INTRODUCTION

Crocodilians (crocodiles, caimans, alligators and gharials) are considered keystone species of their ecosystems due to the different roles they play; they might as well serve as potential sentinels of environmental contamination because they tend to bioaccumulate contaminants throughout their long-life span (^{Milnes and Guillette, 2008}; ^{Somaweera et al., 2020}). Hence the importance of monitoring the health status of wild crocodilians.

One key aspect to assess the health status of wild species is the evaluation of hematologic parameters. As in other reptiles, crocodilian blood cells are erythrocytes, thrombocytes and leukocytes, all nucleated. Leukocytes are classified as granulocytes (heterophils, eosinophils, basophils) and mononuclear cells (lymphocytes, monocytes, azurophils). Heterophils are the equivalent of mammalian neutrophils; eosinophils and basophils have no clear function in reptiles; lymphocytes and monocytes have similar morphology and function to those of mammals; azurophils are unique to reptiles with similar morphology and function to granulocytes and monocytes (^{Montali, 1988}; ^{Dotson et al., 1995}; ^{Alleman et al., 1999}; ^{Stacy et al., 2011}).

In crocodilians, the examination of blood cell morphology through the evaluation of stained peripheral blood smears, along with the determination of the leukocyte differential count, are essential for the accurate diagnosis of some pathological processes (^{Campbell, 2004}; ^{Rossini and García, 2010}). Any change in the morphology of peripheral blood cells can be an indicator of specific diseases (^{Stacy et al., 2011}; Campbell, 2012).

Blood cell morphology varies greatly among reptile species, including those within the same genus (^{Stacy and Whitaker, 2000}; Stacy et al., 2011). In addition, factors such as age, environmental conditions, particularly variations in temperature, season of the year and wild or captive status, can affect several components of the reptile hemogram (Stacy, 2011). In crocodilians, blood cells have been characterized in the American alligator (Alligator mississippiensis Daudin, 1802) (^{Glassman et al., 1981}; ^{Mateo et al., 1984}), Salt-water crocodile (Crocodylus porosus Schneider, 1801) and Australian fresh-water crocodile (Crocodylus johnstoni Krefft, 1873) (^{Canfield, 1985}), Spectacled caiman (Caiman crocodilus Linnaeus, 1758) (^{Rossini and García, 2010}), Broad-snouted caiman (Caiman latirostris Daudin, 1802) and Yacare caiman (Caiman yacare Daudin, 1802) (^{Moura et al., 1999}; Mussart et al., 2006; ^{Zayas et al., 2011}).

Nonetheless, for Morelet's crocodile (Crocodylus moreletii Duméril and Bibron, 1851), there is no information on blood cell morphology, either in wild or in captive individuals and despite its ecological and economic importance. The Morelet's crocodile is native to Mexico, Guatemala and Belize and is one of the two crocodile species extant in Mexico. In the first half of the 20th century, overhunting of this crocodile for its skin resulted in severe population declines (^{Platt and Thorbjarnarson, 2000}; Cedeño-Vázquez et al., 2012). In the 1970s, the Mexican government banned its hunting and decreed its protection and conservation, and populations gradually started to recover and thrive (Platt and Thorbjarnarson, 2000; ^{Sánchez-Herrera et al., 2011}). In the early 1980s, the International Union for Conservation of Nature (IUCN) Red List of Threatened Species included the Morelet's crocodile with the status of Endangered (Platt and Thorbjarnarson, 2000; Cedeño-Vázquez et al., 2012).

Currently, the Morelet's crocodile is legally protected in Mexico (Secretaría de Medio Ambiente y Recursos Naturales [SEMARNAT], 2010), has the status of Least Concern in the IUCN Red List (International Union for Conservation of Nature [IUCN], 2019) and is included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora with a zero quota for commercial trade in wild specimens (La Convención sobre el Comercio Internacional de Especies Amenazadas de Fauna y Flora Silvestres [CITES], 2019). Because of this, the legal use of the Morelet's crocodile for commercial purposes in Mexico is restricted to individuals raised in captivity in breeding farms known as Wildlife Conservation Units (^{Sánchez-Herrera et al., 2011}; Cedeño-Vázquez et al., 2012); there, individuals are also used for research purposes regarding farming and captive management.

Since blood cell morphology varies greatly among reptiles within the same genus and many factors influence the components of their hemogram, we hypothesized that the Morelet's crocodile would have some particularities regarding other crocodilians. Therefore, the objective of this study was to describe the morphological characteristics of peripheral blood cells and to determine the leukocyte differential count of healthy farmed Morelet's crocodile females and males, subadults and adults in the breeding and non-breeding seasons, to have information that can be used as a reference to assess the health status of captive and wild crocodiles. In the latter, this information could also be used to evaluate the effect of environmental contaminants on their hematologic parameters and therefore on the health of wild populations and their ecosystems.

MATERIALS AND METHODS

Animal experimentation was performed in compliance with the guidelines of the Bioethics and Animal Welfare Commission from the School of Veterinary Medicine, University of Veracruz (Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana) in Veracruz, Mexico.

RESULTS

The length/diameter of each peripheral blood cell type, as well as the diameter of their nucleus, were similar in subadult and adult crocodiles, males and females, in both seasons. Hence, data from both sizes and sexes were combined and an average was obtained for each cell type. In general, erythrocytes were the largest blood cells in Morelet's crocodile, while lymphocytes were the smallest. Among leukocytes, eosinophils were the largest. The morphological characteristics of the peripheral blood cells evaluated are described in Table 1 and shown in Figure 1 (a - h).

Figure 1 Morelet's crocodile (Crocodylus moreletii) peripheral blood cells stained with a Romanowsky-type staining, observed at 100x magnification. The arrows point to the indicated cell type. a) Erythrocyte. b) Lymphocyte. c) Heterophil. d) Eosinophil. e) Basophil. f) Monocyte. g) Azurophil. h) Thrombocyte. Bar = 10 μm. 

Regarding the leukocyte differential, in the general population of crocodiles, lymphocytes were the most abundant, followed by heterophils and azurophils, while basophils were the least numerous (Table 2).

When the size of the crocodiles was considered, lymphocytes were the most numerous in subadults and lymphocytes and heterophils in adults, while basophils were the least numerous in both sizes (Table 3). Subadult females had a lower percentage of heterophils in the non-breeding season and a higher percentage of eosinophils and monocytes in the breeding season when compared to adult females (p < 0.05; Table 3). Likewise, subadult males had a higher percentage of lymphocytes and lower of heterophils in the non-breeding season and a higher percentage of azurophils in the breeding season when compared to adult males (p < 0.05; Table 3).

Regarding the sex of the crocodiles, lymphocytes were the most numerous in females and lymphocytes and heterophils in males, while basophils were the least numerous in both sexes (Table 3). No difference was found between female and male, subadult and adult individuals (p > 0.05; Table 3).

Concerning to the season of the year, lymphocytes and heterophils were the most abundant in the non-breeding season and lymphocytes in the breeding season; in both seasons basophils were the least numerous (Table 3). Subadult males had a higher percentage of lymphocytes and lower of eosinophils and azurophils in the non-breeding season (p < 0.05; Table 3). Adult males had a higher percentage of heterophils and lower of eosinophils and basophils in the non-breeding season (p < 0.05; Table 3). No difference was found between seasons in subadult and adult females (p > 0.05; Table 3).

DISCUSSION

Wildlife management requires the assessment of the health status of the individuals (^{Johnstone et al., 2012}), both in captivity and in the wild. In higher vertebrates, health assessment and disease diagnosis require knowledge on the normal hematologic parameters, as they provide information particularly on the functioning of the immune system of individuals and populations (^{Stacy et al., 2011}; ^{Latorre et al., 2016}). In crocodilians, hematologic parameters can also serve to evaluate their exposure to environmental contaminants or toxicants, which would make them potential indicators of ecosystem health (^{Milnes and Guillette, 2008}; Latorre et al., 2016). That is the case for the American alligator, which is considered as an indicator of wetland health throughout its distribution range (Milnes and Guillette, 2008).

In reptiles, routine hematologic evaluation includes assessment of blood cell morphology and leukocyte differential count, among other indicators (^{Stacy et al., 2011}). Since blood cell morphology and count vary greatly among reptilians, it is necessary to know the normal characteristics and ranges for each species to detect sick individuals (Stacy et al., 2011; ^{Mumm et al., 2019}). Unlike several crocodilians in which the characteristics of their blood cells have been reported, there is no information available in the Morelet's crocodile, either captive or wild individuals. Thus, this study is the first to report the morphological characteristics of peripheral blood cells and the leukocyte differential of Morelet's crocodile females, males, subadults and adults in the non-breeding and breeding seasons.

Erythrocytes of Morelet's crocodile showed similar morphology to those from other crocodilians; however, they were larger than the erythrocytes from species such as American alligator (^{Glassman et al., 1981}; ^{Mateo et al., 1984}), Broad-snouted and Yacare caiman (^{Moura et al., 1999}; ^{Barboza et al., 2007}; ^{Rossini and García, 2010}; ^{Zayas et al., 2011}), Smooth-fronted caiman (Paleosuchus trigonatus Schneider, 1801) (^{Rojas, 2005}), Spectacled caiman (Rossini, 2004; Rossini and García, 2010) and Chinese alligator (Alligator sinensis Fauvel, 1879) (^{Peng et al., 2018}). These variations in erythrocyte size could result from differences between species, the size of the animals or the handling of the samples in each study (^{Stacy and Whitaker, 2000}; Stacy et al., 2011).

In general, changes in the morphology and number of erythrocytes are indicative of pathological conditions, particularly anemia (^{Saggese, 2009}). In reptiles, the presence of poikilocytosis (abnormality in erythrocyte morphology) can be normal in healthy animals; however, a high number of poikilocytes is associated with septicemia and severe chronic infection (^{Martínez-Silvestre, 2011}). Also, diagnosis of anemia can help identify diseases before they become life-threatening conditions (^{Campbell and Ellis, 2007}; ^{Sykes and Klaphake, 2008}).

Leukocytes of the Morelet's crocodile had similar morphology to those from the American alligator (^{Glassman et al., 1981}), Yacare caiman (^{Oliveira et al., 1998}; ^{Moura et al., 1999}), Siamese crocodile (Crocodylus siamensis Schneider, 1801) (^{Sakulwira et al., 2004}) and Broad-snouted caiman (^{Zayas et al., 2011}). However, their size showed some variations when compared to those from other crocodilians. Thus, in comparison with the Morelet's crocodile, the Broad-snouted caiman had larger lymphocytes, basophils and monocytes but smaller heterophils and eosinophils (Zayas et al., 2011); the American alligator had larger lymphocytes and smaller eosinophils (Glassman et al., 1981); and the Spectacled caiman had larger basophils and smaller heterophils and eosinophils (^{Rossini and García, 2010}).

In the present study, a type of leukocyte that exhibited the same morphological characteristics as azurophils (^{Montali, 1988}; ^{Dotson et al., 1995}; ^{Alleman et al., 1999}; ^{Stacy et al., 2011}) was observed in the peripheral blood of the Morelet's crocodile and therefore considered as such, separated from monocytes; this was also reported in the Nile crocodile (Crocodylus niloticus Laurenti, 1768) (^{Lovely et al., 2007}). No other study was found that reported morphological characteristics or size of azurophils in other crocodilians. Only ^{Moura et al. (1999)} in the Yacare caiman mentioned the monocytes azurophils, with similar morphology to the azurophils from this study.

Of all leukocytes, eosinophils were the largest ones in the Morelet's crocodile, contrary to reports of heterophils being the largest leukocytes in the Yacare and Broad-snouted caiman (Mussart et al., 2006) and monocytes in the Broad-snouted caiman (^{Zayas et al., 2011}). On the other hand, lymphocytes were the smallest blood cells in the Morelet's crocodile, like what was observed in the Yacare and Broad-snouted caiman (Mussart et al., 2006).

Thrombocytes of the Morelet's crocodile showed similar morphology to those from other crocodilians; likewise, they had a similar size to those from the American alligator (^{Glassman et al., 1981}), but were smaller than thrombocytes from the Spectacled and Broad-snouted caiman (^{Rossini and García, 2010}; ^{Zayas et al., 2011}).

Regarding the leukocyte differential, lymphocytes were the most numerous in the general population of Morelet's crocodile in this study, accounting for up to 77 % of leukocytes. Similar results were reported in this same species (^{Padilla et al., 2011}) and in Yacare and Broad-snouted caiman (Mussart et al., 2006; ^{Zayas et al., 2011}), all in captivity. In most reptiles, lymphocytes compose up to 80 % of leukocytes (^{Sypek and Borysenko, 1988}; ^{Divers et al., 1996}; ^{Troiano et al., 1997}; ^{Alleman et al., 1999}; ^{Lamirande et al., 1999}), however, lymphocytes reached 95 % in farmed Nile crocodiles (^{Makinde and Alemu, 1991}) and 85 % in wild Nile crocodiles (^{Lovely et al., 2007}) but only 42 % in captive Orinoco crocodiles (Crocodylus intermedius Graves, 1819) (^{Manzanilla et al., 2011}). These differences in lymphocyte percentages could be due to species-specific particularities, as these studies reported values from apparently healthy individuals.

In reptiles, an increase in lymphocytes is associated with inflammation, parasitic and viral infections, leukemia and wound cicatrization (^{Martínez-Silvestre et al., 2011}). On the other hand, a decrease in lymphocytes is usually observed during winter, even in tropical regions (^{Alleman et al., 1999}; ^{Lamirande et al., 1999}), and it is also associated with immunosuppression, stress and chronic malnutrition (^{Strik et al., 2007}). Broad-snouted caimans exposed in ovo and in vivo to insecticides had decreased lymphocyte numbers (^{Latorre et al., 2016}).

In this study, heterophils were the second most abundant leukocytes in the Morelet's crocodile, accounting for up to 59 %, similar to one result obtained previously in this same species (^{Padilla et al., 2011}). Although the percentage of heterophils in reptiles varies within species, they constitute more than 50 % of leukocytes in the peripheral blood (^{Mateo et al., 1984}; ^{Montali, 1988}; ^{Manzanilla et al., 2011}). Nonetheless, heterophils only reached 20 % (^{Makinde and Alemu, 1991}) and 39 % (^{Lovely et al., 2007}) in farmed and wild Nile crocodiles, respectively, and 34 % in Broad-snouted caimans (^{Zayas et al., 2011}), despite being the second most numerous leukocytes in these species. Unlike the present study, in the American alligator (Mateo et al., 1984; ^{Schoeb et al., 2002}), Mugger crocodile (Crocodylus palustris Lesson, 1831) (^{Stacy and Whitaker, 2000}) and Orinoco crocodile (Manzanilla et al., 2011) heterophils were the predominant leukocytes.

Heterophils are phagocytic cells and the first to arrive at the site of infection (^{Strik et al., 2007}; ^{Martínez-Silvestre et al., 2011}). Increases in the number of heterophils are associated with inflammation, infection, tissue damage, stress and neoplasias (Martínez-Silvestre et al., 2011). American alligators exposed to an organochlorine miticide/ pesticide (^{Woodward et al., 1993}) and young Broad-snouted caimans exposed to a glyphosate-based herbicide (^{Latorre et al. 2013}) or to an insecticide in ovo and in vivo (Latorre et al., 2016) had increased heterophil counts. However, a physiological increase in heterophils results from the season of the year, with the highest numbers observed in the summer (Martínez-Silvestre et al., 2011). On the other hand, a decrease in the number of heterophils is associated with viral infections (^{Alleman et al., 1992}).

Azurophils were the third most abundant leukocytes in Morelet's crocodiles in this study, constituting up to 33 %. This result was contrary to the 2 % obtained previously in this same species (^{Padilla et al., 2011}). In Nile crocodiles, azurophils were also the third most abundant leukocyte, accounting for up to 21 % (^{Lovely et al., 2007}). No other study considered azurophils as a leukocyte type in crocodilians. One reason for this could be that azurophils are sometimes considered as a type of monocyte and included in the monocyte count in the leukocyte differential (^{Tully and Mitchel, 2012}). Thus, the result from the present study disagrees with information that indicates that azurophils are found in low percentages in reptiles other than snakes and that increased numbers frequently occur in chronic disease states (^{Stacy et al., 2011}) or associated with inflammation and bacterial and parasitic infections (^{Martínez-Silvestre et al., 2011}). The crocodiles from this study were apparently healthy, with no sign of chronic disease; therefore, azurophils may be normally more abundant in the Morelet's crocodile than in other crocodilians. These results also suggest that azurophils be considered separately from monocytes during the hematologic evaluation of crocodilians.

Eosinophils reached 23 % of leukocytes in the Morelet's crocodile. According to ^{Stacy et al. (2011)}, eosinophils compose 7 % to 20 % of leukocytes in healthy reptiles and their circulating numbers vary with species and seasonal changes. This information agrees with the results obtained in the Orinoco crocodile, with up to 14 % eosinophils (^{Manzanilla et al., 2011}), and in the Nile crocodile, with up to 16 % (^{Lovely et al., 2007}) and 8 % (^{Makinde and Alemu, 1991}). Nonetheless, contrary results were reported previously in the Morelet's crocodile, with only 2.9 % eosinophils (^{Padilla et al., 2011}), and in the Broad-snouted caiman, with up to 6 % (^{Zayas et al., 2011}). Although eosinophil function in reptiles remains unclear, an abnormally high eosinophil percentage has been associated with antigenic stimulation, particularly endo- and ectoparasitism (^{Mead and Borysenko, 1984}; ^{Campbell, 2004}). However, since the crocodiles from the present study were apparently healthy and dewormed before blood sampling, the slightly higher percentage of eosinophils observed in them with respect to the normal range in healthy reptiles might be normal in farmed Morelet's crocodile. On the other hand, a lower eosinophil count was observed in American alligators exposed to an organochlorine miticide/ pesticide (^{Woodward et al., 1993}).

Basophils accounted for up to 5 % of leukocytes in Morelet's crocodiles in this study. This result was similar to that obtained in Orinoco crocodiles (3.0 ± 2.1 %; ^{Manzanilla et al., 2011}) but lower than the result in Broad-snouted caimans (up to 10 %; ^{Zayas et al., 2011}), Nile crocodiles (up to 16 %; ^{Lovely et al., 2007}) and Morelet's crocodiles (18.6 %; ^{Padilla et al., 2011}). Nonetheless, all these results can be considered normal because the percentage of basophils ranges from 0 % to 40 % among reptile species (^{Sypek and Borysenko, 1988}; ^{Marks and Citino, 1990}). An increase in the number of basophils is associated with intestinal parasites, hemoparasites and viral infections (^{Innis et al., 2007}).

In this study, monocytes represented up to 15 % of total leukocytes in the Morelet's crocodile. It has been reported that monocytes usually compose 0 % to 10 % of leukocytes (^{Sypek and Borysenko, 1988}; ^{Campbell, 2004}), which agrees with the results obtained in the Nile crocodile (^{Lovely et al., 2007}), Morelet's crocodile (^{Padilla et al., 2011}), Broad-snouted caiman (^{Zayas et al., 2011}) and Orinoco crocodile (^{Manzanilla et al., 2011}). However, in these two last species the percentage of monocytes included the cell type considered as azurophils in the present study. Broad-snouted caimans exposed in ovo and in vivo to insecticides had increased monocyte counts (^{Latorre et al., 2016}). In general, increased numbers of monocytes are associated with inflammatory diseases (^{Strik et al., 2007}).

All these results of the leukocyte differential indicate the existence of variations among crocodilian species, as has been mentioned by different authors. Nonetheless, besides these species-specific differences, the variations in the leukocyte differential could be attributed to diverse levels of antigenic stimulation within populations, i.e., differences between wild and captive animals could be due to the presence of parasites in wild animals and stress and husbandry in farmed individuals (^{Stacy and Whitaker, 2000}; Stacy et al., 2011). In addition, factors such as the environment (particularly temperature and rainfall), geographical location, age, sex, genetic variations, breeding status, diet and sample handling make the results of reptile peripheral blood evaluation highly variable concerning cell morphology and concentration (Stacy et al., 2011; ^{Zayas et al., 2011}; ^{Campbell, 2012}). Therefore, to be considered significant, total and differential leukocyte count must differ from normal reference values at least twice as high or as low (Campbell, 2012). In reptiles, an increase in the leukocyte count has been observed in bacterial or parasitic infections, stress and exposure to toxicants from the environment (^{Woodward et al., 1993}; ^{Schoeb et al., 2002}; ^{Martínez-Silvestre et al., 2011}).

Concerning the size of the crocodiles, the predominance of lymphocytes over the other leukocytes in subadult Morelet's crocodiles in this study agreed with what has been reported in subadult Nile crocodiles (^{Lovely et al., 2007}); however, it differed from the result in captive subadult Mugger crocodiles, in which heterophils were the most numerous leukocytes (^{Stacy and Whitaker, 2000}). On the other hand, basophils were the least abundant leukocytes in subadult individuals in this study; this was also observed in captive subadult Mugger crocodiles (Stacy and Whitaker, 2000); however, this result was different to that in subadult Nile crocodiles, in which monocytes were the least numerous (Lovely et al., 2007). In adult Morelet's crocodiles, heterophils and lymphocytes were the predominant leukocytes, as well as reported for captive adult Mugger crocodiles (Stacy and Whitaker, 2000). In the latter study, adult crocodiles had fewer lymphocytes than juveniles and subadults (Stacy and Whitaker, 2000).

Regarding the sex of the crocodiles, in this study lymphocytes were the most numerous leukocytes in females and both lymphocytes and heterophils in males. Similar results were obtained in captive Mugger crocodiles, as subadult females had more heterophils and lymphocytes than males, whereas adult females had more lymphocytes but fewer heterophils than males (^{Stacy and Whitaker, 2000}). Under the same environmental conditions, females usually have more lymphocytes than their conspecific males of the same age (^{Sypek and Borysenko, 1988}). In Nile crocodiles, the percentage of eosinophils was lower in males than in females, with no difference in the other leukocytes (^{Lovely et al., 2007}). In Orinoco crocodiles, females had fewer basophils and more monocytes than males (^{Manzanilla et al., 2011}).

Concerning the season of the year, there were differences in the percentage of some leukocytes in the non-breeding and breeding seasons in subadult and adult males. Male subadults had more lymphocytes and eosinophils but fewer azurophils in the non-breeding than in the breeding season. Male adults had more heterophils and eosinophils but fewer basophils in the non-breeding than in the breeding season.

As mentioned previously, the immune response in reptiles is highly influenced by the environment, particularly low temperature, which can suppress or inhibit such response (^{Martínez-Silvestre et al., 2011}; ^{Stacy et al., 2011}).

Blood cell morphology and leukocyte differential of the Morelet's crocodile showed both similarities and differences regarding other crocodilians. However, a comparison of blood cell characteristics and leukocyte counts between different populations of Morelet's crocodile was not possible in this study because of the lack of data available on this species, despite its ecological and economic importance in its area of distribution. Therefore, this study contributes novel information towards the establishment of reference values for hematologic evaluation of Morelet's crocodiles. The data generated in this study can be used to diagnose specific diseases associated with abnormal blood cell morphology or increased or decreased leukocyte counts. Nonetheless, it is necessary to conduct more research in farmed and wild individuals to establish differences that may be due to the management of the animals, in order to make an accurate diagnosis of the health status of captive and wild populations. The diagnosis in the crocodiles of some blood disorders related to environmental contamination could also be used to assess the quality of the water used in farmed animals or the ecosystem health in the case of wild individuals.

CONCLUSIONS

The differences observed in blood cell morphology and leukocyte differential count between Morelet's crocodiles from this study and other crocodilians clearly indicate the need to have hematologic information on each species in order to establish specific reference values. Since the animals from this study were apparently healthy, the results obtained here could serve as reference for farmed and wild Morelet's crocodiles. More research is needed in Morelet's crocodile to complement these results in order to establish hematologic reference values for this species, both in the wild and under captivity, as well as to determine which factors might affect and modify the leukocyte differential in crocodilians.