Volume 86, Issue 5 p. 600-613
RESEARCH ARTICLE
Open Access

Morphological and cytochemical characteristics of Varanus niloticus (Squamata, Varanidae) blood cells

Soha A. Soliman

Corresponding Author

Soha A. Soliman

Department of Histology, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt

Correspondence

Soha A. Soliman, Department of Histology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt.

Email: [email protected]

Hanan H. Abd-Elhafeez, Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt.

Email: [email protected]

Catrin S. Rutland, School of Veterinary Medicine and Science, Faculty of Medicine and Health Science, University of Nottingham, Nottingham, UK.

Email: [email protected]

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Hanan H. Abd-Elhafeez

Corresponding Author

Hanan H. Abd-Elhafeez

Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt

Correspondence

Soha A. Soliman, Department of Histology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt.

Email: [email protected]

Hanan H. Abd-Elhafeez, Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt.

Email: [email protected]

Catrin S. Rutland, School of Veterinary Medicine and Science, Faculty of Medicine and Health Science, University of Nottingham, Nottingham, UK.

Email: [email protected]

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Nor-Elhoda Mohamed

Nor-Elhoda Mohamed

Faculty of Science, Biomedicine Branch, University of Science & Technology in Zewail City, Cairo, Egypt

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Barakat M. Alrashdi

Barakat M. Alrashdi

Biology Department, College of Science, Jouf University, Sakaka, Saudi Arabia

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Abdullah A. A. Alghamdi

Abdullah A. A. Alghamdi

Department of Biology, Faculty of Science, Al-Baha University, Al-Baha, Saudi Arabia

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Ahmed Elmansi

Ahmed Elmansi

Biology Department, Faculty of Science, King Khalid University, Abha, Saudi Arabia

Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt

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Abdallah S. Salah

Abdallah S. Salah

Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Kafrelsheikh, Egypt

Institute of Aquaculture, University of Stirling, Stirling, UK

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Samir A. A. El-Gendy

Samir A. A. El-Gendy

Department of Anatomy and Embryology, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt

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Catrin S. Rutland

Corresponding Author

Catrin S. Rutland

School of Veterinary Medicine and Science, Faculty of Medicine and Health Science, University of Nottingham, Nottingham, UK

Correspondence

Soha A. Soliman, Department of Histology, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt.

Email: [email protected]

Hanan H. Abd-Elhafeez, Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut 71526, Egypt.

Email: [email protected]

Catrin S. Rutland, School of Veterinary Medicine and Science, Faculty of Medicine and Health Science, University of Nottingham, Nottingham, UK.

Email: [email protected]

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Diaa Massoud

Diaa Massoud

Biology Department, College of Science, Jouf University, Sakaka, Saudi Arabia

Department of Zoology, Faculty of Science, Fayoum University, Fayoum, Egypt

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First published: 01 February 2023
Review Editor: Alberto Diaspro

Abstract

Varanus niloticus is a lizard residing within the Varanidae family. To date no studies detailing its blood morphology and characteristics have been conducted. This study used histologically stained blood and bone marrow samples to visualize the cells and their characteristics. The erythrocytes were nucleated, these nuclei were located in the middle of the elliptical cells. Hemoglobin filled the erythrocyte cytoplasm. Eosinophils were large cells with lobed nuclei and spherical acidophilic granules. Large granulocytes called heterophils were present and characterized by their fusiform/pleomorphic cytoplasmic granules. Small spherical granulocytes, known as basophils, presented with round, deeply stained metachromatic granules that gave the cytoplasm a dusty or cobblestoned appearance which was able to cover the nucleus, which in turn had an unusual shape. Thrombocytes ranged in shape from ellipsoidal to fusiform. They featured an elliptical, centrally located nucleus and a pale cytoplasm, with small vacuoles, and fine acidophilic granulation. The smallest variety of non-granular leukocytes was the lymphocytes. Their cytoplasm was sparse, finely granular, light blue, had tiny cytoplasmic projections, featuring a high nucleus: cytoplasm ratio. Larger and smaller sized populations of lymphocytes were distinguished, with the larger cells similar in size to azurophils. In general, the pleomorphic monocytes were the biggest mononuclear leucocytes, displaying cytoplasmic projections. Their nuclei were ovoid, kidney- or bean-shaped, with vacuolated and granular cytoplasms. Round cells were common among the monocytic azurophils, and they had a granular cytoplasm, and their nuclei were typically eccentric. The present research identifies the cell types and morphologies within the Varanus niloticus.

Highlights

  • H&E, PAS, toluidine blue, methylene blue, and Safranin O stains provided morphological and morphometric descriptions of Varanus niloticus blood cells from blood smears and bone marrow.
  • The Varanus niloticus had nucleated erythrocytes and white blood cells, mostly granulocytes (heterophils, eosinophils, and basophils) and mononuclear cells (azurophils, lymphocytes, and monocytes).
  • Aquatic vertebrate Varanus niloticus had larger erythrocytes than terrestrial counterparts.
  • Blood cell morphological and cytochemical features were similar to other reptilian species, with some species-specific differences, which likely accommodate differing environmental conditions.
  • These results may help clinical researchers track the pathological conditions and support conservation of these wild animals.

1 INTRODUCTION

There are currently over 9000 species of reptiles, which are cold-blooded vertebrates (Pérez-García, 2022). The River Nile and Sub-Saharan Africa are home to Varanus niloticus (Bayless, 2002). It is a member of the Varanidae family and is the longest lizard in Africa. Varanus niloticus is also commonly referred to as the Nile monitor and the African savanna monitor. As a result of the pet trade invasion, Varanus niloticus has spread to many other regions, including Catalonia, Spain and the United States, and has since established itself as a native species, (Soler & Martinez Silvestre, 2013; Wood et al., 2016). In its native habitat, Varanus niloticus lives in a wide variety of environments such as desert fringes, grasslands, rainforests, rivers, marshes, ponds, lakes, and seashores (Enge et al., 2004; Sunmonu et al., 2019).

There are known species differences in the hematological characteristics between mammals, birds, and reptiles. Similar to avian blood, which contains erythrocytes, thrombocytes, and leucocytes, reptilian blood also contains these cell types (Fontes Pinto et al., 2018). Reptilian blood also contains granulocytes such as lymphocytes and monocytes, and granulocytes including heterophils, eosinophils, and basophils (Arizza et al., 2014; Stacy et al., 2011). Blood cells have been used to help differentiate between different reptilian species and understand unique features affecting the physiology of each species. For example, the mangrove-dwelling monitor (Varanus indicus) and the savannah monitor (Varanus exanthematicus), have both had their blood cell counts and sizes compared to their body sizes. The metabolic activities of blood cells have helped explain the variations between their size and number (Frydlova et al., 2013). Studying reptilian blood from a phylogenetic perspective also advances our understanding of the evolutionary development of both non-avian reptiles and the reptile clade as a whole.

The current study uses a variety of stains, including hematoxylin and eosin, Periodic Acid Schiff, toluidine blue, methylene blue, and Safranin O, to examine the morphology and cytochemistry of Varanus niloticus blood cells from blood smears and bone marrow.

2 METHODS AND MATERIALS

2.1 Ethics and sample collection

The conduct of this study and its methodologies were overseen by Institutional Aquatic Animal Care and Use Committee IAACUC of Kafrelsheikh University, Approval number: IAACUC-KSU-2022-27. Six male Nile monitors were kept in a controlled environment for two days in the animal facilities within the Faculty of Veterinary Medicine's Histology Department laboratory, South Valley University's (SVU), Egypt. Euthanasia was conducted on the third day using ethically approved techniques by a trained veterinary professional using intraperitoneal administration of sodium pentobarbital 60–120 mg/kg (Shaker & Ibrahium, 2021). All of the animals were deemed healthy, based on visual health assessments. To confirm maturity, snout-to-vent lengths <38 cm, and weights of 10 ± 2 kg were confirmed (Moustafa et al., 2013). Blood was taken from three Nile monitors, and femur bone marrow samples were taken from the other three for histological processing and embedding in paraffin.

2.2 Blood sample collection

Alcohol was used to disinfect the collection site, and a syringe used to draw 2 mm of blood from the ventral coccygeal vein in accordance with the previously described protocol (Salakij et al., 2014). For further processing and staining, the collected blood was immediately transferred into anticoagulant coated tubes (potassium ethylenediamine tetra acetic acid) to enable morphological analysis (Moustafa et al., 2013).

2.3 Cytomorphological analysis

For cytomorphological studies, blood smears were prepared immediately on grease-free slides, fixed using methanol and left to dry. Each slide was then stained with either hematoxylin and eosin (H&E), Periodic Acid Schiff (PAS), Safranin O, toluidine blue or methylene blue.

The smears were dehydrated through ascending grades of alcohol 70% 80%, 90%, and 100%, 3 min per concentrations. The protocols for stain were previously described (Suvarna et al., 2013). Each stained blood smear was examined using an oil-immersion lens under a Leitz Dialux 20 microscope (Germany), and pictures were taken with a Canon digital camera (Canon PowerShot A95, Japan).

2.4 Bone marrow sample collection, staining and visualization

Femurs were removed and cut longitudinally, and Worbel- Moustafa fixative was applied for 24 h (Abd-Elhafeez et al., 2017a, 2017b). Postfixation, decalcification was undertaken in 10% EDTA (in 0.1 M Tris/HCl buffer, pH 7.4) for 30 days, with the EDTA changed every 7 days until the bone became soft. The femurs were then washed four times in 0.1 M sodium phosphate buffer (pH 7.2) for 15 min each. All of the components of each buffer were as described previously (Abd-Elhafeez et al., 2021; Suvarna et al., 2013).

The dehydrated fixed and decalcified specimens were cleared in methyl benzoate, embedded in paraplast paraffin, and dehydrated in an ascending series of ethanol alcohol (Sigma Aldrich), as described previously (Abd-Elhafeez et al., 2021). Hematoxylin and eosin and Safranin O stains were used of stain paraffin sections of each femur using techniques previously described (Suvarna et al., 2013).

2.5 Morphometric evaluation and measurements

Evaluations were undertaken on 50 erythrocytes and 30 of each of the white blood cells from across the samples using a light microscope with an oil immersion lens (Leitz Dialux 20 microscope, Germany). Image J (http://fiji.sc/Fiji) was used to measure the morphological parameters including nucleus and cell diameters, in addition to cell cross sectional areas. The outcomes were collated into Excel (Microsoft, USA) and presented as mean ± standard deviation (SD). White blood cell and nuclei sizes were statistically compared using one-way Analysis of variance with post hoc testing.

3 RESULTS

Qualitative and quantitative investigations were undertaken on the blood samples and femur bone marrow. The morphological measurements of the erythrocytes and leucocytes are shown in Tables 1 and 2, respectively.

TABLE 1. Morphometric measurements of erythrocytes
Measurement Cell Nucleus
Width (μm) 10.0 ± 0.8 (8.4–11.5) 3.8 ± 0.2 (3.2–4.2)
Length (μm) 18.0 ± 0.9 (16.2–19.9) 6.2 ± 0.3 (5.0–6.8)
Cross-sectional area (μm2) 141.1 ± 14.1 (112.8–172.6) 18.2 ± 1.5 (14.2–21.4)
Length/width 1.8 ± 0.2 (1.5–2.4) 1.6 ± 0.1 (1.3–1.9)
Nucleus cross section/cell cross section 0.13 ± 0.01 (0.09–0.16)
  • Note: All measurements were expressed as means ± SD. N = 50 cells. Numbers in brackets represent minimum to maximum range.
TABLE 2. Morphometric measurements of white blood cells
Cell type Cell diameter Nucleus diameter
Heterophil 19.8 ± 1.9 (15.5–23.3) 8.0 ± 1.2 (5.9–10.0)
Eosinophil 17.3 ± 1.4 (15.1–19.4) 7.5 ± 1.2 (5.1–9.4)
Azurophil 15.5 ± 1.9 (11.5–19.0) 9.8 ± 1.4 (7.5–12.4)
Monocyte 14.2 ± 2.0 (11.2–18.9) 10.3 ± 1.2 (8.0–12.9)
Basophil 11.7 ± 2.0 (8.4–15.9) 9.0 ± 1.1 (7.4–11.5)
Lymphocyte 10.5 ± 1.8 (7.5–13.9) 8.8 ± 1.7 (5.8–12.4)
Thrombocyte 9.1 ± 1.3 (7.1–12.8) 6.2 ± 0.9 (4.8–7.8)
  • Note: Presented largest through to smallest cell size. N = 30 cells and their nuclei measured per cell type. All measurements were expressed as mean ± SD μm. Numbers on brackets indicate minimum-maximum range.

The largest cells were the heterophils at 19.8 ± 1.9 μm, the eosinophils, azurophils, monocytes, basophils, and lymphocytes decreased in size through to the smallest cells which were the thrombocytes at 9.1 ± 1.3 μm. In terms of cell diameter, the basophils, eosinophils, heterophils, azurophils, lymphocytes, monocytes and thrombocytes were all significantly different in size to each other (p values ranged from 0.01 to 0.0001; Table 2).

Nucleus size was not directly related to cell size with monocytes containing the largest nuclei at 10.3 ± 1.2 μm, followed by the azurophils, basophils, lymphocytes, heterophils, eosinophils and finally the thrombocytes at 6.2 ± 0.9 μm. The basophil nuclei and lymphocyte nuclei sizes were not significantly different to each other (p = .0671), neither were the eosinophils and heterophils (p = .143) or the azurophils and monocytes (p = .015). In contrast all of the other cell nuclei sizes were significantly different to each other (p = .01 through to p < .0001; Table 2).

3.1 Morphological description of cell types

Erythrocytes: The Varanus niloticus had nucleated erythrocytes, with the nucleus located in the middle of the elliptical shaped erythrocytes. Hemoglobin filled the homogeneous cytoplasm of erythrocytes (Figure 1a). Erythrocyte cell and nuclear widths, lengths, cross-sectional areas and nucleus: cell size ratio is presented in Table 1.

Details are in the caption following the image
Morphological features of granulocytic white blood cells using H&E. (a, b) Varanus niloticus erythrocytes (arrows) were nucleated, these were located within the center of the elliptically shaped cells Erythrocytes had a homogeneous cytoplasm that was filled with hemoglobin. Eosinophils (e) were large cells with spherical acidophilic granules and a lobed nucleus. Small lymphocytes were present (ly). (c, d) Heterophils (h) were large granulocytes that were identified by their fusiform/pleomorphic cytoplasmic granules. Monocytes (m) were large pleomorphic cells and had indented bean-shaped nuclei and a vacuolated cytoplasm. (e, f) Thrombocytes (t) were ellipsoidal to fusiform in shape, with a pale cytoplasm and an elliptical, centrally located nucleus

Eosinophils: The large eosinophil cells contained lobed nuclei and spherical acidophilic granules (Figure 1a,b).

Heterophils: Fusiform/pleomorphic cytoplasmic granules were used to identify the large granulocytes known as heterophils (Figure 1c,d).

Thrombocytes: The majority of the thrombocytes were elliptical in shape, though some were occasionally circular. The thrombocytes also had small vacuoles and finite acidophilic granulation within their cytoplasms. The thrombocytes ranged in shape from ellipsoidal through to fusiform. They contained an elliptical, central nucleus with a pale stained cytoplasm (Figure 1e,f).

Azurophils: Monocytic azurophils were usually rounded cells. Their cytoplasms were granular, and their nuclei were frequently eccentric (Figure 2a–c).

Details are in the caption following the image
Morphological features of non-granulocytic white blood cells using H&E. (a–c) Azurophils were often round monocytic cells. They usually had eccentric nuclei and the cytoplasm was granular. Thrombocyte (t). (d, e) Monocytes, a mononuclear, pleomorphic cells with cytoplasmic projections. Their nuclei were ovoid or indented kidney-bean shaped and they had a granular and vacuolated cytoplasm. (f) Small lymphocytes had a high nucleus: cytoplasm ratio

Monocytes: In general, monocytes were the biggest mononuclear leucocytes within this species. They exhibited cytoplasmic projections and were pleomorphic cells. Their nuclei were kidney- or bean-shaped and ovoid in shape. They had a granular and vacuolated cytoplasm (Figure 2d,e).

Lymphocytes: The smallest variety of non-granular leukocytes in this species was the lymphocytes. Their nucleus to cytoplasm ratio was high (cell diameter was 10.5 ± 1.8 μm whereas the nucleus was 8.8 ± 1.7 μm), and their cytoplasm was sparse, finely granular, and stained pale blue (Figure 2f). Additionally, these cells exhibited tiny cytoplasmic projections. Two populations of lymphocytes were distinguished, large and small (Figure 3a–d). The small lymphocytes typically had rounded, uneven surfaces. Their nuclei were positioned centrally or eccentrically. The large lymphocytes were similar in size to the azurophils. Using PAS, the azurophils typically had eccentric nuclei and granular cytoplasms (Figure 3e–h).

Details are in the caption following the image
Morphological features of non-granulocytic white blood cells using PAS. (a–d) Small lymphocytes (LY) had a high nuclear: cytoplasmic ratio. The cytoplasm was faintly stained using PAS. The cell surfaces had irregular outline and each nucleus was either centrally or eccentrically located. Large lymphocytes (lly) were faintly stained using PAS. Thrombocytes (t) had PAS-positive granules (arrows). Monocyte (m), thrombocyte (t), and immature granulocytes (Igr) with a segmented nucleus and a difficult to differentiate granular cytoplasm. (e–h) Azurophils usually had eccentric nuclei with a granular cytoplasm which stained faintly with PAS

Basophils: Small spherical granulocytes, the basophils, had round, intensely stained metachromatic granules that were able to cover the nucleus and gave the cytoplasm a dusty or cobblestone appearance. The basophils had irregular shaped nuclei.

3.2 Histological staining observations

PAS: The heterophils had cytoplasm with a PAS-positive affinity but PAS-negative granules. Monocytes had granular, barely PAS-stained cytoplasm (Figure 4a–c), whereas in the eosinophils, PAS reactivity was present (Figure 4d).

Details are in the caption following the image
Morphological features of granulocytic white blood cells using PAS. (a–c) The cytoplasm of the heterophils (h) exhibited PAS-positive affinities while their granules were PAS-negative. Monocytes (m) had a granular, faintly stained, cytoplasm when using PAS. (d) Eosinophils € exhibited PAS reactivity

Toluidine blue: The heterophil cytoplasm with its granules was barely stained following exposure to toluidine blue, whereas the nuclei had no affinity for it (Figure 5a–d). The cytoplasms of the eosinophils were barely stained while their granules had no affinity for toluidine blue (Figure 5e). Immature granulocytes had segmented nuclei and granular cytoplasms that were difficult to distinguish, and a low affinity for toluidine blue (Figure 5f). Meanwhile, small lymphocytes expressed positive staining (Figure 6a,b) and the azurophils had an eccentric nucleus, a granular, vacuolated cytoplasm all of which stained with toluidine blue (Figure 6c). Monocytes contained an indented kidney-shaped nucleus and granular, vacuolated cytoplasm stained by toluidine blue (Figure 6d).

Details are in the caption following the image
Morphological features of granulocytic white blood cells using toluidine blue. (a–d) Heterophil granules had no affinity for toluidine blue with light cytoplasmic staining. Monocyte (m) and lymphocyte (ly). (e) Eosinophil (e) granules had no affinity for toluidine blue while their cytoplasm was faintly stained. (f) Immature granulocytes (Igr) contained a segmented nucleus and a difficult to differentiate granular cytoplasm which exhibited a low affinity for toluidine blue
Details are in the caption following the image
Morphological features of non-granulocytic white blood cells using toluidine blue. (a, b) Small lymphocytes (ly) stained with toluidine blue. (c) Azurophils had eccentric nuclei and their cytoplasms were granular and vacuolated and stained by toluidine blue. (d) Monocytes had an indented kidney-shaped nucleus and a granular, vacuolated cytoplasm stained by toluidine blue

Methylene blue: Small spherical granulocytes known as basophils had round, deeply stained metachromatic granules that give the cytoplasm a dusty or cobblestone appearance and may cover the nucleus. Basophils had an odd-looking nucleus (Figure 7a–j). Methylene blue gave a faint blue stain to the granulocytes (Figure 8a,b). Granulocytes (likely eosinophils) reacted to methylene blue metachromatically (Figure 8c) and small lymphocytes had very faint metachromatic characteristics (Figure 8d,e). Metachromatic reactivity was also present in polychromatic RBCs (Figure 8f).

Details are in the caption following the image
Morphological features of basophil white blood cells using methylene blue. (a–j) Basophils were identified as small spherical granulocytes with fine deep staining around the metachromatic granules that gave the cytoplasm a dust-like or cobblestone appearance which might obscure the nucleus. Basophils had eccentric nuclei
Details are in the caption following the image
Morphological features of white blood cells using methylene blue. (a, b) The granulocytes stained faintly blue following application of methylene blue. (c) Granulocytes (probably eosinophils) also exhibited metachromatic reactivity for methylene blue indicating that they contain basic amino acid residues, which are essential for glycosaminoglycan binding. (d, e) Small lymphocytes exhibited slight metachromatic features. (f) Polychromatic RBCs exhibited metachromatic reactivity

Safranin O: The erythrocytes were strongly Safranin O-positive, whereas the thrombocytes were generally not stained (Figure 9a), except for positively stained cytoplasm present in some thrombocytes (Figure 9c,d). Granular Safranin O-positive cytoplasmic staining was also present in basophils (Figure 9b), the granulocytes contained tiny Safranin O-stained granules, and the lymphocytes were positively stained (Figure 9c,d).

Details are in the caption following the image
Morphological features of blood cells using Safranin O. (a) RBCs (arrows) stained strongly positive following the application of Safranin O, however thrombocytes (t) were negative. (b) Basophils exhibited a granular Safranin O-positive cytoplasm. (c, d) Safranin O fine granules were detected in the granulocytes (gr), and some thrombocytes (t) had a Safranin O-positive cytoplasm (arrow). Lymphocytes (ly) were also stained by Safranin O

3.3 Bone marrow characteristics

The hematopoietic tissue within the femur was investigated regarding their morphological characteristics. Hematopoietic tissues were located within the marrow cavity of each femur, which was surrounded by bone spicules (Figure 10a). In the hematopoietic tissues, monocytic and granulocytic precursors were present (Figure 10b–l and Figure 11a–d).

Details are in the caption following the image
Morphological features of hematopoietic tissues using H&E. Paraffin sections of femurs stained by H&E. (a) The marrow cavity (m) of the femur contained hematopoietic tissues which was surrounded by bone spicules (b). (b–l) Hematopoietic tissues contained monocytic precursors (arrowheads) and granulocytic precursors (arrows). Note the presence of RBCs
Details are in the caption following the image
Morphological features of blood cells using Safranin O. Paraffin sections of femurs stained using Safranin O. (a) The marrow cavity (m) of the femur contained hematopoietic tissues surrounded by bone spicules (b). (b–d) Hematopoietic tissues contained monocytic precursors (arrowheads) and granulocytic precursors (arrows). Note the presence of RBCs

4 DISCUSSION

Hematological studies are very rare despite the global popularity of reptilian research. Because of this, the current study is the first to describe the blood profile of the Egyptian Nile monitor (Varanus niloticus). The goal of the current study was to describe the cytochemical and morphometric properties of this species' blood cells in order to provide detailed information that may aid in monitoring initiatives and to support protection and conservation plans. Additionally, it might offer important details about species life histories and the phylogenetic relationships among various vertebrates (Martins et al., 2020).

Erythrocytes, leukocytes, and thrombocytes were the main components found within the blood of the Nile monitors (Varanus niloticus). The Nile monitor, similar to other reptiles, had nucleated erythrocytes, these are not observed in mammals. From an evolutionary perspective, mature nucleated erythrocytes are a distinguishing trait of lower vertebrates. The loss of erythrocyte nuclei and other cytoplasmic organelles is thought to facilitate mammalian endothermy by allowing for efficient gas exchange and increasing oxygen-carrying capacity, making mammals the only vertebrates to have enucleated erythrocytes in the mature state (Anderson et al., 2018). However, there are a few unusual, rare cases where fish and amphibians, non-mammalian vertebrates, do not have nuclei (Emmel, 1924; Mueller et al., 2008; Wingstrand, 1956).

Reptile erythrocytes resemble bird erythrocytes in both appearance and function, but they are larger in reptiles (Claver & Quaglia, 2009). The erythrocytes of the Nile monitor were flattened with an elliptical shape, this observation concurred with studies carried out on other reptilian species (Parida et al., 2014; Salakij et al., 2014; Stacy et al., 2011). The nuclei with the erythrocytes were positioned in the middle of the cell and contained hemoglobin. It was previously reported that erythrocyte nuclei in reptiles appeared to be rounded with irregular margins (Claver & Quaglia, 2009), this was also observed in V. niloticus in the present study. The relatively large size of the V. niloticus erythrocyte in this study (at 18 μm long) was also consistent with the larger size seen in ectotherms (reptiles, fish, amphibians) compared to endotherms (bird, mammals; Hawkey et al., 1991). This is also thought to be due to lower oxygen demands and mass-specific resting metabolic rates seen in ectoderms. The erythrocytes of reptiles also contain hemoglobin tetramers, like those of other vertebrates, which aid in the exchange of oxygen and carbon dioxide in tissue and organs. In fact, the lower metabolic rates of reptiles has been highlighted as a possible reason for erythrocyte longevity, in excess of 600 days (Altland & Brace, 1962). This compares to birds such as the duck, pigeon, and chicken with their high metabolism with erythrocytes living just 35–45 days on average (Rodnan et al., 1957). Meanwhile mammalian red blood cells have an average longevity of 22 days in mice, 100 days in people, 120 days in dogs (Rodnan et al., 1957).

White blood cells from reptiles have been classified based on their shapes rather than their functions (Claver & Quaglia, 2009). Eosinophils, heterophils, and basophils made up the Nile monitor's granulocytic leucocytes, while lymphocytes and monocytes made up the agranulocytes. In several species of reptiles and birds, the heterophils replaced the neutrophils, as previously reported (Blofield et al., 1992; Sacchi et al., 2020; Stacy et al., 2011).

In terms of blood morphometrics, several researchers have noted that reptiles are a heterogeneous group because they show significant variations between orders and even within the same family members (Louei Monfared, 2014; Ozzetti et al., 2013; Starck et al., 2017; Zayas et al., 2011). The morphometric measurements of V. niloticus red blood cells in the present study showed that the nuclei were 18.0 ± 0.9 μm long, with an average cross-sectional area of 141.1 ± 14.1 μm. In the desert monitor (V. griseus), the nuclei were 26.1 ± 0.3 μm and the RBCs were 130.3 ± 2.0 μm (Arıkan & Çicek, 2014; Arıkan & Çiçek, 2010). According to earlier studies the marked variations in erythrocyte sizes and their nuclei may be attributed to a variety of factors, including environmental conditions, health status, breeding time, daily activity, hibernation, and lifestyle (González-Morales et al., 2015; Sykes & Klaphake, 2008). The Nile monitor is well adapted to both an aquatic and terrestrial lifestyle and is typically found near water streams of the Nile and its tributaries. A more terrestrial-adapted species is the desert monitor (V. griseus). Our findings were in line with earlier studies that claimed that the aquatic vertebrates have larger erythrocytes than terrestrial counterparts (Aldrich et al., 2006; Arıkan, 2014; Fleischle et al., 2019).

The Nile monitor (V. niloticus) has blood that is primarily made up of erythrocytes, leucocytes, thrombocytes, and liquid plasma, similar to the blood compositions observed in other reptiles. Despite the fact that mammalian plasma is typically colorless, reptiles have yellowish-green plasma, which is likely due to higher concentrations of riboflavin and carotenoids (Dessauer, 1970).

Non-mammalian thrombocytes function in a manner similar to platelets in mammalian vertebrates, thrombocytes therefore play a hemostatic role (Martin & Wagner, 2019; Stacy & Harr, 2021). In the bird thrombocytes are also round to oval, nucleated, with a pale cytoplasm, and smaller than erythrocytes (Campbell & Joshua Dein, 1984). The present research showed that the Nile monitor (V. niloticus) also produced thrombocytes that were small, ellipsoidal to fusiform in shape, with a pale cytoplasm, containing an elliptical nucleus in the middle. Additionally, the cytoplasm contains small vacuoles and finite acidophilic granulations. In terms of function, the thrombocytes of lower vertebrates were analogous to the platelet of mammalian species (Levin, 2019; Peng et al., 2018). Nucleated thrombocytes were recorded previously in many reptilian species (Alleman et al., 1992; Arıkan & Çicek, 2014; Giori et al., 2020; Louei Monfared, 2014; Metin et al., 2006). Only mammals have exhibited enucleated platelets while reptiles and birds have nucleated thrombocytes, which are arguably functionally less efficient than mammalian platelets (Martin & Wagner, 2019; Schmaier et al., 2011). The platelets of mammals are relatively short-lived as the life spans range between 10 days in humans and 5 days in mice (Johnson et al., 2018; Lebois & Josefsson, 2016). Taking in our consideration the mean erythrocyte lifespan in reptiles ranges between 600 to 800 days while in humans it is only 120 days, thrombocytes have a longer lifespan of thrombocytes in reptiles (Louis et al., 2020; Olsson et al., 2020). It is assumed that the slow metabolic rate of reptiles is closely related to the exceedingly slow turnover of erythrocytes comparing to other vertebrates (Khalaf et al., 2020; Stacy et al., 2011). It is worth mentioning that the thrombocytes in fish, amphibians, reptiles, and birds have a membrane-bound canalicular system which plays an essential role in their function (Chamut & Osvaldo, 2018; Levin, 2019; Selvadurai & Hamilton, 2018). This system is also found in mammalian platelets and has been linked to their origin in the megakaryocyte cytoplasm (Levin, 2019; Martin & Wagner, 2019; Stalker et al., 2012). Our morphometric analysis in the Nile monitor (V. niloticus) indicated that the smallest cells were the thrombocytes (9.07 ± 1.33 μm) which helps facilitate its role in maintaining blood hemostasis, blood clotting formation and the initiation of wound healing (Martin & Wagner, 2019; Peng et al., 2018). Additionally, these cells may exhibit some endocytic and phagocytic abilities (Silva et al., 2005).

Primarily due to a lack of functional knowledge, reptilian leucocytes are categorized according to appearance rather than function, as such their nomenclatures are tentative (de Carvalho et al., 2017; Stacy et al., 2011). The present research showed that heterophils appeared to be the second-most prevalent cell type in the Nile monitor after erythrocytes. Their defining features were that they were relatively large cells with a transparent cytoplasm. Reptilian structural and cytochemical studies revealed that heterophils function in a similar way to mammalian neutrophils, phagocytizing foreign particles and bacterial invaders (Arıkan, 2014; Fingerhut et al., 2020; Zimmerman et al., 2010). It was also interesting to note that heterophils were not found in any of the five species of Turkish lacertid lizards (Arıkan et al., 2009). The eosinophils in the present study were relatively large cells with lobed nuclei and spherical acidophilic granules. Even though the primary role of eosinophils in reptiles remains unclear, recent research has shown that helminthes and protozoan parasitic infections cause an abnormal increases in eosinophil numbers (Bessa et al., 2020; Mendoza-Roldan et al., 2020). The basophils in the Nile monitor were small, spherical granulocytes with small, round, metachromatic granules that were deeply stained. Some species of reptiles, like the green turtle, loggerhead turtles, bobtail lizards, and other European lizards, only occasionally have these cells (Casal & Orós, 2007; Moller et al., 2016; Sacchi et al., 2011). The primary role of basophils is long-term protection against chronic illness and the inflammatory response it triggers (Hawkey et al., 1989). Additionally, they may aid in the recovery and elimination of hemoparasites such as trypanosomes and haemogregarines from the reptilian body (Strik et al., 2007).

The Nile monitor's (V. niloticus) lymphocytes resembled those of other vertebrates previously studied. The smallest variety of the non-granular leukocytes, they had a large nucleus to cytoplasm ratio, following staining the cytoplasm was a light blue color. Lymphocytes are involved in immune responses and hematopoietic growth factor production (Arıkan, 2014). The present investigations showed that the largest mononuclear leucocyte with cytoplasmic projections was the monocyte. Their nuclei were kidney- or bean-shaped or ovoid in shape. These cells have previously, in reptiles, exhibited phagocytic traits and become active under conditions of persistent inflammation (Stacy et al., 2011). Azurophils, a different kind of granulocyte only found in reptiles, were also discovered in V. niloticus in the present investigation. These cells have also been observed in a variety of reptilian species, including lizards, snakes, crocodiles, and occasionally turtles (García-De la Peña et al., 2020). They are similar to monocytes in many ways, but evidence from several studies has shown they exhibit stronger phagocytotic affinities toward bacterial particles (de Carvalho et al., 2017).

In conclusion, the Nile Monitor's (V. niloticus) blood cells have morphological and cytochemical characteristics that are similar to those of other reptilian species. There are, however, some species-specific variations, which likely account for variations in their environmental circumstances. These findings may help in develop appropriate conservation and identification strategies for protecting endangered species and informing evolutionary biology, as well as providing clinically relevant research to track pathological conditions of this lizard.

ACKNOWLEDGMENTS

The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University (grant number G.R.P.1/29/43) and the School of Veterinary Medicine and Science, University of Nottingham for funding this work.

    CONFLICT OF INTEREST STATEMENT

    The authors declare no conflicts of interest.

    DATA AVAILABILITY STATEMENT

    The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.