Open Veterinary Journal, (2024), Vol. 14(11): 2893-2900

Research Article

10.5455/OVJ.2024.v14.i11.18

Hematological and biochemical parameters in the blood of working Belgian Shepherd dogs: An age-related study

Nejra Hadžimusić* and Dženita Hadžijunuzović-Alagić

Department for Clinical Sciences, University of Sarajevo—Veterinary Faculty, Sarajevo, Bosnia and Herzegovina

*Corresponding Author: Nejra Hadžimusić. Department for Clinical Sciences, University of Sarajevo—Veterinary Faculty, Sarajevo, Bosnia and Herzegovina. Email: nejra.hadzimusic [at] vfs.unsa.ba

Submitted: 13/08/2024 Accepted: 07/10/2024 Published: 30/11/2024

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Abstract

Background: Age-related changes in physiological parameters are crucial in understanding the health and performance of working dogs, particularly those in demanding roles such as military and law enforcement. However, limited research exists on how aging affects the hematological and biochemical health of these dogs.

Aim: This study aims to characterize age-related variations in hematological and biochemical parameters in working Belgian Shepherd dogs to provide insights that could inform health management strategies for these animals.

Methods: Blood samples were collected from 26 male Belgian Malinois working dogs, categorized into three age groups: adults (2–6 years), seniors (7–10 years), and geriatrics (11+ years). Comprehensive hematological and biochemical analyses were conducted. Analysis of complete blood count was performed for a total of 16 parameters: red blood cell, white blood cell, packed cell volume, hemoglobin, platelet, neutrophil, basophil, leukocyte, monocyte, lymphocyte, and eosinophil counts. Mean cell volume, mean cell hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, and mean platelet volume were subsequently determined. The biochemistry parameters included glucose, creatinine, urea, blood urea nitrogen:creatinive ratio, phosphorus, calcium, sodium, potassium, Na:K ratio, chloride, total protein, albumin, globulin, albumin:globulin ratio, as well as alanine aminotransferase, alkaline phosphatase, gamma glutamyl transeferase, total cholesterol, amylase, and lipase.

Results: Significant age-related changes were observed in various parameters. Older dogs exhibited decreased creatinine and increased phosphorus and potassium levels, indicating potential changes in muscle mass, renal function, and electrolyte balance. Additionally, the albumin-to-globulin ratio decreased with age, reflecting shifts in protein synthesis and immune function.

Conclusion: The study highlights important age-related variations in hematological and biochemical parameters in working Belgian Shepherd dogs. These findings emphasize the need for age-specific health management strategies to maintain the health, performance, and longevity of these dogs, thereby enhancing their effectiveness in critical service roles.

Keywords: Age-related changes, Hematological parameters, Biochemical markers, Belgian Shepherd dogs, Working dogs.

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Introduction

Dogs have long been integral to various sectors of society, fulfilling roles that range from assistance, such as service and guide dogs, to therapeutic, detection, and protection tasks. Dogs play crucial roles in search-and-rescue operations, explosive detection, and law enforcement. The modern concept of police dogs emerged in the early 20th century, and they are now vital for crime prevention and securing areas of interest. The military also employs dogs for scouting and detection tasks, including explosives and narcotics (Bray et al., 2021). The Belgian Malinois is particularly valued for its intelligence, adaptability, and high success rate in military training, making it well-suited for various tasks (Spinella et al., 2022). Numerous studies have established that various hematologic and biochemical analytes, including packed cell volume, hemoglobin concentration, red blood cells, alkaline phosphatase (ALP), albumin, globulins, calcium, and phosphate, are influenced by age and undergo changes during the first year of life, with most parameters reaching a plateau around that time (Nielsen et al., 2010). Additionally, values can vary later in life; for example, a longitudinal study on Beagles observed changes in alanine aminotransferase (ALT), aspartate aminotransferase activities, glucose, and triglyceride levels as the dogs aged (Nielsen et al., 2010).

In terms of breed-related differences, research has demonstrated that puppies of German Shepherds, Golden Retrievers, and Beagles exhibit significant variations in their hematologic profiles, underscoring the necessity for breed-specific reference intervals (Nielsen et al., 2010). Sighthounds are known to possess distinct hematologic and biochemical characteristics compared to the broader dog population (Fayos et al., 2005; Nielsen et al., 2010). Other breeds also display inherent hematologic and serum biochemical differences, such as microcytosis in Shiba Inus and hyperlipoproteinemia in miniature Schnauzers (Fayos et al., 2005; Nielsen et al., 2010). Some breeds, like Cavalier King Charles Spaniels, may have both quantitative and inherent differences, exemplified by idiopathic macrothrombocytopenia (Singh and Lamb, 2005). Therefore, breed is a crucial factor in determining relevant population-based reference intervals and should be carefully considered in breed-specific studies that include hematologic and biochemical parameters. Recent research has delved into the biochemical aspects of aging, a topic gaining relevance with the increase in the elderly population and consequent rise in healthcare costs. Understanding the aging process is vital for mitigating age-related diseases, with studies highlighting the significant impact of biological mechanisms on lifespan across species (Polsky et al., 2022). During aging, numerous changes occur in the bone marrow, which may have clinical implications. Differences in the marrow between younger and older subjects have been demonstrated in both animal models and humans, involving alterations in overall cell number, lineage differentiation, cellular composition, and the function of hematopoietic stem cells (Groarke and Young, 2019). Such insights are not only critical for enhancing the selection and performance of working dogs but also for ensuring their well-being throughout their service life, thereby contributing to our society’s security and protection.

It is crucial, therefore, to assess the impact of aging on the physiological traits of clinically healthy dogs. Objective measures such as complete blood counts (CBC) and serum biochemistry analyses are generally more precise and specific than physical examinations for assessing physiological health.

Moreover, the breed appears to play a significant role in determining the relevance of population-specific reference intervals and should be taken into account during health examinations that involve hematologic and biochemical parameters. Additionally, various external factors, such as environment, season, and lifestyle, as well as internal factors like age, sex, and neutering status, also influence these parameters (Lee et al., 2020). The focus on Belgian Shepherds, known for their intelligence, resilience, and adaptability, provided valuable insights into how aging impacts a breed renowned for its physical capabilities (Spinella et al., 2022). These aspects are essential for understanding the specific requirements and management of working dogs, especially as they age and their operational effectiveness and health are evaluated. Given the rising interest in the biochemical processes of aging—prompted by an increasing elderly population and the subsequent escalation in healthcare costs—understanding the aging process becomes crucial for reducing the prevalence of age-related diseases. The study by Polsky et al. (2022) on stress-induced biological aging and the work by Groarke and Young (2019) on aging and hematopoiesis highlights the profound influence of biological mechanisms on the lifespan across different species, including changes in the bone marrow that have clinical significance. These changes encompass variations in cell number, lineage differentiation, cellular composition, and the functionality of hematopoietic stem cells observed across younger and older subjects in both animal models and humans.

Thus, the aim of this research was to characterize the variations in hematological and serum biochemical indicators associated with aging in working Belgian Shepherd dogs.

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Materials and Methods

Blood analysis

Blood samples of 26 working male Belgian Malinois, aged 3 to 13 years, were taken as a part of an ongoing yearly evaluation to track their health condition. Dogs were categorized into three groups based on their age. Group I consisted of adult dogs aged between 2 and 6 years (n=10); Group II was comprised of senior dogs aged between 7 and 10 years (n=10), while Group III included geriatric dogs older than 11 years (n=6). Dogs exhibiting no clinical symptoms of disease and demonstrating normal levels of vitality were selected for the study. Blood samples were collected from v. cephalica antebrachi externa into Ethylenediaminetetraacetic acid (EDTA) tubes. Analysis of CBC was performed for a total of 16 parameters: red blood cell, white blood cell, packed cell volume, hemoglobin, and platelet, neutrophil, basophil, leukocyte, monocyte, lymphocyte, and eosinophil counts. Mean cell volume (MCV), mean cell hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, and mean platelet volume (MPV) were subsequently determined. The biochemistry parameters included glucose, creatinine, urea, blood urea nitrogen:creatinive ratio, phosphorus, calcium, sodium, potassium, Na:K ratio, chloride, total protein, albumin, globulin, albumin:globulin ratio, as well as ALT, ALP, gamma glutamyl transeferase, total cholesterol, amylase, and lipase. The analysis of hematological parameters was conducted using a ProCyte Dx Hematology Analyzer (IDEXX, Netherlands), while biochemistry parameters were conducted using Catalyst One Chemistry Analyzer (IDEXX, Netherlands).

All dogs received care in compliance with the “Declaration on the Rights of Animals” (UNESCO, 1978) and the “Universal Declaration on Animal Welfare” (WSPA, 2000).

Data analysis

The analysis of the results was carried out using SPSS software, Version 17.0, to calculate the mean values of examined hematological and biochemistry parameters and to determine statistically significant differences according to age.

Ethical approval

In our study, we utilized dogs that were brought in for routine annual examinations as part of their regular health check-ups. This systematic approach ensured that the animals were not subjected to any unnecessary stress or invasive procedures beyond what is standard in their care. All procedures adhered to ethical guidelines, prioritizing the well-being of the animals involved.

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Results

To assess the variations in physiological differences of Belgian Shepherd dogs based on their age, we examined the CBC outcomes for each age group (Table 1) and biochemistry values (Table 2).

All hematology parameters remained within the reference range; however, numerous values were significantly different between the investigated age groups (Table 1). The RBC count in Group II was significantly higher compared to Group III. HCT and HGB levels followed a similar trend, with Group II showing higher values compared to Group III.

MCV, MCH, and MCHC values showed no significant differences among the groups, with values remaining steady across all three groups.

Regarding reticulocyte percentages and counts, no significant differences were observed. Similarly, WBC counts were not significantly different between the groups, although Group III had a slightly higher mean (11.89 ± 5.94 × 10^9/l) compared to Groups I and II (9.38 ± 2.47 × 10^9/l and 9.39 ± 2.89 × 10^9/l, respectively).

Significant differences were seen in the distribution of white blood cells. Neutrophils (%) were highest in Group III (78.7% ± 1.77%), significantly differing from Group I (68.3% ± 7.97%) and Group II (73.03% ± 8.49%). Lymphocyte percentages were lowest in Group III (12.95% ± 1.74%), with Group I (20.8% ± 4.59%) showing the highest values. Monocyte percentages were also significantly lower in Group II (4.67% ± 1.07%) compared to Groups I and III.

Platelet counts were significantly higher in Group III (312.33 ± 61.53 × 10^9/l) compared to Group I (208 ± 96.84 × 10^9/l), while platelet distribution width (PDW) and MPV were significantly higher in Group II.

The values of the biochemical parameters are presented in Table 2 and are as follows: The glucose concentrations were similar between the groups, with Group II showing slightly higher values (5.92 ± 0.42 mmol/l) compared to Group I (5.73 ± 0.68 mmol/l) and Group III (5.62 ± 0.57 mmol/l). Creatinine levels were significantly higher in Group I (88.60 ± 13.56 µmol/l) compared to Group II (78.6 ± 9.24 µmol/l), while Group III (74.67 ± 20.76 µmol/l) showed values that overlapped with both groups. There were no significant differences in urea levels among the groups.

Phosphorus levels were significantly higher in Group III (1.30 ± 0.15 mmol/l) compared to both Groups I and II (0.91 ± 0.32 and 0.98 ± 0.23 mmol/l, respectively). Calcium levels were also significantly higher in Group III (2.57 ± 0.16 mmol/l) compared to Groups I and II, which showed gradually increasing values (2.47 ± 0.08 and 2.54 ± 0.1 mmol/l, respectively).

For electrolytes, sodium and chloride concentrations were similar between the groups. Potassium was significantly higher in Groups II and III (4.29 ± 0.42 and 4.6 ± 0.42 mmol/l, respectively) compared to Group I (3.83 ± 0.24 mmol/l), leading to a significant reduction in the Na:K ratio in Groups II and III compared to Group I.

Total protein and globulin concentrations showed no significant differences between the groups, although albumin levels were lower in Group III (29.83 ± 2.14 g/l) compared to Group II (32 ± 1.83 g/l).

For liver enzymes, ALT levels were elevated in Group III (142.17 ± 195.70 U/l) compared to both other groups. ALP and GGT showed no significant differences between the groups.

Cholesterol concentrations were significantly higher in Group III (5.70 ± 1.46 mmol/l) compared to Groups I and II, with Group II (4.46 ± 0.79 mmol/l) showing slightly higher values than Group I (4.20 ± 0.60 mmol/l). Lipase and amylase levels were highest in Group III, although no statistically significant differences were observed between the groups.

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Discussion

A significantly lower RBC value was determined in the group of geriatric dogs compared to seniors. Hematocrit value also decreased within Group III. Geriatric dogs had lower HGB values compared to both adults and senior dogs. RDW is frequently utilized alongside other parameters such as HCT, HGB, and MCV to aid in the differential diagnosis and categorization of anemias and bone marrow disorders. RDW offers greater precision in detecting anisocytosis compared to MCV due to its sensitivity to the standard deviation of erythrocyte volume variability. This means that anisocytosis can be identified as either high or low, regardless of the average volume of the RBC population being the same (Miglio et al., 2023). The significance of assessing RDW has escalated in human medicine due to its broad clinical utility. Research has revealed that anisocytosis is associated with cardiovascular diseases, infections, cancer, endocrine disorders, pulmonary diseases, gastrointestinal and liver disease, kidney diseases, sepsis, neurological disorders, fractures, and various other acute or chronic ailments (Miglio et al., 2023; Xanthopoulos et al., 2022). Consequently, RDW is now recognized as an independent and robust predictor of negative health outcomes in the wider population. Given the importance outlined above, variations in RDW as dogs age are of exceptional significance.

Table 1. Hematology parameters tested.

Among the white blood cells, the only parameter exhibiting a statistically significant difference was the monocyte count, which was higher in the geriatric dogs group compared to the senior group, as well as in adults compared to senior dogs (Table 1). In the study by Bruno et al. (2022), it was noted that monocyte levels were higher in elderly dogs (over 10 years of age) compared to adults aged between 2 and 6 years. The authors note that these changes were associated with physiological, immunological, and biological alterations in the body as a result of aging. This finding corresponds with our results. Platelet counts increased with age. The increase was statistically significant in geriatric dogs compared to adults (Table 1). The finding that platelet counts increase with advancing age has also been reported by Bruno et al. (2022). Jain et al. (2013) stated that these changes were correlated with physiological, immunological, and biological alterations in the body due to aging. This increase may be attributed to decreased bone marrow function, splenomegaly, and a reduction in RBC production as discussed by Bruno et al. (2022) and Radakovich et al. (2017), a reduction that was also observed in our study (Table 1).

The average platelet size is represented by the MPV, while the variability in platelet size is indicated by the PDW (Schneider and Mischke, 2016). Platelet indices (PDW and MPV) in three groups of dogs show significant changes. Thus, the PDW value was significantly lower in geriatric dogs compared to seniors, while the MPV value was higher in seniors compared to both adult and geriatric dogs (Table 1).

Table 2. Biochemistry parameters tested.

Platelet indices, along with platelet count, indicate that these parameters can provide pathogenetic insights into various human diseases. Increased MPV values are typically associated with a higher production rate of platelets. For instance, elevated MPV values were observed in cases of canine babesiosis, indicating heightened platelet production in infected dogs. Additionally, increased MPV levels were linked to normal or elevated numbers of megakaryocytes in the bone marrow cytology of dogs with immune-mediated thrombocytopenia (Schneider and Mischke, 2016). However, some studies have also reported decreased MPV (microthrombocytosis) in severe thrombocytopenia, which may indicate primary immune-mediated thrombocytopenia Schneider and Mischke, 2016). Additionally, elevated MPV levels in humans have been recognized as independent predictors of in-hospital deep venous thrombosis (Cil et al., 2012). In veterinary medicine, MPV may be more susceptible to excitement caused by blood sample collection, which could lead to an increased adrenaline-mediated release of platelets, especially from the spleen. This potential release of platelets from the spleen can also affect other platelet parameters, particularly platelet size. A recent experimental study in mice showed that splenic platelets are larger than those found in circulation. In thrombocytopenic human patients, significantly higher MPV and PDW values were found in cases of hyperdestructive thrombocytopenia compared to hypoproductive thrombocytopenia, indicating that these parameters can provide clinical insights into the underlying causes of thrombocytopenia, particularly in relation to immune-mediated destruction (Schneider and Mischke, 2016). This study showed a significantly higher value of Plateletcrit in geriatric dogs compared to seniors (Table 1). Plateletcrit (PCT) determines the percentage of the blood volume that consists of platelets. A decline in PCT and PLT was noted in dogs with experimentally induced endotoxemia (Schneider and Mischke, 2016).

Table 1 demonstrates that MCV, MCH, MCHC, Ret, ret hemoglobin values showed no significant differences across the studied dog groups.

According to the serum biochemical profiles, significant differences were observed in the concentration of some parameters (Table 2). Creatinine levels are highest in Group I and decrease with age. This decline could suggest a reduction in muscle mass or changes in kidney function as the dogs age. The significant difference between Groups I and II indicates this trend, but Group III shows variability. The data indicate that older dogs exhibit decreased creatinine levels and increased phosphorus and potassium levels, suggesting potential changes in muscle mass, renal function, and electrolyte balance. Furthermore, the albumin-to-globulin ratio tends to decrease with age, which may reflect shifts in protein synthesis and immune function. These observations align with previous studies on aging in both animals and humans, highlighting the importance of regular health assessments for working dogs.

Phosphorus levels are significantly higher in Group III compared to Groups I and II. This increase could be related to dietary factors, renal function, or bone metabolism changes as the dogs age. Calcium levels show a slight but significant increase with age, indicating changes in bone metabolism or dietary calcium absorption efficiency in older dogs. Potassium levels increase significantly with age, particularly from Group I to Groups II and III. This rise might be associated with changes in renal function or muscle metabolism. The Na:K ratio decreases with age, reflecting the higher increase in potassium relative to sodium. This decrease may indicate shifts in electrolyte balance as the dogs age. Albumin levels show slight fluctuations with age, peaking in Group II. This could be due to variations in protein synthesis or liver function. Globulin levels increase with age, particularly in Group III, reflecting possible changes in immune function or protein metabolism. However, the increase was not statistically significant. A previous study indicated that elderly individuals with chronic inflammation tend to have higher concentrations of globulin and lower levels of albumin in their total protein composition (Lee et al., 2020). Specifically, elevated serum globulin levels are closely linked to cancer, chronic liver disease, and diabetes mellitus, while reduced albumin levels are associated with malnutrition, chronic infection, and nephrotic disease, and serve as a primary physiological indicator of aging. Additionally, another previous study reported a decreased albumin/globulin ratio in older dogs. In this regard, our research shows that albumin: globulin ratio decreases with age, particularly in Group III, suggesting a relative increase in globulin levels compared to albumin. Specifically, elevated serum globulin levels are closely linked to cancer, chronic liver disease, and diabetes mellitus, while reduced albumin levels are associated with malnutrition, chronic infection, and nephrotic disease, and serve as a primary physiological indicator of aging. Additionally, another previous study reported a decreased albumin/globulin ratio in older dogs. Furthermore, another previous study reported a decreased albumin/globulin ratio in older dogs, attributed to elevated levels of immunoglobulin A (Blount et al., 2005).

Some studies have shown that a decline in glucose concentration was observed in older dogs in both middle and large-sized groups. However, previous research has found varying patterns of serum glucose changes with aging. One study reported an increase in serum glucose levels with age (Strasser et al., 1993), attributing this to decreased organ sensitivity to concentration changes, reduced organ production, and increased insulin resistance. Conversely, other studies demonstrated that glucose levels decrease with age (Blount et al., 2005). In our case, however, glucose values did not change significantly.

ALT is primarily associated with liver function. Our findings indicate that, in this population of working dogs, age does not appear to influence serum ALT levels significantly. This suggests that liver health remains consistent across different life stages in the cohort we studied. ALP is an enzyme related to various physiological processes, including bone turnover and bile metabolism. The lack of significant variation in ALP levels with age suggests that factors such as diet, exercise, and overall health status may play a more critical role than age alone in determining ALP concentrations in working dogs. Similar to ALT and ALP, our results show no significant differences in GGT levels with age. This finding reinforces the idea that age-related physiological changes may not substantially impact liver enzyme levels in this particular breed and working context.

In our study, we observed higher total cholesterol levels in dogs within Group III. While the median cholesterol levels in geriatric dogs were within the reference interval, the observed trends toward hypercholesterolemia might suggest a higher prevalence of hypothyroidism or the potential presence of nephrotic syndrome, especially given the declining albumin levels (Table 2). Future prospective studies, including urinalysis to check for proteinuria and thyroid panels, would be valuable for identifying the causes of elevated cholesterol in aging dogs. However, elevated cholesterol levels can be attributed to high-fat diets, certain diseases, and obesity. The development of these conditions is influenced by various dog characteristics, including age, breed, sex, and whether the dog has been neutered or spayed (Usui et al., 2014). The effect of aging on plasma total cholesterol has been reported in the study by Kawasumi et al. (2014). This suggests that age may influence cholesterol metabolism, potentially leading to variations in serum cholesterol concentrations among different age groups.

Amylase and lipase are crucial enzymes involved in the digestion of carbohydrates and fats, respectively. Elevated serum concentrations of these enzymes can indicate pancreatic function and overall metabolic health in working dogs (Cridge et al., 2022). However, in our study, we did not establish significant differences in enzyme levels based on the age of the dogs. This finding suggests that age-related physiological changes may not have a marked impact on the serum concentrations of amylase and lipase in this population.

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Conclusion

This study provides valuable insights into the age-related variations in hematological and biochemical parameters in working Belgian Shepherd dogs. Our findings reveal significant changes in several key parameters, such as creatinine, phosphorus, calcium, potassium, albumin, and globulin, as the dogs age. These changes are crucial for understanding how aging impacts the physiological health and operational efficiency of these dogs, which are integral to various critical roles in military and law enforcement.

By focusing on Belgian Shepherds, known for their intelligence and adaptability, we have highlighted the importance of age-specific health management strategies. This research contributes to the broader understanding of aging in working dogs, aiming to enhance their health, performance, and longevity. Ensuring the well-being of these dogs not only improves their effectiveness in service roles but also supports societal safety and security.

Overall, our study emphasizes the need for continuous monitoring and tailored health care for aging working dogs, providing a foundation for better management practices and improved quality of life for these vital members of our communities.

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Acknowledgments

The authors did not receive any specific support or assistance from individuals or organizations during the preparation of this research.

Conflict of interest

The authors declare that they have no conflict of interest.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contributions

NH and DžHA contributed equally to this work. NH was responsible for the conceptualization, data analysis, and drafting of the manuscript. DžHA contributed to the research methodology, data collection, and revision of the manuscript. Both authors reviewed and approved the final version of the manuscript.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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