Acta Herpetologica 16(2): 99-108, 2021 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.36253/a_h-11339 Hematological values of wild Caiman latirostris (Daudin, 1802) in the Atlantic Rainforest in Pernambuco, Brazil Luciana C. Rameh-De-Albuquerque1, Alexandre P. Zanotti1, Denisson S. Souza1, George T. Diniz2, Paulo B. Mascarenhas-Junior3,4,5,*, Ednilza M. Santos³, Jozelia M.S. Correia3 1 Parque Estadual de Dois Irmãos, Recife, Pernambuco 52171-011, Brazil 2 Fiocruz, Fundação Oswaldo Cruz - Instituto Ageu Magalhães, Recife, Pernambuco 50670-420, Brazil 3 Laboratório Interdisciplinar de Anfíbios e Répteis da Universidade Federal Rural de Pernambuco, Recife, Pernambuco 52171-900, Brazil 4 Programa de Pós-graduação em Biologia Animal da Universidade Federal de Pernambuco, Recife, Pernambuco 50670-901, Brazil 5 Centro Universitário Brasileiro, UNIBRA, Recife, Pernambuco 50050-230, Brazil *Corresponding author. E-mail: paulobragam16@gmail.com Submitted on: 2021, 14th June; Revised on: 2021, 18th October; Accepted on: 2021, 29th October Editor: Daniele Pellitteri-Rosa Abstract. Hematological studies in crocodilians are important tools in the evolutionary diagnosis and control of sick- nesses, such as anaemia, malnutrition, dehydration, inflammation, and parasitism, among others. We aimed to obtain reference intervals for the hemogram of Caiman latirostris in wild populations that inhabit Recife’s Metropolitan Region, Pernambuco. We obtained blood samples from 42 caimans, from different sexes (22 males and 20 females) and ages classes (eight hatchlings, 24 subadults and 10 adults) in two areas of Atlantic Rainforest domain. We found that hematological parameters were included within the reference intervals for other crocodilian species. It was pos- sible to observe differences between the areas for the mean corpuscular volume values, suggesting a possible difference between adult and juvenile individuals in the two study areas. When comparing sexes, there was no significant differ- ence between the study parameters, but it was possible to observe differences in the mean corpuscular volume, mean corpuscular hemoglobin and hemoglobin in the Estação Ecológica de Tapacurá region. Although small differences have been observed between the two populations, we can infer that the hematological parameters are similar. We can use this information to evaluate animal’s health in nature and for comparations with captive individuals, allowing the establishment of ideal maintenance conditions and assisting in the identification of possible pathologies. Keywords. Broad-snouted Caiman, crocodilians, hematimetric indices, hematology. INTRODUCTION The broad-snouted caiman (Caiman latirostris) occurs in rivers, mangroves and flooded areas in Argen- tina, Bolivia, Brazil, Paraguay, and Uruguay. In Brazil, this species is found in the Cerrado, Caatinga, Atlantic Forest and Pampas biomes, from the coastal region of Rio Grande do Norte, distributed through the basins of the rivers São Francisco and Paraná/Paraguay to Rio Grande do Sul (Coutinho et al., 2013). In the wild, these caimans can live in large aggregations or in small groups and are always present in the mangrove areas of rivers, lagoons, wetlands, and lakes (Filogonio et al., 2010). This species, classified as Least Concern (LC), presents a large geographical area of distribution and an apparent ability to colonise anthropic environments (Júnior et al., 2018). However, anthropic pressure caused by the constant growth of human populations which, to a certain extent, triggers the destruction of their habitat, mainly through the drainage of water bodies, deforestation, pollution, 100 L.C. Rameh-De-Albuquerque et alii intensive pesticide use, as well as illegal hunting in cer- tain regions. These impacts can affect connectivity and consequently gene flow between populations on a micro and macrogeographic scale, with such processes result- ing in the decline of populations (Coutinho et al, 2013; Bassetti and Verdade, 2014). Reference hematological and blood biochemistry values are necessary for detecting the effects of envi- ronmental, infectious, parasitic, or toxicological stress in these animals, providing information on their health and therefore, can be used as a rapid diagnostic tool (Heatley and Russel, 2019). Hematological studies in wild or captive broad-snouted caimans have a scien- tific, educative and production improvement outcomes, and can be applied to conservation, reproduction, and reintroduction projects (Barboza et al., 2006; Adelakun et al., 2017). Blood analysis is a relatively non-invasive method which can provide important clinical data as well as information about the physiological conditions and health of animals (Padilha et al., 2011; Adelakun et al., 2017). The hemogram is comprised of the determina- tion of the total erythrocyte count, hematocrit, hemo- globin concentration and hematological indices such as the mean corpuscular volume, mean corpuscular hemo- globin and mean corpuscular hemoglobin concentration (Saggese, 2009; Heatley and Russel, 2019). The eryth- rogram data serve to aid in the evolutionary diagnosis, characterisation, and control of sicknesses, such as anae- mia, malnutrition, dehydration, inflammation, and para- sitism among others (Barboza et al., 2007; Saggese, 2009; Heatley and Russel, 2019). The clinical interpretation of the leukogram is chal- lenging for many reasons, primarily because of the strict reference intervals, including the total leukocyte count and smear cell percentages which are not available for all species of healthy reptiles (Campbell, 2006; Saggese, 2009; Zayas et al., 2011). Normal hematological values for reptiles, including crocodilians, when determined by different laboratories, demonstrate ample inter and intraspecific variation due to the differences in blood sample collection, handling of the specimen, analysis techniques, physiological state of the reptiles, age, sex, nutrition, population dynamics, environmental condi- tions and use of anaesthetics (Stacy and Whitaker, 2000; Campbell, 2006; Saggese, 2009; Zayas et al., 2011). Ref- erence intervals can be found for several reptile spe- cies, including crocodilians (Stacy and Whitaker, 2000; Padilha et al., 2011; Zayas et al., 2011). Since the mean hematological parameter values can vary between spe- cies, the reference values obtained for healthy animals can serve as a guide for dealing with sick animals (Stacy and Whitaker, 2000). Lastly, the effects on the leukocyte response to bacterial, fungal, viral, and parasitic infec- tions or stress agents have been minimally investigated for this species (Heatley and Russel, 2019). Thus, when referring to a set of reference values, it is important to determine the conditions in which the blood samples were obtained. Understanding and recognising possible variations in hematological results for reptiles contrib- utes to a critical interpretation of the published reference values and their clinical significance. Thus, the aim of this study was to describe the morphological character- istics of the peripheral blood of C. latirostris and estab- lish erythrogram reference indices for this species in wild protected areas in Recife’s Metropolitan Region. MATERIAL AND METHODS Study area Between 2014 and 2015, we collected samples in water bodies located in the municipalities of Recife and São Lourenço da Mata, both belonging to the Recife’s Metropolitan Region (RMR), the main socio-economic centre of the state of Pernam- buco, Brazil. The region is dominated by Atlantic Forest and the climate is tropical and humid, with autumn-winter rains (Alvar- es et al., 2013). Furthermore, the area has a large water network (Carvalho, 2004) with the Capibaribe river, and great influence on RMR, occupying an area of 7,545.88 km2, which represents 7.6% of Pernambuco (APAC, 2020). In the municipality of São Lourenço da Mata, we collected samples in the Estação Ecológica de Tapacurá (EET) reservoir (8°2’S, 35°11’E), a lentic environment in a rural area, formed by the damming of the Tapacurá river. The water body is approximately 7.5 km2 and is surrounded by sugar cane matri- ces (Almeida and Oliveira, 2009), open areas, agricultural are- as, livestock, and riverside communities (Mascarenhas-Junior et al., 2020). It also has 5.35 km2 of forest fragments, of which 1.72 km2 belong to Conservation Units of Integral Protection (Mata do Camucim, Mata do Toró, Mata do Outeiro de Pedro) (CPRH, 2020) (Fig. 1). In the reservoir areas, there are also con- stant fishing activities, with recurring recordings of bycatch of local fauna (Mascarenhas-Junior et al., 2018; Santos et al., 2020). Approximately 25 km from Tapacurá, we also collected samples from caimans located in the Parque Estadual de Dois Irmãos (PEDI) (8°0’S, 34º56’E), in the municipality of Reci- fe, capital of Pernambuco. The area comprises approximately 11.6 km2 of protected forests (Mata de Dois Irmãos, 3.8 km2; Fazenda Brejo dos Macacos, 7.8 km2) and in these fragments there is approximately 1.4 km2 of water bodies forming the Prata microbasin, the Açude do Meio (~0.024 km2), Açude do Prata (~0.025 km2), Açude de Dentro (~0.015 km2) and Açude de Dois Irmãos (~0.135 km2). Despite this area is in an urban environment with anthropic pressures, the presence of a mature marginal forest aids in the maintenance of the water quality (Lima, 2004). In this study, we only accessed the Açude de Den- tro and Açude de Dois Irmãos. 101Caiman latirostris hematological parameters Data collection Captures were performed bimonthly between 2014 and 2015, with active captures during the nocturnal period and with the aid of aluminium boats. The individuals were identified in the environment through the reflection of eyeballs (tapetum lucidum) from the interception of a beam of concentrated light using a spotlight (Magnusson, 1995). The captures were per- formed manually or with the use of cables snares connected to a telescopic pole (up to 5 m). Additionally, aquatic funnel traps with baits to attract the caimans were used. All physical post- capture restraint was performed using adhesive tapes. The caimans were evaluated in terms of trauma, body scars and the absence of clinical signs and symptoms of diseases. Fol- lowing this evaluation, blood samples were collected through supravertebral occipital venous sinus punctures using 20-gauge needles without anticoagulants and disposable needles. Imme- diately following the collection of samples, blood smears were prepared, air dried and stained with modified May-Grünwald- Giemsa colouration (Rosenfeld, 1947). A small volume of blood (0.5 mL) was collected in a microtube containing heparin for the determination of hematological parameters. The total hemo- cyte count (THC), total leukocyte count (TLC) and total throm- bocyte count (TTC) were performed manually in a Neubauer chamber after 10 mL of blood being diluted in 2.0 mL of Natt and Herrick solution. The hematocrit (HT) was determined using the microhematocrit method, through the centrifugation of microcapillaries at 10000 rpm (Coles, 1986) and the hemo- globin concentration (HB) was determined using the cyan- methemoglobin method, mixing 20 mL of blood with 2.5 mL of Drabkin solution (Labtest Diagnostica®). Through conventional calculations, hematometric indices of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpus- cular hemoglobin concentration (MCHC) were calculated. To count the differential leukocytes, a total of 100 cells were count- ed using a microscope under immersion (1000X). After the collection of the blood material, the individuals were measured, weighed, sexed, and marked. The size class was determined through the snout-vent length (SVL), following the proposal by Leiva et al. (2019): Class I or hatchlings (< 25cm); Class II or subadults (25 cm to 67.9 cm); Class III (68 cm to 99.9 cm) and Class IV (over 99 cm) or adults. The weight was determined using scales with a limit of 40 kg. Furthermore, sex was determined for larger individuals using a cloacal palpation technique (Yanosky, 1990) and, in smaller caimans, surgical tweezers were inserted into the cloaca to separate the edges for Fig. 1. Recife’s Metropolitan Region (RMR) with Tapacurá reservoir and Parque Estadual de Dois Irmãos highlighted in white lines. 102 L.C. Rameh-De-Albuquerque et alii the observation of the genitalia (Webb et al., 1984). Lastly, all the individuals were marked with cuts in the scales of the single and double crests and with the subcutaneous implantation of microchips. At the end of data collection, caimans were released back into the water bodies from where they were captured. Data analysis To test the normality and homogeneity of the variables included in the study, we used Shapiro Wilk and Bartlett tests, respectively. For the analyses of the quantitative vari- ables, we used a Bartlett test for homogeneity and the Shap- iro Wilk test for the normality of the variables included in the study. The mean differences for the independent variables were evaluated using an ANOVA followed by Tukey’s post-hoc test when observing the presumption of homogeneity. Otherwise, Kruskal-Wallis tests were used followed by the post-hoc test using Fisher’s criteria of the lowest significant difference (LSD). A student t test was also used to observe when there was a presumption of normality, and when there was not, a Mann- Whitney test was used to evaluate the medians of the variables included in the study. We used categorical analyses for the comparative analysis between the qualitative variables, such as Pearson’s χ2 test andm when necessary, Fisher’s exact test. All the conclusions were taken at a significance level of 5%. The R software (R Core Team, 2021) was used for the evaluation of the study results. RESULTS Cells morphological aspects The erythrocytes presented an elliptical shape, with abundant cytoplasm, acidophilic, pinkish in colour, occupying approximately 80% of the cell. The nucleus presented condensed chromatin, basophilia, predomi- nantly elliptical and occupies a central position in the cell. Larger ery throcy tes with spherical nuclei and more rounded forms were also occasionally found (Fig. 2a). No intracellular inclusions or hemoparasites were observed. The thrombocytes were predominantly elliptical with abundant cytoplasm, hyaline, also presenting a less elongated shape with little cytoplasm. Eventually, azuro- philic granules were observed in the cytoplasm. The pre- dominantly elliptical nucleus can present morphological variations, with chamfers, indentation or grooves, violet in colour with a central location (Fig. 2a, 2f and 2h). The lymphocytes presented a large variation in terms of size and shape. The presence of irregularly shaped or spherical cells was common. The cytoplasm is scarce, basophilic, with azurophilic granules, commonly exhibiting cytoplasmic projections (Fig. 2a, 2b and 2g). The monocytes presented a spherical to oval shape, often presenting an irregular outline (Fig. 2b, 2c and 2i). The pale to moderately basophilic cytoplasm may con- tain cytoplasmic vacuoles of varying sizes. The nucleus may be spherical, oval or even U-shaped, generally with irregular outlines, occupying an off-centre position. The eosinophils generally have a spherical shape. The cytoplasm is abundant and homogenously filled by acidophilic granules which are compact, spherical, oval or slightly elongated, pinkish in colour with a relatively homogenous colour. The nucleus is violet in colour and is generally spherical or lenticular and is in an off-centre position and may often be bilobulated (Fig. 2d and 2f). The heterophils are large and are spherical, oval or irregular. The cytoplasm is generally abundant, full of compact granules whose acidophilia varies in its inten- sity, depending on the granule aspect and is of a dark pink or salmon colour. In terms of their morphology, the granules have varying aspects, both in terms of their colour intensity and their shape, where they can be spherical, fusiform with intense colouration or even stick, drumstick or oval shaped. There is a clear predom- inance of fusiforms compared to the others. The spheri- cal nucleus is located off-centre or peripherally (Fig. 2d, 2e and 2g). The basophils have a spherical shape and are smaller when compared to the other granulated leukocytes. The cytoplasm presents strongly basophilic spherical gran- Fig. 2. Photomicrographs of Caiman latirostris blood cells coloured with modified May-Grunwald-Giemsa (Rosenfield, 1947) (mag- nification 1000X). (a) Mature erythrocytes in high quantity, lym- phocytes (arrow), thrombocytes (head of arrow); (b) monocytes (arrow), lymphocyte (head of arrow); (c) monocyte; (d) eosinophil (arrow), heterophil (head of arrow); (e) heterophil; (f ) eosinophil; (g) heterophile (arrow), thrombocytes (head of arrow), lymphocyte (outlined arrow); (h) thrombocytes; (i) monocytes with vacuoles. 103Caiman latirostris hematological parameters ules of varying sizes and when located on top of the nucleus it can be impossible to distinguish between their outlines. General parameters of caimans Blood samples from 42 caimans were collected, with 22 from PEDI and 20 from EET. The sex proportions of the captured individuals were 22 males (PEDI = 12, EET = 10, 52.38%), 18 females (PEDI = 10, EET = 8, 42.86%) and 2 undetermined individuals (4.76%). From the total number of animals, 10 were adults (PEDI = 3, EET = 7, 23.9%), eight hatchlings (PEDI = 5, EET = 3, 19.0%) and 24 subadults (PEDI = 14, EET = 10, 57.1%). Between the two locations it was possible to observe differences in the capture of different age class- es, with a greater number of adults in the EET and more subadults in PEDI. The biometric and hematological parameters col- lected for Caiman latirostris in the two locations are presented in Table 1. The SVL varied from 17.30 cm to 100.04 cm and the weight varied from 114.2 g to 51,380 g. It was possible to observe differences in the hemato- logical parameters for the different areas for the values of MCV (P = 0.04), thrombocytes (P ≤ 0.01), monocytes (P = 0.01), basophils (P = 0.01) and eosinophils (P = 0.02; Table 1). The hematological data and their reference inter- vals for the different age groups are established in Table 2 and Table 3. The MCV values did not indicate differ- ence between adult and subadult individuals (P = 0.06). In the comparisons between the areas, differences in the values for hatchling thrombocytes (P = 0.04) and adult eosinophils (P = 0.03) were identified (Table 2 and Table 3). For the comparison between the sexes in the sam- ples, there was no significant difference between the hematological parameters, with the only significant dif- ference being for TL (P = 0.04). However, when perform- ing this evaluation between individuals of the same area, it was possible to observe differences in the red blood cells (P = 0.02), MCV (P < 0.01), MCH (P = 0.01) and monocytes (P = 0.01) in Tapacurá and for the hemo- globin values in the PEDI (P = 0.02) with higher values for red blood cells in females. The other parameters, MCV, MCH, monocytes and hemoglobin, were higher for males (Table 4 and Table 5). DISCUSSION In general, the hematological parameters found for Caiman latirostris in wild environments in the State of Pernambuco fall within the reference intervals for other species of crocodilians (Stacy and Whitaker, 2000; Padil- Table 1. Hematological reference values for wild Caiman latirostris and differences between the two study areas (Pernambuco, Brazil). SD (Standard deviation), P (p value), SVL (Snout-Vent Length), TL (Total Length), TEC (Total Erythrocyte Count), TLC (Total Leukocyte Count), HB (Hemoglobin), Ht (Hematocrit), MVC (Mean corpuscular volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), TTC (Total Thrombocyte Count). Parameter PEDI (n = 22) EET (n = 20) P Mean SD Range Mean SD Range SVL (cm) 47.2 36.14 17.3-100 58.88 25.5 17.8-100.04 0.09 TL (cm) 81.77 41.62 36-209 106.31 60.64 28-211 0.30 Weight (g) 5287 11934 125-51380 8140 12030 114.2-38300 0.42 TEC (103 cells/mm3) 178.86 73.58 30-355 219.75 63.58 120-345 0.06 TLC (103 cells/mm3) 5.23 2.87 0.75-11.75 3.84 2.01 1.25-8.25 0.07 HB (g/dl) 6.85 0.82 5.2-8.0 7.22 0.6 5.8-8 0.11 Ht (%) 20.18 6.13 3-29 21.5 3.61 15-29 0.40 MCV (fl) 1.53 1.18 0.76-5.45 1.02 0.18 0.75-1.42 0.04 MCH (pg) 0.5 0.4 0.22-2 0.35 0.09 0.22-0.59 0.15 MCHC (%) 42.66 40.91 25-223.3 33.9 4.33 22.3-41.76 0.67 TTC (103 cells/mm3) 2.98 1.94 0.5-8.37 4.5 1.59 1.5-7 0.01 Lymphocytes (%) 54.32 12.37 33-81 51.5 12.95 31-77 0.48 Monocytes (%) 5.18 2.72 1.0-9.0 3 2.18 0-9 0.01 Basophils (%) 2.73 1.75 0-5 1.4 1.79 0-8 0.01 Heterophils (%) 34.82 12.94 6.0-60.0 41.8 13.83 9.0-63.0 0.10 Eosinophils (%) 2.95 1.73 0-6 1.8 1.4 0-5 0.02 104 L.C. Rameh-De-Albuquerque et alii ha et al., 2011; Zayas et al., 2011; Bassetti and Verdade, 2014). Some factors that may have affected the results obtained in previous studies may be related to the meth- odology used, the environmental conditions and the diet of the population sampled. Among the hematological parameters, it was pos- sible to observe differences between the areas in the MCV values. These values suggest a possible difference between adult individuals and subadults between the two study areas. The causes of physiological changes in Table 2. Hematological reference values for different age groups of wild Caiman latirostris (Parque Estadual de Dois Irmãos, Pernambuco, Brazil). SD (Standard deviation), P (p value), SVL (Snout-Vent Length), TEC (Total Erythrocyte Count), TLC (Total Leukocyte Count), HB (Hemoglobin), Ht (Hematocrit), MVC (Mean corpuscular volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), TTC (Total Thrombocyte Count). Parameter Adults (n = 3) Hatchlings (n = 5) Subadults (n = 14) P Mean SD Range Mean SD Range Mean SD Range SVL (cm) 99.13 62.39 70-109 22.17 6.48 17.3-21.0 44.25 4.83 32.4-50.0 <0.01 Weight (g) 23553 21138 3834-51380 330 370.63 125-1140 1799 965.94 166-3210 <0.01 TEC (103 cells/mm3) 171.25 101.77 30-255 175.71 26.21 150-210 183.64 88.29 55-355 0.28 TLC (103 cells/mm3) 5.87 3.05 1.5-8.5 4.86 1.04 3.5-6.5 5.23 3.69 0.7-11.75 0.34 HB (g/dl) 6.45 0.7 6-7.5 7.11 0.73 5.8-8 6.84 0.91 5.2-8 0.44 Ht (%) 18.5 3.42 14-22 21.57 4.72 12-26 19.91 7.71 47178 0.80 MCV (fl) 1.88 1.87 0.76-4.67 1.24 0.28 0.80-1.73 1.58 1.31 0.82-5.45 0.22 MCH (pg) 0.73 0.85 0.26-2 0.41 0.03 0.37-0.46 0.48 0.3 0.22-1.21 0.16 MCHC (%) 35.45 5.74 30.5-42.86 34.21 6.8 26.54-48.34 50.66 57.78 25-223 0.86 TTC (103 cells/mm3) 2.75 2.01 0.75-5.5 2.54 0.82 1-3.5 3.35 2.45 0.5-8.37 0.04 Lymphocytes (%) 43.25 8.88 35-55 59.29 8.12 48-68 55.18 13.82 33-81 0.22 Monocytes (%) 5.25 3.5 1-9 4.57 2.94 2-9 5.55 2.5 2-9 0.07 Basophils (%) 3.25 2.22 0-5 2.29 1.5 0-5 2.82 1.83 0-5 0.11 Heterophils (%) 44 7.12 38-52 30.43 9.25 18-45 34.27 15.41 6-60 0.28 Eosinophils (%) 4.25 0.96 3-5 3.43 1.62 2-6 2.18 1.72 0-4 0.03 Table 3. Hematological reference values for different age groups of wild Caiman latirostris (Estação Ecológica de Tapacurá, Pernambuco, Brazil). SD (Standard deviation), P (p value), SVL (Snout-Vent Length), TEC (Total Erythrocyte Count), TLC (Total Leukocyte Count), HB (Hemoglobin), Ht (Hematocrit), MVC (Mean corpuscular volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), TTC (Total Thrombocyte Count). Parameter Adults (n = 7) Hatchlings (n = 3) Subadults (n = 10) P Mean SD Range Mean SD Range Mean SD Range SVL (cm) 74.71 18.39 70-100.4 41.1 35.12 17.8-24.0 44.24 14.97 59.0-67.5 <0.01 Weight (g) 15495 13641 1015-38300 709.73 827.8 114-216 829 512.35 540-1740 <0.01 TEC (103 cells/mm3) 223.64 47.17 130-290 246.25 70.28 185-345 190 89.93 120-345 0.28 TLC (103 cells/mm3) 4.34 1.92 1.75-7.75 3.19 0.24 3-3.5 3.25 2.9 1.25-8.25 0.34 HB (g/dl) 7.19 0.65 5.8-8 7.4 0.54 6.9-8 7.12 0.61 6.2-7.8 0.44 Ht (%) 21.45 3.24 15-26 23.25 4.35 19-29 20.2 4.02 16-26 0.80 MCV (fl) 0.98 0.15 0.75-1.26 0.96 0.08 0.84-1.02 1.15 0.25 0.75-1.42 0.22 MCH (pg) 0.33 0.05 0.27-0.45 0.31 0.06 0.22-0.37 9.42 0.13 0.22-0.59 0.16 MCHC (%) 33.91 3.37 28.33-33.33 31.33 6.16 22.33-36.31 35.92 4.51 30-41.76 0.86 TTC (103 cells/mm3) 4.02 1.5 1.5-6 6.12 0.77 5.25-7 4.25 1.61 1.75-5.75 0.04 Lymphocytes (%) 54.09 13.68 37-77 53.5 14.84 32-66 44.2 8.64 31-55 0.22 Monocytes (%) 2.18 1.54 0-5 4.25 1.5 3-6 3.8 3.27 1-9 0.07 Basophils (%) 1 1.1 0-3 1 0 1 2.6 3.13 0-8 0.11 Heterophils (%) 40.36 15.59 9-59 39 16.06 30-63 47.2 7.6 35-54 0.28 Eosinophils (%) 1.45 0.82 0-3 2.25 2.22 0-5 2.2 1.79 0-4 0.03 105Caiman latirostris hematological parameters erythrogram parameters for reptiles are numerous. With increasing age, the total erythrocyte count, the MCV, MCH, MCHC, hematocrit and hemoglobin, tend to increase (Heatley and Russel, 2019). A relative increase in the total erythrocyte count, hemoglobin and/or hematocrit occurs in males of some Table 4. Hematological reference values for male and female wild Caiman latirostris (Parque Estadual de Dois Irmãos, Pernambuco, Brazil). SD (Standard deviation), P (p value), SVL (Snout-Vent Lenght), TL (Total Length), TEC (Total Erythrocyte Count), TLC (Total Leukocyte Count), HB (Hemoglobin), Ht (Hematocrit), MVC (Mean corpuscular volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), TTC (Total Thrombocyte Count). Parameter Males (n=12) Females (n=10) P Mean SD Range Mean SD Range SVL (cm) 37.77 25.43 17.3-100 49.51 13.3 42-86.50 0.07 TL (cm) 74.21 51.07 36-209 90.85 26.18 75.5-163 0.11 Weight (g) 5944 14631 125-51380 4498 8332 166-28000 0.22 TEC (103 cells/mm3) 203.33 39.9 150-255 149.5 94.5 30-355 0.12 TLC (103 cells/mm3) 5.77 2.3 2.75-11 4.57 3.45 0.75-11.7 0.36 HB (g/dl) 7.21 0.69 5.8-8 6.43 0.78 5.2-8 0.02 Ht (%) 22.25 4.22 12-28 17.7 7.3 3-29 0.10 MCV (fl) 1.12 0.27 0.76-1.73 2.01 1.64 0.82-5.45 0.12 MCH (pg) 0.36 0.07 0.26-0.46 0.67 0.56 0.22-2 0.21 MCHC (%) 33.3 5.6 25.71-48.34 53.9 60.09 25-223 0.62 TTC (103 cells/mm3) 2.77 1.13 1-5.75 3.23 2.67 0.5-8.37 0.69 Lymphocytes (%) 56.17 12.38 38-81 52.1 12.64 33-73 0.46 Monocytes (%) 5.08 3 1-9 5.3 2.5 2-9 0.85 Basophils (%) 2.67 1.56 0-5 2.8 2.04 0-5 0.87 Heterophils (%) 32.92 13.06 6-52 37.1 13.09 17-60 0.46 Eosinophils (%) 3.16 1.99 0-6 2.7 1.42 0-4 0.53 Table 5. Hematological reference values for male and female wild Caiman latirostris (Estação Ecológica de Tapacurá, Pernambuco, Brazil). SD (Standard deviation), P (p value), SVL (Snout-Vent Lenght), TL (Total Length), TEC (Total Erythrocyte Count), TLC (Total Leukocyte Count), HB (Hemoglobin), Ht (Hematocrit), MVC (Mean corpuscular volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), TTC (Total Thrombocyte Count). Parameter Males (n=10) Females (n=8) P Mean SD Interval Mean SD Interval SVL (cm) 67.73 27.31 28.5-100.04 59.75 14.14 41-73.5 0.49 TL (cm) 104.33 78.14 28-211 124.3 25.24 82-147 0.50 Weight (g) 12032 15975 540-38300 5584 4447 1015-11260 0.57 TEC (103 cells/mm3) 192.5 67.17 120-345 259.37 46.17 195-345 0.02 TLC (103 cells/mm3) 3.72 2.34 1.25-8.25 4.15 1.9 1.75-7.75 0.67 HB (g/dl) 7.04 0.69 5.8-8 7.5 0.44 7-8 0.11 Ht (%) 20.8 4.29 15-29 22.75 2.87 18-26 0.27 MCV (fl) 1.12 0.17 0.84-1.42 0.89 0.11 0.75-1.02 0.00 MCH (pg) 0.39 0.1 0.22-0.59 0.29 0.04 0.22-0.35 0.01 MCHC (%) 34.21 5.54 22.33-41.76 33.28 3.16 30-40 0.66 TTC (103 cells/mm3) 4.62 1.66 1.75-7 4.09 1.67 1.5-6 0.51 Lymphocytes (%) 47.1 10.88 31-66 58.75 12.52 44-77 0.06 Monocytes (%) 3.9 2.28 1-9 1.5 1.07 0-3 0.01 Basophils (%) 1.8 2.3 0-8 1.25 1.2 0-3 0.49 Heterophils (%) 45 10.87 30-59 36.25 15.56 9-54 0.20 Eosinophils (%) 2.2 1.69 0-5 2.25 0.89 0-3 0.15 106 L.C. Rameh-De-Albuquerque et alii reptile species. However, many species may not corre- spond to this expectation (Heatley and Russel, 2019). In this study, most parameters did not present significant differences between males and females, but we observed differences in the MCV, MCH and hemoglobin values, which were higher in males, whereas the total erythro- cyte count was higher in females in the EET region. The main aims of the blood smear readings include the differentiation of cell types, the evaluation of cell morphology, the observation of anomalies in cell mor- phology and the observation of cell inclusions or extra- cellular anomalies, such as hemoparasites (Heatley and Russel, 2019). In this study, no intracellular inclusions nor hemoparasites were found. This study corroborates the findings of Basset (2016) for C. latirostris and Moura et al. (1999) for Caiman yac- are, in relation to the types of cells found in peripheral blood. The leukocytes were classified as lymphocytes, azurophilic monocytes, eosinophils, heterophils and basophils. For many authors, azurophils are a variation of monocytes, as can be observed (Zayas et al., 2011), for the same species, whereas other researchers recommend that these cells could be counted separately in snakes but should be grouped for other reptile species (Moura et al., 1999). In this study all the cells were grouped as as monocytes, and we observed we observed differences in the number of monocytes between areas, with the PEDI presenting higher values compared to the EET. Likewise, when comparing sexes within the same area, males in the EET presented higher values than females. In relation to the morphological characteristics of the other leukocytes, this study corroborates the find- ings described for other species of crocodilians (Moura et al., 1999; Stacy and Whitaker, 2000; Padilha et al., 2011; Zayas et al., 2011). The reptile heterophile has larg- er cytoplasmic granules, despite being fewer in number, compared to lizards and snakes (Clever and Quaglia, 2009; Sacchi et al., 2011). The lymphocytes are the most common leukocytes, accounting for up to 80% of the differential count in healthy reptiles (Heatley and Russel, 2019). In this study the lymphocytes were the most numerous leukocytes, followed by heterophils, monocytes, eosinophils, and basophils, which differs from the results found by Zayas et al. (2011), for the same species. They observed a higher number of lymphocytes followed by heterophils. How- ever, they found more basophils compared to monocytes and eosinophils. Stress factors may be responsible for the increase in total leukocyte count and differential count; however, the extent of these changes has not been com- prehensively investigated in reptiles. With a few excep- tions, we lack fundamental knowledge on the timing of inflammation and associated cellular responses in rep- tiles (Heatley and Russel, 2019). Although we attempted, in this study, to relate the differences found between ages, sex and sample areas, additional factors should be considered, as the influ- ence of environmental parameters such as climate, sea- sonal period, nutritional state and population dynamics (Moura et al., 1999; Stacy and Whitaker, 2000; Heatley and Russel, 2019). This study corroborates the idea presented by Bas- set (2016) where the results obtained from experiments involving blood hematology and biochemistry param- eters should always be considered as a diagnostic tool for an animal’s or even of a population’s state of health. As affirmed by Stacy and Whitaker (2000) in their study on Crocodylus palustris, the differences found may not be a true reflection of the differences between spe- cies, therefore making it difficult to come to any firm conclusion about the biological significance of these dif- ferences. However, it is still worth considering this data when interpreting the blood parameters of one species, since these are the best data available currently. ACKNOWLEDGMENTS We would like to thank the PEDI management and the EET manager and crew for making field trips and sample collections possible. We also thank the Fundação de Amparo a Ciência no Estado de Pernambuco (FACE- PE) for funding’s (APQ 0245/2.04-15), the Sistema de Autorização e Informação em Biodiversidade (SISBIO) for the sampling license (39929-2) and Ethics and Ani- mal Use Committee from Universidade Federal Rural de Pernambuco (CEUA- UFRPE) for authorizing the execu- tion of the project (license number 068/2014). 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Acta Herpetologica Vol. 16, n. 2 - December 2021 Firenze University Press A new species of the genus Noblella (Amphibia: Strabomantidae) from Ecuador, with new information for Noblella worleyae Carolina Reyes-Puig1,2,3,4,*, Juan M. Guayasamin 2,5 Claudia Koch6, David Brito-Zapata1, Matthijs Hollanders7, Melissa Costales8, Diego F. Cisneros-Heredia1,2,3 So close so different: what makes the difference? Dario Ottonello1,7,*, Stefania D’Angelo2, Fabrizio Oneto3,6, Stefano Malavasi1, Marco Alberto Luca Zuffi4, Filippo Spadola5 Hematological values of wild Caiman latirostris (Daudin, 1802) in the Atlantic Rainforest in Pernambuco, Brazil Luciana C. Rameh-De-Albuquerque1, Alexandre P. Zanotti1, Denisson S. Souza1, George T. Diniz2, Paulo B. Mascarenhas-Junior3,4,5,*, Ednilza M. Santos³, Jozelia M. S. Correia3 Bone histology of Broad-snouted Caiman Caiman latirostris (Crocodylia: Alligatoridae) as tool for morphophysiological inferences in Crocodylia Paulo Braga Mascarenhas-Junior1,2,3,6, Luis Antonio Bochetti Bassetti4, Juliana Manso Sayão5,6 Is the Northern Spectacled Salamander Salamandrina perspicillata aposematic? A preliminary test with clay models Giacomo Barbieri, Andrea Costa, Sebastiano Salvidio* Sexual size dimorphism in the tail length of the Caspian Whip Snakes, Dolichophis caspius (Serpentes, Colubridae), in south-western Hungary György Dudás1, Krisztián Frank2* Semi-automated photo-identification of Bahamian Racers (Cubophis vudii vudii) Sebastian Hoefer1,*, Andreu Rotger2, Sophie Mills1, Nathan J. Robinson1,3