinically relevant shifts in these parameters a question that would require year-round sampling. Extending the dataset to include renal function markers such as SDMA, iron studies, and coagulation profiles would broaden clinical utility. And replicating the work in other tropical cities Ho Chi Minh City, Jakarta, Lagos would test how geographically generalisable these breed-climate intervals are.

Acknowledgement

Acknowledgements Institutional SupportThe Manila College of Veterinary Sciences provided laboratory access, analyser calibration, and veterinary nursing support for all blood collection sessions.

References

1. Weiss DJ, Wardrop KJ, editors. Schalm's veterinary hematology. 6th ed. Ames (IA): Wiley-Blackwell; 2010.
2. Friedrichs KR, Harr KE, Freeman KP, Szladovits B, Walton RM, Barnhart KF, et al. ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species. Vet Clin Pathol. 2012; 41(4):441-453.
3. Zaldívar-López S, Marín LM, Iazbik MC, WestendorfStingle N, Hensley S, Couto CG. Clinical pathology of Greyhounds and other sighthounds. Vet Clin Pathol. 2011; 40(4):414-425.
4. Harper EJ, Hackett RM, Wilkinson J, Heaton PR. Agerelated variations in haematologic and plasma biochemical test results in Beagles and Labrador Retrievers. J Am Vet Med Assoc. 2003; 223(10):14361442.
5. Kiral FK, Seyrek K, Pasa S, Yenisey C. Seasonal variations in haematological values and cellular immunity in dogs. Ankara Univ Vet Fak Derg. 2005; 52(3):191-195.
6. Piccione G, Casella S, Lutri L, Vazzana I, Ferrantelli V, Caola G. Reference values for some haematological, haematochemical, and electrophoretic parameters in the Cirneco dell'Etna dog. J Vet Diagn Invest. 2010; 22(2):271-274.
7. Geffré A, Concordet D, Braun JP, Trumel C. Reference value advisor: a new freeware set of macroinstructions to calculate reference intervals with Microsoft Excel. Vet Clin Pathol. 2011; 40(1):107-112.
8. Philippine Atmospheric, Geophysical and Astronomical Services Administration (PAGASA). Climate of the Philippines. Quezon City: PAGASA; 2021.
9. Cruz SI, Reyes MA. Companion animal demographics in Metro Manila: a veterinary clinic-based survey. Philipp J Vet Med. 2022; 59(1):34-42.
10. Pedersen HD, Häggström J, Falk T, Jørgensen L, Olsen LH, Iversen L, et al. Auscultation in mild mitral regurgitation in dogs: observer variation and effects of physical maneuvers. J Vet Intern Med. 1999; 13(2):5664.
11. Rossi G, Giordano A, Pezzia F, Kjelgaard-Hansen M, Paltrinieri S. Serum paraoxonase 1 activity in dogs: preanalytical and analytical validation and establishment of reference intervals. Vet Clin Pathol. 2013;42(2):190199.
12. O'Brien MA, McMichael MA, Le Boedec K. Reference intervals and age-related changes for venous blood gas variables in clinically healthy neonatal puppies. J Am Vet Med Assoc. 2014; 245(10):1129-1135.
13. R Core Team. R: a language and environment for statistical computing. Vienna (Austria): R Foundation for Statistical Computing; 2023.
14. Horn PS, Pesce AJ. Reference intervals: a user's guide. 1st ed. Washington (DC): AACC Press; 2005.
15. Kaneko JJ, Harvey JW, Bruss ML, editors. Clinical biochemistry of domestic animals. 6th ed. Burlington (MA): Academic Press; 2008.
16. Willard MD, Tvedten H, editors. Small animal clinical diagnosis by laboratory methods. 5th ed. St. Louis (MO): Elsevier Saunders; 2012.
17. Mori A, Lee P, Takemitsu H, Iwasaki E, Kimura N, Yagishita M, et al. Influence of body weight on plasma biochemistry values in clinically healthy dogs. Vet Res Commun. 2011; 35(4):223-235.