BRIEF COMMUNICATION

Hematologic values and blood biochemistry of anesthetizedpudu (Pudu puda) in a semi-captive population

O. Alejandro Aleuy & Miguel Manríquez &

Rodrigo Jiménez & Verónica Arnés & Mirela Noro

Received: 22 April 2012 /Accepted: 6 February 2013 /Published online: 23 February 2013# Springer-Verlag London 2013

Abstract Southern pudu (Pudu puda) is one of the smallestdeer in the world, categorized as vulnerable by the IUCN.Establishment of reference values for hematologic and bio-chemical guidelines is fundamental for monitoring healthand physiological status of animals. The aim of this studywas to describe ranges for hematological and plasma bio-chemical guidelines in pudu. Blood samples were collectedfrom 29 anesthetized pudus (21 female and 8 males; 5juveniles and 24 adults) from a semi-captive population.Hematologic parameters, plasma concentrations of energy,protein and mineral guidelines, as well activity of enzymesmarkers of hepatic and muscular damage were determined.Mean, median, and reference interval hematologic and bio-chemistry guidelines were presented. No significant differ-ences between genders for any parameter were observed,except plasma calcium concentrations. The plasma concen-trations of total protein, globulin, triacylglyceride, and chlo-rine were higher in adult than young pudus. Values obtainedin this study will be employed to build a database forestablishing reference limits for this species.

Keywords Hematology . Clinical biochemistry . Pudu .

Captive . Pudu puda

The southern pudu (Pudu puda) is one of the smallest deerin the world with an adult size of about 40 cm in height and9 kg in weight (Silva-Rodríguez et al. 2011). The puduinhabits the South American temperate rainforest and iscategorized as vulnerable. The distribution and density ofpudus populations have declined during the last decades.The main causes for the decline are forest loss, predation bydomestic dogs, and poaching (Silva-Rodríguez et al. 2010).The current scenario has made necessary the development ofex situ and in situ conservation initiatives. Therefore, cap-tures, translocations, rehabilitations of injured pudus, andreleases from both breeding centers and confiscated animalsare an increasingly common practice (Silva-Rodríguez et al.2011). In order to not affect the health of wild populationsand improve the management of captive animals, it becomesnecessary to gather information that allows the establish-ment of physiological responses in this species (Silva-Rodríguez et al. 2011).

The blood participates directly or indirectly in almost allbiochemical processes in the body; therefore, the correctevaluation of biochemical and hematological values is im-portant for monitoring health status and diagnosis of dis-eases (Kaneko et al. 2008; Boes 2010). Many articles onhematological and biochemical serum values in differentdeer species have been published (Chapple et al. 1991;English and Lepherd 1981; Marco and Lavin 1999; Mautzet al. 1980; Uhart et al. 2003; Vengušt et al. 2006; Muneratoet al. 2010; Szabo et al. 2005), but there is no informationabout hematology and clinical biochemistry on the southernpudu. The objective of this study was to describe hemato-logical values and plasma concentrations of biochemicalvariables in semi-captive pudus population.

The study was conducted between late fall and earlywinter of 2009 in 29 pudus (8.4±1.7 kg bw, 21 femalesand 8 males, 5 juvenile animals (>3 months and ≤1 year),and 24 adults (>1 year), maintained in a 2-ha enclosure inthe province of Valdivia (39°43′ S, 73°01′ W), Chile.

O. A. AleuyCentro de Rehabilitación de Fauna Silvestre (CEREFAS), Institutode Patología Animal, Universidad Austral de Chile, Casilla 567,Valdivia, Chile

M. Manríquez :R. Jiménez :V. ArnésInstituto Ciencias Clínicas Veterinarias,Universidad Austral de Chile (UACh), Casilla 567,Valdivia, Chile

M. Noro (*)Faculdade de Agronomia e Medicina Veterinaria,Universidade de Passo Fundo (UPF),Passo Fundo, RS, Brasile-mail: mirelanoro@gmail.com

Comp Clin Pathol (2013) 22:529–533DOI 10.1007/s00580-013-1700-1

Animals were fed shrubs (Fuchsia magellanica andAristotelia chilensis) and balanced supplement (min crudeprotein 13 %, ether extract 3 %, max crude fiber 15 %) andprovided with water ad libitum. Each animal was herded to asmall enclosure (9 m2), captured by net, and anesthetizedwith 0.6 mg/kg i.m. of xylacin (Xilacina®, 2 %, CentrovetLab) and 6 mg/kg i.m. of zolazepam/tiletamine (Zoletil 50®,Virvac Lab SA) given by hand syringe. Once in lateralrecumbency, blood samples were obtained from the lateralsaphenous vein using a 2-mL tube with EDTA for hemato-logical analysis and a 3-mL heparin tube for serum bio-chemistry. Samples for biochemical analysis werecentrifuged at 800×g for 10 min and the serum obtainedwas frozen at −20 °C for later examination.

Hematological analyses were performed at the ClinicalPathology Laboratory located in the Veterinary MedicalTeaching Hospital, Universidad Austral de Chile. Hematocritwas measured using the microhematocrit capillary methodafter centrifuging for 5 min at 16,000×g. The hemoglobinconcentration was determined by cyanomethemoglobinmethod using Hitachi®, Photometer 4020 (BoehringerMannhein). Erythrocyte and leucocyte counts wereobtained with a Neubauer chamber. Differential leucocytecounts and erythrocyte morphology were performed onblood-stained smears using a Romanowski stain (Corzap1, Hemogram®) at 1,000× (Olympus CX31®). Meancorpuscular volume and mean corpuscular hemoglobinconcentrations were calculated by Wintrobe indexes.Fibrinogen was determined by a refractometer after plasmaprecipitation at 56 °C by 3 min and centrifugation for 5 minat 16,000×g.

Plasma concentration of total protein, albumin, globulin,urea, total bilirubin, glucose, triacylglycerides, cholesterol, cre-atinine, phosphorus, and chlorine was analyzed using Humankits (Human®) and lactate using Sentinel kit (Sentinel CH®).Plasma activities of aspartate aminotransferase (AST; EC,2.6.1.1), alanine aminotransferase (ALT; EC, 2.6.1.2), gammaglutamyl transferase (GGT; EC, 2.3.2.2), alkaline phosphatase(SAP; EC, 3.1.3.1), amylase (EC, 3.2.1.1), and creatine kinase(CK; EC, 2.7.3.2) were analyzed by Human kits; lipase (EC,3.1.1.3) by Sentinel kits; and glutathione peroxidase (GPx; EC,1.11.1.9) by Ransel (Randox®). All biochemical analysis wasquantified using an autoanalyzer (Metrolab 2300®, WienerLab). Calcium, magnesium, potassium, and sodium plasmaconcentrations were determined by atomic absorption spec-trometry (Solaar Series S®, Thermo).

The distribution of the data was analyzed and referenceintervals were estimated according the American Society ofVeterinary Clinical Pathology (Friedrichs et al. 2012) usingthe software MedCalc v.12.1.4 (MedCalc Software,Belgium). Differences between sexes and ages were testedusing a t test for that fitted a normal distribution and anonparametric Mann–Whitney U test otherwise, with

significance set at P<0.05. Statistical analysis was performedusing the Statistix 8.0 (NH Analytical Software, Roseville,MN, USA). Some values were measured in fewer than the 29total samples owing to insufficient volume of blood.

At microscopical examination, it draws attention the sickleshape of its red blood cells (Fig. 1). In other deer species suchas mule deer (Odocoileus hemionus), white-tailed deer(Odocoileus virginianus), and chital deer (Axis axis), thatshape takes place in vitro and is mediated by O2 and pH actionon the different hemoglobin types (Boes 2010). The erythro-cytes were small, with 2.5 to 5.0 μL length and 1.0 to 2.1 μLwidth, observations on several deer species have revealed adirect relationship between erythrocyte size and body size inmembers of the Cervidae (Boes 2010).

Values for erythrocytes, hematocrit, and hemoglobinobtained (Table 1) were lower than those reported in otherdeer species such as gray brocket deer (Mazama gouazou-bira), marsh deer (Blastocerus dichotomus), white-taileddeer (O. virginianus), chital deer (A. axis), and pampas deer(Ozotoceros bezoarticus celer), both physically and chemi-cally restrained (Chapple et al. 1991; English and Lepherd1981; Munerato et al. 2010; Szabo et al. 2005; Uhart et al.2003; Vengušt et al. 2006; Boes 2010). These can be relatedto the small size of the species and the confidence that semi-captive animals have to human presence, reducing the effectof spleen contraction and release of red cell into the circu-lation (Vengušt et al. 2006). Furthermore, probably therelaxin effect of xylacine used in our anesthetic protocolmay bypass the excitement stage and results in a resting ornear resting level of these values (Munerato et al. 2010).

The leukocyte morphology was similar to that described forother deers (Chapple et al. 1991; English and Lepherd 1981;Uhart et al. 2003). The leukocyte count was 8,176±2,139cells/μL, with predominance of lymphocytes. The neutrophilsshowed three to five segments and monocytes were the largestcells with 9.2 to 12.8 μL in diameter. Hematological parametershowed no significant differences between genders or age.

The values of plasma concentration and biochemical activ-ities are summarized in Table 2. No significant differences for

Fig. 1 “Sickle” form of pudu erythrocytes. ×1,000

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any parameter were observed between genders, except calce-mia (female=2.2±0.3 mmol/L, male=2.5±0.3 mmol/L; P<0.05), probably due to pregnancy of some females, that couldreduce plasma concentrations of calcium (Kaneko et al. 2008).The plasma concentrations of total protein, globulin, triacyl-glyceride, and chlorine were higher in adult than young pudus(Fig. 2).

Total plasma protein, albumin, globulin, and fibrinogenwere lower than that found in other deer species, unlike ureathat showed higher concentrations (Mautz et al. 1980; Uhart etal. 2003). This result might reflect the presence of a balancedsupplement in diet and the high protein content of plants insouthern Chile, which increases the production and absorptionof ruminal ammonium and, thus, the consequent increase ofthe ureagenesis in liver (Kaneko et al. 2008).

The creatinine plasma concentration was lower than infree range O. bezoarticus celer and O. virginianus andslightly higher than captive M. gouazoubira probably dueto nutrition and exercise of pudu under semi-captivity andthe direct relationship between muscular mass and creati-nine muscle production (Marco and Lavin 1999; Szabo et al.2005). It is important to consider that animals in this studyhad the possibility to maintain individual (i.e., feeding,exploration) and social (i.e., reproduction, breeding, andterritoriality) behaviors, without human intervention, thatcould have influence on more muscular animals.

Glycemia as well as AST and CK activities were higherthan that reported in other deer species. Moreover, lactateplasma concentration was higher than bovine or sheep

reference values (Kaneko et al. 2008). These results maybe related to the comparative nervous temperament of pudu,where the hyperglycemic effect of both, epinephrine re-leased in a physically stressed animal and the use of α-2-adrenergic agonists (xylazine) in chemical immobilization,can produce variation in glucose and muscular metabolism(Munerato et al. 2010; Vengušt et al. 2006). Elevations ofglycemia and serum muscle enzyme activity in deer that arenot used to handling have been reported previously(Chapple et al. 1991; English and Lepherd 1981) and couldbe an important factor in pudu considering their sensibilityto stressful stimuli, even in short handling as those made inthis study.

The higher GGT activity compared with cows and sheepsuggests a possible subclinical presence of Fasciola ormycotoxins in the diet. Postmortem findings relative tothose conditions were found in individuals belonging tothe same enclosure, though this would have to be investi-gated for further details. However, SAP plasma activity waswithin the range observed for sheep, but higher than bovinereference values (Kaneko et al. 2008).

The mineral (Mg, P, Ca, Ca/P, Na, K, Cl, GPx) plasmaconcentrations are within reported ranges for mammals.Moreover, GPx blood activity was within reference values(>130 U/g Hb), indicating an adequate selenium status ofanimals (Kaneko et al. 2008).

Due to the short sampling period, it was not possible todetermine seasonal differences in plasma concentrations ofminerals. However, the strong seasonality demonstrated in

Table 1 Hematology values for semi-captive southern pudu (adults and juveniles and males and females) at the province of Valdivia, Chile

Analyte N Mean SD Median Min Max RI (LL–UL) 90 % CI LL 90 % CI UL N outliers Dist Method

Erythrocytes (106/μL) 29 5.80 1.23 5.57 3.45 7.80 3.17–8.45 2.63–3.90 7.73–8.98 2.00 G R

Hematocrit (%) 29 36.0 3.80 37.0 27.0 43.0 29–44 27–31 42–46 1.00 G P

Hemoglobin (g/L) 29 133 17.5 135 104 175 96–169 87–107 159–179 1.00 G R

MCHC (g/L) 29 364 19.9 368 328 408 321–407 311–336 396–416 3.00 G R

MCV (fL) 29 60.0 14.0 61.0 35.0 87.0 30–89 23–38 81–96 2.00 G R

Leukocytes (μL) 29 8,176 2,139 8,200 3,800 11,250 3,732–12,706 2,411–4,845 11,595–14,322 0.00 G R

Lymphocytes (%) 29 73.8 10.6 76.5 47.0 92.0 52–98 46–61 92–104 1.00 G R

Lymphocytes (μL) 29 6,018 1,924 5,956 2,660 9,200 1,996–10,065 1,132–3,055 9,029–10,893 1.00 G R

Neutrophils (%) 28 22.7 9.80 21.50 8.00 47.00 3.0–42.0 0–9 37–47 0.00 G P

Neutrophils (μL) 29 1,730 845 1,575 0.0 3,302 73–3,386 0–526 2,934–3,839 0.00 G P

Monocytes (%) 29 0.17 0.47 0.0 0.0 2.00 0–2 − − 0.00 NG NP

Monocytes (μL) 29 13.8 38.7 0.0 0.0 166.0 0–166 − − 0.00 NG NP

Eosinophils (%) 29 3.00 2.87 2.00 0.0 10.00 0–9 0.0 0–10 0.00 G P

Eosinophils (μL) 29 222 214 177 0.0 800 0–645 0.0 481–792 1.00 G R

Basophils (%) 29 0.14 0.44 0.0 0.0 2.00 0–2 − − 0.00 NG NP

Basophils (μL) 29 8.50 29.10 0.0 0.0 141 0–141 − − 3.00 NG NP

Fibrinogen (g/L) 29 2.30 1.20 2.50 1.00 5.00 1.00 5.00 − 1.00 G NP

SD standard deviation, Min minimum, Max maximum, RI reference interval, LL low limit, UL upper limit, Dist distribution, G Gaussian, NG non-Gaussian, P parametric, NP nonparametric, R robust, MCHC mean cell hemoglobin concentration, MCV mean cell volume

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Fig. 2 Dot plot of plasmaconcentrations of total protein,globulin, triacylglyceride, andchlorine in adult (n=23) andjuvenile pudus (n=5). The shorthorizontal line is the mean foreach age

Table 2 Serum biochemistry values for semi-captive southern pudu (adults and juveniles and males and females) at the province of Valdivia, Chile

Analyte N Mean SD Median Min Max RI (LL–UL) 90 % CI LL 90 % CI UL N outliers Dist Method

Total protein (g/L) 27 77.9 8.22 76.0 62.0 96.0 58–94 54–64 88–100 4.00 G R

Albumin (g/L) 28 32.0 4.23 31.0 22.0 42.00 23.7–40.3 21.4–26.0 38.0–42.6 0.00 G P

Globulin (g/L) 27 42.0 6.40 44.0 28.0 54.00 28–57 24–34 53–60 4.00 G R

Total bilirubin (μmol/L) 28 1.72 0.63 1.50 0.74 3.08 0.29–3.09 0–0.64 2.55–3.41 2.00 G R

Urea (mmol/L) 28 9.29 3.24 9.35 4.23 15.77 2.32–15.91 0.88–4.08 14.08–17.67 0.00 G R

Creatinine (μmol/L) 28 114 16 114 89 151 79–148 72–89 138–157 1.00 G R

Triacylgliceride (mmol/L) 28 0.49 0.22 0.44 0.16 1.03 0.05–0.92 0–0.17 0.80–1.04 0.00 G P

Cholesterol (mmol/L) 29 1.70 0.38 1.67 0.93 2.46 0.90–2.51 0.70–1.12 2.27–2.72 2.00 G R

Lactate (mmol/L) 27 6.50 0.36 6.51 5.89 7.26 5.73–7.25 5.55–5.93 7.05–7.46 0.00 G R

Glucose (mmol/L) 29 10.30 3.76 9.45 3.33 18.96 2.9–17.6 0.90–4.92 15.6–19.65 0.00 G P

Calcium (mmol/L) 26 2.33 0.27 2.32 1.80 2.85 1.75–2.90 1.61–1.91 2.74–3.04 1.00 G R

Phosphorus (mmol/L) 28 2.62 0.68 2.50 1.43 4.25 1.28–3.95 0.91–1.66 3.58–4.32 0.00 G P

Ca/P 25 0.92 0.27 0.88 0.32 1.47 0.35–1.50 0.22–0.49 1.30–1.64 0.00 G R

Magnesium (mmol/L) 26 1.07 0.13 1.07 0.83 1.28 0.80–1.35 0.73–0.88 1.28–1.42 0.00 G R

Sodium (mmol/L) 21 150 5.91 150 140 161 139–162 135–142 158–166 0.00 G R

Potassium (mmol/L) 17 4.12 0.38 4.15 3.55 4.90 3.27–4.95 3.01–3.57 4.65–5.21 1.00 G R

Chloride (mmol/L) 28 117 15.20 116 94.0 156 83–146 76–94 136–157 1.00 G R

GPx (U/g Hb) 19 343 67 329 235 477 >203 174–250 − 1.00 G R#

AST (U/L) 29 151 43 134 87 258 46–240 28–74 206–266 0.00 G R

ALT (U/L) 29 75 22 76 35 115 30.3–120.5 19.2–42.5 108.5–131.2 0.00 G R

GGT (U/L) 29 59.5 9.80 59.0 39.0 81.0 38–80 33–45 74–86 4.00 G R

ALP (U/L) 28 263 5 259 112 494 38–481 0–93 417–537 2.00 G R

Amylase (U/L) 28 444 96 433 264 648 224–632 171–282 568–700 3.00 G R

Lipase (U/L) 27 16.4 2.32 16.00 13.00 21.00 11.0–21.0 9.8–12.2 19.1–22.9 4.00 G R

CK (U/L) 29 315 144 266 34 666 34–597 0–113 517–677 2.00 G P

SD standard deviation, Min minimum, Max maximum, RI reference interval, LL low limit, UL upper limit, Dist distribution, G Gaussian, NG non-Gaussian, P parametric, NP nonparametric, R robust, # left side, Ca/Pi calcium/phosphorus, GPx glutathione peroxidase, AST aspartateaminotransferase, ALT alanine aminotransferase, GGT gamma glutamyl transferase, ALP alkaline phosphatase, CK creatine kinase

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those variables in several deer species obligates to evaluatethis factor in a future research.

The captivity and feeding conditions permit that valuesobtained could be used as a tool to determine the physiologicalresponse of pudu in different ex situ and in situ research/con-servation initiatives.

Acknowledgments We thank to Eduardo Silva-Rodriguez by hishelpful comment on the manuscripts. The study was funded by theDirection of Research and Development (DID) from UniversidadAustral de Chile.

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