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Research in Veterinary Science 86 (2009) 146–151

Biochemical and hematological reference ranges for Amazonfreshwater turtle, Podocnemis expansa (Reptilia: Pelomedusidae),

with morphologic assessment of blood cells

A.A. Oliveira-Junior a, M. Tavares-Dias b,*, J.L. Marcon a

a Department of Physiological Sciences, Institute of Biological Sciences, Federal University of Amazonas, Av. Gal. Rodrigo Octavio Jordao Ramos,

3000, 69.077-000 Manaus, Amazonas State, Brazilb Health and Biotechnology Institute, Federal University of Amazonas, Estrada Coari-Mamia, 305, 69.460-000 Coari, Amazonas State, Brazil

Accepted 12 May 2008

Abstract

Blood parameters are useful to measure physiological disturbances in chelonians. Thus they can provide important information forthe diagnosis and prognosis of diseases. The aim of this paper was to determine the reference range of plasma glucose, total protein,triglycerides, cholesterol and urea levels, as well as hematocrit, hemoglobin, red blood cell, thrombocyte and white blood cell countsfor healthy Podocnemis expansa (Schweigger, 1812), bred in a turtle farm in the Amazonas State, Brazil. Plasma glucose, hemoglobin,mean corpuscular volume (MCV), lymphocytes and heterophils were the parameters with the smallest variations. Significant (p < 0.001)correlations between red blood cells count and hematocrit and hemoglobin concentration were found. Turtle P. expansa had lympho-cytes, azurophils, heterophils, eosinophils and basophils with morphologic features similar to the ones of others turtles species. No inves-tigated blood parameter was influenced by sex. In further studies, the established reference ranges might be useful for the healthassessment of this turtle species.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Blood; Physiology; Hematology; Leukocytes; Chelonian; Turtles

1. Introduction

The giant turtle from Amazon, Podocnemis expansa

(Schweigger, 1812), is the largest freshwater chelonian ofSouth America, measuring from 75.0 to 107.0 cm in length.This turtle species has a wide geographic distribution inAmazon, inhabiting rivers, lagoons, swamps and floodplains of Brazil, Bolivia, Colombia, Ecuador, Guyana,Peru and Venezuela (Viana et al., 2004). In Brazil, P.

expansa turtles have been used for human feeding sincethe beginning of colonization (Mundim et al., 1999),because its meat and eggs are very appreciated by theAmazon people who live near the forests and rivers. The

0034-5288/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.rvsc.2008.05.015

* Corresponding author. Tel./fax: +55 9735612363.E-mail address: mtavaresdias@ufam.edu.br (M. Tavares-Dias).

Amazon turtle is at risk of extinction (Diniz and Santos,1997), and therefore it is protected by federal law. As aconsequence, the culture of the Amazon turtle was intro-duced in the Amazonas State by environmental authoritiesin 1999, in an attempt to preserve the natural populationsof this species. However, despite all these facts, there is stilllittle information on the physiology of P. expansa, espe-cially regarding their hematological profile.

For wild P. expansa population from Araguaia River(GO, Brazil) some serum biochemical parameters (Mun-dim et al., 1999) and leukocytes percentage (Oliveiraet al., 2000) have been reported, whereas for captive turtlesonly serum biochemical values have been reported (Santoset al., 2005). For wild P. expansa from Amazon, biochem-ical plasma and red blood cells parameters have been stud-ied in a population from Purus River (Marcon et al., 2004).

A.A. Oliveira-Junior et al. / Research in Veterinary Science 86 (2009) 146–151 147

Therefore, no reference range for such hematologicalparameters has yet been established for this important tur-tle species, and data regarding leukocytes are still extremelymodest.

In chelonian species, intraspecific variation has beenreported in different turtle species, because variabilitymay be expected among different individuals (Andersonet al., 1997; Troiano and Silva, 1998; Pires et al., 2006),due to some internal and external factors such as the qual-ity of the aquatic environment, genetic variation, nutri-tional status, sex and age. Within chelonian species,normal values may also vary with geographic localizationof the turtle, age or size, sex, season, diet and activity (Tay-lor and Jacobson, 1982; Bolten and Bjorndal, 1992; Ander-son et al., 1997; Peterson, 2002; Pires et al., 2006; Casal andOros, 2007). In addition, some turtles are capable of greatadaptations (Peterson, 2002), which might make difficult acomparison among chelonian species with very differentlifestyles.

To help assess the causes of disease (Munro et al., 1998;Knotkova et al., 2005, Casal and Oros, 2007) and to eval-uate populations that are potentially at risk, the establish-ment of reference ranges are needed. In addition, assessingthe physiological status of endangered species is a high pri-ority because it helps to develop an appropriate manage-ment for wild as well as for captivity individuals (Boltenand Bjorndal, 1992; Pires et al., 2006). Therefore, the pres-ent study determined the reference ranges of twenty-twoblood parameters for farmed P. expansa turtles. Thus, thisis the most complete report regarding hematologicalparameters for this important chelonian species fromAmazon.

2. Materials and methods

2.1. Turtles and culture conditions

Twenty-eight specimens of healthy P. expansa (9 malesand 19 females) collected from a commercial farm in Man-aus (Amazon State, Brazil) were used for the determinationof blood parameters. The turtles were maintained in pondsof 470 m2. They were feed daily with aquatic macrophites(Pistia stratiotes and Azolla filiculoides) and pelleted fishmeal with 34.0% of crude protein. When the blood sampleswere collected, the water temperature was 28.2 �C and oxy-gen concentration was 3.55 mg/L.

2.2. Blood parameters determination

Each turtle was quickly caught and while it was physi-cally restrained, a blood sample was collected from the cau-dal vessel with syringes coated with sodium heparin(2.500 UI/mL). These samples were used for determiningthe hematological parameters. Red blood cell counts(RBC) were determined optically with a Neubauer cham-ber using a stain solution (38.0 g sodium citrate, 20.0 mL37–40% formalin, 0.2 g toluidine blue, distilled water in

q.s.p 1000.0 mL). Hematocrit (Hct) was determined bymicrohematocrit method, and hemoglobin concentration(Hb) by cyanomethaemoglobin method. From these pri-mary indices resulted the secondary Wintrobe indices, themean corpuscular volume (MCV) and the mean corpuscu-lar hemoglobin concentration (MCHC).

In order to control as many variables as possible and toproduce good-quality blood smears, they were made imme-diately after blood collection and actively air-dried to avoidthrombocyte clumping and morphologic changes. In addi-tion, the blood smears were soon stained with a combina-tion of May Grunwald–Giemsa–Wright (Tavares-Diasand Moraes, 2003). Blood smears were used for totalthrombocyte (TT), total white blood cell (WBC) counts(Tavares-Dias et al., 2008), and WBC differential relativeand absolute counts. Two hundred leukocytes werecounted for each blood smear for differential relative andabsolute counts. They were classified as lymphocytes, azur-ophils, heterophils, eosinophils and basophils. The bloodwas centrifuged and the plasma was used for determiningglucose, total protein, urea, triglycerides and cholesterol,which were measured by commercial kits (Doles, Brazil).

2.3. Statistical analysis

The normality of the data was evaluated by applying theKolmogorov–Smirnov test. The outliers were identifiedand discarded, thus the remaining data yielded a normaldistribution (Horn et al., 2001). For each normally distrib-uted parameter analyzed the mean, the standard deviationand reference ranges (defined as 95% confidence intervals)were calculated. In addition, linear regression was per-formed between RBC, hematocrit and hemoglobin concen-tration, and significance testing was set at 0.05.

3. Results

In order to establish reference ranges, no turtle withlesions on the plastron or carapace was used. The turtlesranged from 0.5 to 12.8 Kg (4.3 ± 3.3 Kg) in weight,straight-line carapace length ranged from 19.0 to 53.0 cm(33.6 ± 10.3 cm) and plastron length ranged from 16.0 to45.5 cm (28.0 ± 9.0).

Reference ranges established for biochemical and redblood cells parameters of P. expansa are given in Table 1.No hematological parameters were influenced by sex. Thenumber of samples tested for each blood parameter is indi-cated at the table. Triglycerides and cholesterol were theparameters with the highest variation, while glucose, hemo-globin and MCV the variables with the smallest variation.Positive correlations (p < 0.001) of red cells count withhematocrit [Hct = 3.319 + (79.052) � (RBC)] and hemo-globin [Hb = 0.623 + (20.607) � (RBC)] (Fig. 1), as wellas between hematocrit and hemoglobin [Hb = 1.066 +(0.209) � (Hct)] (Fig. 2) were found.

In blood smears, mature erythrocytes were homoge-neous in size, shape and color. They were typically oval,

Table 1Biochemical parameters and red blood cells reference range for turtle P. expansa (N = 28), bred in central Amazon

Parameters N Reference range Mean ± SD Min/Max

Total protein (g/dL) 28 3.0–4.0 3.5 ± 1.3 1.2–5.9Glucose (mg/dL) 28 84.9–97.7 91.3 ± 17.7 63.3–134.6Triglycerides (mg/dL) 24 24.6–40.1 35.4 ± 19.7 11.9–70.9Cholesterol (mg/dL) 26 51.0–65.1 58.1 ± 18.3 22.2–90.8Urea (mg/dL) 25 3.0–5.1 4.0 ± 2.6 1.0–9.9RBC (� 106/lL) 28 0.25–0.30 0.28 ± 0.07 0.16–0.45Hct (%) 28 22.6–27.7 25.1 ± 6.9 15.0–41.0Hb (g/dL) 28 5.8–7.2 6.5 ± 1.2 4.1–11.8MCV (fL) 28 866.7–977.9 922.3 ± 150.2 625.0–1250.0MCHC (g/dL) 28 24.2–28.2 26.2 ± 5.4 13.4–42.9

Min = minimum; Max = maximum.

RBC (x 10 μL)6

0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

Hem

og

lob

in (

g/d

L)

0

2

4

6

8

10

12

14

Fig. 1. Relationship of red blood cells (RBC) with hemoglobin (r = 0.845;p < 0.01) in farmed P. expansa (N = 28).

Hematocrit (%)

10 15 20 25 30 35 40 45

Hem

oglo

bin

(g/d

L)

0

2

4

6

8

10

12

14

Fig. 2. Relationship between hematocrit and hemoglobin concentration(r = 0.691; p < 0.001) in farmed P. expansa (N = 28).

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with an oval to irregularly round nuclei that containeddense and dark-staining chromatin. The cytoplasm ofmature erythrocytes was round and acidophilic. Intracyto-plasmic bodies were found in 50% of turtles’ erythrocytes.

Polychromatophilic erythrocytes were also seen. Theircytoplasm was more basophilic. Polychromatophilic eryth-rocyte count was 1.5 ± 1.1% (range from 0.0 to 4.0%) ofthe mature erythrocytes. Parasites of RBC were notobserved in any turtle.

The TT and WBC counts are showed in Table 2. Lym-phocytes and heterophils were the WBC with the smallestvariation. Thrombocytes are round shaped cells, but occa-sionally they can be oval. Their cytoplasm is hyaline andhas no granules, with the nucleus accompanying the shapeof the cell (Fig. 3A). Azurophils are cells with different sizesand shapes, sometimes vacuolated, with the cytoplasm con-taining fine azurophilic granules that varies in amount. Thenucleus is frequently eccentric and spherical (Fig. 3B).Lymphocytes are round and small, with basophilic cyto-plasm and a round nucleus due to its high relationship withthe cytoplasm (Fig. 3C). Eosinophils are round and ofassorted sizes, but relatively smaller than heterophils. Theircytoplasm is rich in eosinophilic granules, and the nucleusis generally eccentric and rarely segmented (Fig. 3D). Het-erophils are predominantly round, with a cytoplasm rich ineosinophilic and basophilic granules. Its nucleus is small,eccentric and occasionally segmented (Fig. 3E). Basophilsare round, with different sizes and a cytoplasm rich in baso-philic granules, which obscure the rounded nucleus(Fig. 3F). Occasionally, degranulated eosinophils, heter-ophils and basophils are also found.

For P. expansa, no difference significant (p > 0.05)between females and males was found for the bloodparameters.

4. Discussion

Studies of physiologic indicators of turtles kept in cap-tivity may provide information about the animals’ healthand therefore, may be used as a quick tool for diagnosis(Troiano and Silva, 1998; Pires et al., 2006). Farmed turtlesmay have values for blood biochemical parameters differ-ent from wild turtles (Bolten and Bjorndal, 1992; Chanda-var and Naik, 2004; Santos et al., 2005), due to artificialdiets or stressful conditions (Bolten and Bjorndal, 1992;Santos et al., 2005). In the present study, plasma glucose

Table 2Total thrombocyte (TT) and white blood cell counts reference ranges for turtles P. expansa (N = 28), bred in central Amazon

Parameters N Reference range Mean ± SD Min/Max

TT (lL) 28 3348.0–4767.0 4058.0 ± 1915.0 2080.0–9900.0WBC (lL) 27 5186.0–6958.0 6072.0 ± 2349.0 2480.0–10450.0Lymphocytes (lL) 24 730.0–996.0 863.0 ± 332.0 343.0–1402.0Lymphocytes (%) 28 14.0–16.7 15.4 ± 3.6 9.0–23.0Azurophils (lL) 25 298.0–458.0 378.0 ± 204.0 101.0–792.0Azurophils (%) 28 5.4–7.2 6.3 ± 2.5 3.0–11.0Heterophils (lL) 27 2482.0–3285.0 2883.0 ± 1064.0 1.017–5141.0Heterophils (%) 28 44.6–51.0 48.0 ± 8.6 35.0–63.0Eosinophils (lL) 27 1124.0–1621.0 1373.0 ± 658.0 504.0–2835.0Eosinophils (%) 28 20.6–24.7 22.6 ± 5.6 13.0–34.0Basophils (lL) 27 364.0–618.0 491.0 ± 337.0 105.0–1386.0Basophils (%) 28 6.7–9.0 7.8 ± 3.0 4.0–14.0

Min = minimum; Max = maximum.

Fig. 3. Blood cells of turtle P. expansa. Thrombocytes (A), azurophil (B),lymphocyte (C), eosinophil (D), heterophil (E) and basophil (F) stainedwith May Grunwald–Giemsa–Wright. Bars = 11 lm.

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concentration was higher than serum glucose concentrationfor wild P. expansa (Mundim et al., 1999; Marcon et al.,2004). However, it was lower than the concentration forthis same species also bred in captivity (Santos et al.,2005). In contrast, plasma protein concentration was simi-lar to the level of serum protein concentration for P.

expansa from Araguaia River (Mundim et al., 1999) andthe ones farmed in Goias State, Brazil (Santos et al., 2005).

In farmed P. expansa, plasma triglyceride and urea lev-els were similar to values reported for this same speciesfree-ranging (Marcon et al., 2004), but cholesterol levelswere higher. In contrast, these values were lower thanserum levels reported by Santos et al. (2005), for P. expansa

also bred in captivity. Farmed turtles may have anincreased cholesterol level due to artificial diet. Besides,farmed turtles are less active than wild populations.

For the farmed P. expansa of this study, the RBC, Hb,Hct and MCHC were similar to the values reported for thissame chelonian free-ranging (Marcon et al., 2004). Onlythe MCV was higher. This indicates that the studied turtleswere kept in adequate conditions. In addition, a positivecorrelation between RBC, Hct and Hb was found for P.

expansa. This indicates a proportional increment betweenthese parameters, as it has been demonstrated also forsnakes Natrix natrix (Wojtaszek, 1991). Probably this alsooccurs in others reptilian species, including otherschelonian.

It has been reported the presence of small intracytoplas-mic inclusions in erythrocytes of desert tortoise Gopherus

agassizii (Alleman et al., 1992) and loggerhead green turtlesCaretta caretta (Casal and Oros, 2007).These inclusions arecommon in the erythrocytes of healthy chelonians (Casaland Oros, 2007) and has been considered degeneratingorganelles (Alleman et al., 1992). In erythrocytes of Caretta.caretta the intracytoplasmic inclusions have basophilicstaining (Casal and Oros, 2007), and similar feature werefound here to erythrocytes of P. expansa.

Polychromatophilic erythrocytes accounted for >1.0%of the erythrocytes of clinically normal Amazon turtles ofthis study. In contrast, for G. agassizii this count was lessthan 1.0% (Alleman et al., 1992). These results suggest thatthe immature erythrocytes may vary among the turtle spe-cies. Polychromatic erythrocytes seem to be present in theperipheral blood of healthy reptiles, but a higher numberof mature erythrocytes occur in direct proportion to theirmaturational stages. Therefore, these proportions may beused as indicators of erythropoietic activity changes, in aregenerative response that can be modulated by anemicprocesses.

Turtle thrombocytes have glycogen granules (Allemanet al., 1992; Work et al., 1998; Casal and Oros, 2007),which are more abundant in the cytoplasm of phagocytes.Reptilian thrombocytes, besides taking part in hemostasis(Pellizzon et al., 2002; Soslau et al., 2005), are also required

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in the defense mechanisms through phagocytosis (Pellizzonand Lunardi, 2000). Studies reporting total thrombocytecounts are few and far between, which in part can be attrib-uted to the lack of a direct and adequate method for thispurpose (Wojtaszek, 1991; Work et al., 1998). InP. expansa, total thrombocytes count was similar to theones of tortoises Chelonoidis chilensis chilensis (Troianoand Silva, 1998), but was lower than the ones of tortoisesGopherus polyphemus (Taylor and Jacobson, 1982) and C.

caretta (Pires et al., 2006). This difference might be dueto species, different counting methods and/or others fac-tors. Sometimes, even when similar counting methods areused for the same species, differences may still occur,because depending of blood collection, thrombocytes tendto show aggregation. Consequently this can influence thethrombocytes count. In reptilian, it has been reported thatheparin led to aggregation of thrombocytes (Salakij et al.,2002). Aggregates accumulate either on the edges of bloodsmears or on counting field and as a consequence, thethrombocytes cannot be counted. Therefore, the best anti-coagulant needs to be determined for the reptilian speciesbeing evaluated. For P. expansa, sodium heparin at2.500 UI/mL presented the best results, because no aggre-gate or clot was found in blood samples.

In the present work, morphological features of leuko-cytes were similar to those reported for wild P. expansa

(Oliveira et al., 2000), except the mononuclear cells. Itwas found here monocytes with azurophilic granules,which were then called azurophils, while Oliveira et al.(2000) did not report monocytes with azurophilic granules.Nevertheless, rarely have monocytes been identified in dif-ferent turtle species, as well as in other reptiles. Therefore,the classification of leukocytes in chelonians has not beenconsistent within a particular species, either because vari-able criteria have been used to categorize cells or becausecellular lineages have been used uncertainly (Allemanet al., 1992; Work et al., 1998; Pires et al., 2006; Casaland Oros, 2007).

Leukocyte counts can indicate activation of the cellularimmune system (Tavares-Dias and Moraes, 2007). Detec-tion of alterations in leukocytes values and the ability topredict the subsequent health state of chelonians is partic-ularly important when the prophylactic management ofinfections is a problem. In the P. expansa of this presentstudy, WBC counts were lower than in Elseya novaeguineae

(Anderson et al., 1997), C. chilensis chilensis (Troiano andSilva, 1998) and Chelonia mydas (Work et al., 1998), how-ever was higher than in C. caretta (Pires et al., 2006). Infarmed P. expansa, most of the WBC (80.0%) were heter-ophils, basophils and eosinophils. Similar findings havealso been reported for this same turtle species from Ara-guaia River (Oliveira et al., 2000). On the other hand, forC. mydas most of the WBC were lymphocytes (Worket al., 1998) or heterophils, lymphocytes and eosinophils(Samour et al., 1998; for E. novaeguineae (Andersonet al., 1997) and C. caretta (Pires et al., 2006; Casal andOros, 2007) it was lymphocytes and heterophils, and for

C. chilensis chilensis eosinophils, heterophils and lympho-cytes (Troiano and Silva, 1998). Therefore, since all thesespecies were healthy, a greater amount of WBC seems toresult in a more efficient functioning of the immune system.This information increased the knowledge of the potentialrole of leukocytes on these turtle species. Furthermore,cytochemical studies might help in the understanding ofthe elimination of infectious agents.

When comparing values between females and males ofP. expansa, there were no differences significant for anyblood parameters studied. Similar findings were reportedfor C. chilensis chilensis (Troiano and Silva, 1998). Incontrast, for E. novaguineae, Hb and Hct were higherin males than females while cholesterol was higher infemales than males (Anderson et al., 1997) and for C.

mydas, cholesterol levels (Bolten and Bjorndal, 1992)and RBC values (Samour et al., 1998) were higher infemales than males.

The results of this study provide important informationon the reference ranges of blood parameters and cells mor-phology of Amazon turtle, P. expansa. These can be usedfor comparison in further investigations of the health statusof this turtle species, allowing a better understanding oftheir physiology. Therefore, further studies are plannedto determine the effects of age, sex, nutritional status andseasons in wild populations, and also to compare animalsfrom different farms.

Acknowledgments

The authors gratefully acknowledge research grantsfrom FAPEAM/ Fundac�ao de Amparo a Pesquisa doEstado do Amazonas (Grant # 2204/05 and 2203/05) andCNPq/Conselho Nacional de Desenvolvimento Cientıficoe Tecnologico (Grant # 35.0117/20005-5).

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