Research Article Hematology of Wild Caught Dubois s Tree ... · of vertebrates [ ]. ese parameters...

Research ArticleHematology of Wild Caught Duboisrsquos Tree Frog Polypedatesteraiensis Dubois 1986 (Anura Rhacophoridae)

Madhusmita Das and Pravati Kumari Mahapatra

Cell and Developmental Biology Laboratory PG Department of Zoology Utkal University Bhubaneswar Odisha 751 004 India

Correspondence should be addressed to Pravati Kumari Mahapatra mahap pkyahoocom

Received 28 August 2013 Accepted 11 December 2013 Published 30 January 2014

Academic Editors A Baonza H M Chamberlin and H Kuroda

Copyright copy 2014 M Das and P K MahapatraThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Blood was analyzed from eighty (forty males and forty females) adult individuals of Polypedates teraiensis to establish referenceranges for its hematological and serum biochemical parameters The peripheral blood cells were differentiated as erythrocyteslymphocytes eosinophils neutrophils monocytes basophils and thrombocytes with similar morphology to other anuransMorphology of blood cells did not vary according to sexThe hematological investigations includedmorphology andmorphometryof erythrocytes morphometry of leucocytes packed cell volume (PCV) hemoglobin content (Hb) mean corpuscular volume(MCV) mean corpuscular hemoglobin (MCH) mean corpuscular hemoglobin concentration (MCHC) erythrocyte or red bloodcell (RBC) count leukocyte or white blood cell (WBC) count differential leukocyte count and neutrophil to lymphocyte (NL)ratio Besides protein cholesterol glucose urea uric acid and creatinine content of blood serum were assayed Hematologicalparameters that differed significantly between sexes were RBC count length and breadth of RBC neutrophil NL ratio areaoccupied by basophils and diameter of large lymphocyte and eosinophilsThe level of glucose urea and creatinine in blood serumalso significantly differed between sexes

1 Introduction

Hematology is the most straightforward and less invasivetechnique to access the health status of natural populationof vertebrates [1] These parameters allow the detectionof changes in physiological pathological ecological andenvironmental conditions in natural population [2 3] Dif-ferential leucocyte count provides information about theimmunological status of an individual Similarly hematocritreflects the efficiency of oxygen carrying capacityThe plasmaor serum biochemical analyses provide information aboutinternal organs electrolytes proteins and nutritional andmetabolic parameters [4] Amphibians are known to besensitive animals and show physiological variables to acuteenvironmental changes [5] For this reason it has beensuggested that physiological ecology of these animals shouldbe incorporated into conservation plans and monitoringprograms of individual populations [6] But hematologicaland serum biochemical reference ranges exist for most ofthe animal species that receive veterinary care [4] Thoughthere are many hematological reports on anuran species

there is less information available for the rhacophorid species[7 8] Detailed hematological reports on rhacophorid frogPolypedates maculatus have been studied byMahapatra et al[8] Another frog that lives sympatric with P maculatus is Pteraiensiswhich is arboreal andmostly found in bushes plan-tations and gardens and rarely enters human habitation [9]Blood cell profile of the tadpoles of P teraiensiswas describedby us earlier [10] and here we describe the hematological andserum biochemical parameters of adults

2 Materials and Methods

Adults of Polypedates teraiensis (Figure 1(a)) were collectedfrom Chowduar (20∘3110158401110158401015840N 85∘4910158401110158401015840E) Odisha Fortyadult specimens from each sex of the species were utilizedin the present investigation After collection the frogs weremaintained in the terrarium for acclimatization to laboratoryconditions and handled following standardized procedures[11] Blood samples were taken from the ventral abdominalvein Prior to blood collection specimens were weighted (in

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 491415 7 pageshttpdxdoiorg1011552014491415

2 The Scientific World Journal

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Figure 1 (a) Adult Polypedates teraiensis (b) elliptical RBCwith centrally placed nucleus (c) elliptical RBCwith eccentrically placed nucleus(d) circular RBC with centrally placed nucleus (e) spindle-shaped RBC (f) teardrop-shaped cell (g) dividing erythrocytes (h) destructionof erythrocytesrsquo membrane (i) large lymphocyte (j) small lymphocyte (k) monocyte (l) eosinophil (m) tetralobed neutrophil (n) trilobedneutrophil (o) basophil and (p) cluster of thrombocytes (scale bar = 10120583m (b)ndash(p))

grams) and snout to vent length (SVL) was measured (incm) Blood was collected in the morning hours to avoiddiurnal variation Collected blood was transferred from thesyringe to a penicillin vial and kept undisturbed for clottingAfter following retraction of clot the supernatant serumwas pipetted into an eppendorf tube The serum was thenused for all biochemical investigations For other hemato-logical investigations the collected blood was transferredfrom the syringe to a penicillin vial containing pinch ofethylene diamine tetra acetic acid (EDTA) an anticoagulantThe blood was mixed well without frothing Hematologicalinvestigations included morphology and morphometry oferythrocytesmorphometry of leucocytes packed cell volume(PCV) hemoglobin content (Hb) mean corpuscular volume(MCV) mean corpuscular hemoglobin (MCH) mean cor-puscular hemoglobin concentration (MCHC) erythrocyte or

red blood cell (RBC) count leukocyte or white blood cell(WBC) count differential leukocyte count and neutrophilto lymphocyte (NL) ratio Serum biochemical parametersincluded estimation of total protein cholesterol glucoseurea uric acid and creatinine content

For morphology and morphometry of blood cells bloodsmears were prepared using push slide technique The driedblood smears were stained with Giemsarsquos stain and observedunder light microscope (Hund H500 WETZLAR) Differenttypes of blood cells present in the smear were identifiedfollowing Turner [12] and Heatley and Johnson [13] Slideswere viewed in zigzag pattern covering all parts of the bloodsmear and leukocytes were counted in each field of view until100 cells were counted Blood cells were photographed withthe help of a Canon EOS 450 122 Mega pixel camera (EF-S 18-55 1S Kit) and measured using an ocular micrometer

The Scientific World Journal 3

which was standardized against a stage micrometer (ERMAJapan made) Formulae used by Arserim and Mermer [14]were followed for measurement of blood cells For the otherhematological investigations procedures described byCoppoet al [15] were followed

Chemicals used for the serumbiochemical studieswere ofanalytical grade Bovine serum albumin (BSA) was obtainedfrom SIGMA Chemicals Co USA Folin-phenol reagentwas obtained from Sisco Research Laboratory MumbaiIndia Eco-pak glucose kit autozyme urea enzymatic kitinfinite liquid uric acid kit and autozyme creatinine kitwere procured from Accurex Biomedical Private LimitedMumbai India All other chemicals were of the highestpurified grade available For the estimation of total proteinand total cholesterol regular laboratory methods that isthose of Lowry et al [16] and Rosenthal et al [17] werefollowed respectively For the estimation of glucose ureauric acid and creatinine the abovementioned laboratory kitswere used

Statistical analysiswas done followingKolmogorov-Smir-nov test 119875 gt 005 averages were compared with studentrsquos 119905-test The significance level was 119875 le 005 Statistical analyseswere performed by SPSS (version 1000)

3 Results and Discussion

In the present study SVL (snout to vent length) in malesranged from 60 to 70 cm with a mean of 642 plusmn 043 cm andin females it ranged from 60 to 92 cm with a mean of 747 plusmn136 cm In case of males the average body weight was 1687plusmn062 g (ranged from 160 to 175 g) and in females averagebody weight was 1937 plusmn 193 g (ranged from 175 to 215 g)Hematological and biochemical parameters investigated inthe present study are represented in Tables 1 and 2

31 Morphology of Blood Cell The characteristics shape ofamphibian erythrocytes is elliptical form [18] Apart from thecharacteristic ellipsoidal shape amphibians are also knownto display wide variation in erythrocyte morphology acrossspecies [7 19 20] In the present study erythrocytes of variousshapes were observedThe different shapes included ellipticalcells with centrally placed nuclei (Figure 1(b)) elliptical cellswith eccentrically placed nuclei (Figure 1(c)) and circularcells with centrally placed nuclei (Figure 1(d)) Ellipsoidalerythrocytes have also been reported in the blood smearof Polypedates maculatus [8] Besides some irregular formssuch as spindle-shaped cells (Figure 1(e)) and teardrop-shaped cells (Figure 1(f)) were observed Such irregular cellswere also observed in the tadpole of this species and itwas attributed to anemic state [10] Dividing erythrocytes(Figure 1(g)) and erythrocytes with damaged cell membrane(Figure 1(h)) were also evident in the present study

The lymphocytes observed were of two distinct formsthat is large lymphocytes (Figure 1(i)) and small lymphocytes(Figure 1(j)) Both lymphocytes were rounded in shapeHowever there was difference in size The nuclei wererounded in shape both in large and small lymphocytesand occupied the entire cell leaving a narrow rim of light

violet cytoplasm towards the periphery Monocytes wererounded in shape and had eccentrically placed indentednuclei (Figure 1(k)) Eosinophils were also rounded in shapewith segmented nuclei Eosinophils had bilobed nucleilocated at one end of the cells (Figure 1(l)) Neutrophils werelarge and rounded in shape They were easily distinguishedfrom other cells due to their lobular and segmented nucleiNeutrophils with tetralobed nuclei (Figure 1(m)) or trilobednuclei (Figure 1(n)) were more common Basophils inves-tigated in this species were round cells having large darkviolet-stained granules over the irregular nuclei as well asentire cells (Figure 1(o))Thrombocytes or platelets were longand oval in shape having large and ovoid nuclei They werefound in clusters on the blood smears (Figure 1(p)) Earlierreports suggest amphibian thrombocytes to be nucleated andspindle-shaped [14]

32 Morphometry of Blood Cells Length and breadth oferythrocytes remained 1687 plusmn 137 120583m and 895 plusmn 012 120583min males and 1977 plusmn 153 120583m and 862 plusmn 034 120583m in femalesrespectively The mean length of erythrocytes in females wasmore than the males and the difference was statistically sig-nificant (119905 = 2922 df = 6 119875 = 0026) Arserim and Mermer[14] have reported larger erythrocytes in case of females(2303 120583m 1459 120583m) than males (2232 120583m 1365 120583m) inRana macrocnemis The mean length and breadth of erythro-cytes in both males and females were found to be less whencompared to other Rhacophorid anurans that is Buergeriabuergeri (198 120583m 13 120583m) Rhacophorus annamensis (20120583m127 120583m) and Rhacophorus schlegelii (216 120583m 133 120583m) asreported by Kuramoto [7] The aspect ratio (Lengthbreadthof RBC) was more in females than males This was alsostatistically significant (119905 = 265 df = 6 119875 = 003)The surface area occupied by the erythrocytes was more infemales than in males (Table 1) but this difference was statis-tically insignificant But higher surface area of erythrocytesin males (24315 plusmn 37841 120583m2) in comparison to females(21058 plusmn 38279 120583m2) has been reported in adult frogs ofPolypedates maculatus [8] The mean value of long axes andshort axes of nuclei remained 615 plusmn 0264 120583m and 50 plusmn008 120583m in males and 75 plusmn 173 120583m and 55 plusmn 090 120583m infemales respectively The surface area of nuclei ranged from2301 to 2585 120583m2 with a mean of 2398 plusmn 127 120583m2 in caseof males In females it ranged from 207 to 4964 120583m2 with amean of 330 plusmn 1280 120583m2 However there was no significantdifference between sexes for the parameters studied on nucleiof RBCs

The erythrocytes were larger in size in tadpoles of thisspecies [10] in comparison to the adults of the presentstudy Snyder and Sheafor [21] have suggested that with theevolutionary development of more efficient cardiovascularsystem and that they are attended by higher vascular resis-tances including smaller capillary radial dimensions whichare attended by smaller RBCsThus decrease in size of RBCsin adult frogs of the present study in comparison to tadpoles[10] is an example of physiological adaptation required fortransition from aquatic to terrestrial mode of life


Table 1 Hematological parameters observed in adult males and females of Polypedates teraiensis

Hematological parametersSex

Male FemaleX plusmn SD (range) X plusmn SD (range)

RBC countlowast 059 plusmn 001 (058ndash061) 062 plusmn 001 (061ndash065)Haemoglobin conc () 595 plusmn 012 (58ndash61) 582 plusmn 029 (55ndash62)Packed cell volume () 5062 plusmn 110 (495ndash520) 5155 plusmn 095 (505ndash525)Mean corpuscular volume (1205833) 85131 plusmn 2712 (81967ndash86666) 82241 plusmn 1331 (80307ndash83333)Mean corpuscular haemoglobin (Pg) 10005 plusmn 366 (9672ndash10517) 9671 plusmn 690 (9076ndash10576)Mean corpuscular haemoglobin conc () 1174 plusmn 017 (1171ndash1195) 1175 plusmn 073 (113ndash1277)RBC length (120583m)lowast 1687 plusmn 13 (155ndash185) 1977 plusmn 153 (180ndash215)RBC breadth (120583m)lowast 895 plusmn 012 (88ndash91) 862 plusmn 034 (82ndash89)Lengthbreadth of RBC 188 plusmn 016 (177ndash210) 229 plusmn 018 (202ndash241)Surface area of erythrocyte (120583m2) 11777 plusmn 92 (10760ndash12698) 15301 plusmn 3511 (12152ndash19135)Length of RBCrsquos nucleus (120583m) 615 plusmn 026 (59ndash65) 75 plusmn 173 (59ndash95)Breadth of RBCrsquos nucleus (120583m) 50 plusmn 008 (49ndash51) 55 plusmn 090 (45ndash67)Lengthbreadth of RBCrsquos nucleus 122 plusmn 004 (118ndash127) 135 plusmn 015 (116ndash152)Surface area of RBCrsquos nucleus (120583m2) 2398 plusmn 127 (2301ndash2585) 330 plusmn 1280 (207ndash4964)WBC count (number of WBCmm3) 1212 plusmn 033 (118ndash125) 1215 plusmn 069 (115ndash129)Neutrophil ()lowast 2352 plusmn 130 (220ndash250) 2666 plusmn 116 (250ndash276)Lymphocyte () 5552 plusmn 204 (531ndash580) 5492 plusmn 047 (546ndash556)Basophil () 35 plusmn 024 (32ndash38) 28 plusmn 11 (19ndash42)Eosinophil () 1102 plusmn 112 (100ndash125) 997 plusmn 017 (98ndash102)Monocyte () 642 plusmn 051 (59ndash71) 57 plusmn 046 (50ndash60)Neutrophillymphocyte ratiolowast 042 plusmn 003 (037ndash045) 048 plusmn 002 (045ndash050)Diameter of neutrophil (120583m) 105 plusmn 055 (90ndash115) 105 plusmn 045 (100ndash115)Area occupied by neutrophil (120583m2) 8754 plusmn 795 (6362ndash10381) 8754 plusmn 975 (7855ndash10381)Diameter of large lymphocyte (120583m)lowast 95 plusmn 023 (75ndash115) 92 plusmn 035 (75ndash120)Area occupied by large lymphocyte (120583m2) 7084 plusmn 632 (4415ndash12131) 6644 plusmn 65 (4415ndash11304)Diameter of small lymphocyte (120583m) 525 plusmn 031 (40ndash60) 51 plusmn 065 (41ndash59)Area occupied by small lymphocyte (120583m2) 2143 plusmn 565 (1256ndash2826) 2001 plusmn 598 (1319ndash2732)Diameter of basophil (120583m) 110 plusmn 058 (90ndash135) 105 plusmn 065 (95ndash135)Area occupied by basophil (120583m2)lowast 9498 plusmn 59 (6358ndash14306) 8654 plusmn 975 (7084ndash14306)Diameter of eosinophil (120583m)lowast 115 plusmn 381 (100ndash120) 120 plusmn 031 (95ndash135)Area occupied by eosinophil (120583m2) 10381 plusmn 123 (7850ndash11304) 11304 plusmn 899 (7084ndash14306)Diameter of monocyte (120583m) 115 plusmn 085 (100ndash120) 112 plusmn 056 (100ndash120)Area occupied by monocyte (120583m2) 10381 plusmn 1032 (7850ndash11304) 9847 plusmn 95 (7850ndash11304)119883 mean SD standard deviation Pg pictogram lowastDifference in values between males and females statistically significant

Knowledge on erythrocytes in vertebrates provides uswith much valuable information Snyder and Sheafor [21]have described erythrocytes to be the center piece in theevolution of vertebrate circulatory systemThe measurementof erythrocyte dimensions is often an important componentof standard hematologic survey in amphibians [22] It canbe used for comparison across species [23] and studies ofenvironmental seasonal or altitudinal acclimatization [24ndash26] Measuring erythrocytes can also provide informationregarding the genome size of a species [7 27] In amphibianserythrocyte has long been known to be correlated negativelywith metabolic rates both at the organism level [19 28] andthe tissue level [29]This relationship stems from the fact thatlarger surface-area-to-volume ratios in smaller cells allow formore efficient exchange of oxygenThis idea is exemplified in

intraspecific comparisons of amphibians at different altitudeswhere animals at higher latitudes have smaller erythrocytes[24 30] presumably tomaximize cellular efficiency of oxygentransport and exchange in a low-oxygen environment Someinvestigators have stressed that erythrocyte may be used inploidy determination [31ndash34] Moreover erythrocyte size canalso be used as a diagnostic assay to assess the effects of airpollution in animals [35]

Amongst the leucocytes mean diameter of large lym-phocytes small lymphocytes monocytes and basophilsremained more in males than in females (Table 1) The sizedifference was significant for large lymphocytes only (119905 =226 df = 18 119875 = 0036) The diameter of eosinophils wasmore in females (120 plusmn 031) than in males (115 plusmn 045)and the difference was significant (119905 = 289 df = 18 119875 =


Table 2 Serum biochemical parameters observed in adult males and females of Polypedates teraiensis

Serum biochemical parametersSex


Protein (gdL) 118 plusmn 054 (112ndash124) 1275 plusmn 053 (123ndash135)Cholesterol (gdL) 20 plusmn 008 (19ndash21) 217 plusmn 009 (21ndash23)Glucose (gdL)a 5075 plusmn 537 (440ndash560) 645 plusmn 525 (600ndash720)Urea (gdL)a 7125 plusmn 095 (700ndash720) 800 plusmn 081 (790ndash810)Uric acid (gdL) 495 plusmn 031 (45ndash52) 515 plusmn 012 (50ndash53)Creatinine (gdL)a 31 plusmn 008 (30ndash32) 292 plusmn 009 (28ndash30)X mean SD standard deviation aDifference in values between males and females statistically significant

2893) There was no difference in mean size of neutrophilsin both sexes The area occupied by the large lymphocytessmall lymphocytes basophils and monocytes was more inmales than in females The area occupied by eosinophilswas more in females than in males There was no differencein mean area of neutrophils in both sexes (Table 1) Thedifference was significant for the area of basophils (119905 = 2342df = 18 119875 = 00309) while for other leucocytes the dif-ference remained insignificant The diameter of neutrophils(1698 120583m) basophils (1369 120583m) eosinophils (1630 120583m) andmonocytes (1430 120583m) was more in Rana macracnemis [14]than in the present rhacophorid studied Similarly largerleucocytes (neutrophils = 152 120583m lymphocytes = 136 120583meosinophils = 162 120583m monocytes = 152 120583m and basophils =169 120583m) have been reported in R catesbeiana by Coppo et al[15]

33 RBC Count Hemoglobin Concentration and Red BloodCell Indices Themean number of RBCs in males was 059 plusmn0012millionsmm3 of blood but in females it was 062plusmn0017millionsmm3 of bloodThe number of RBCs remainedmorein females than in males This difference was statisticallysignificant (119905 = 42 df = 6 119875 = 0005) The gram percentageof hemoglobin per 100mL of blood varied from 58 to 61with a mean of 595 plusmn 012 among males In females it rangedfrom 55 to 62100mL with a mean of 582 plusmn 029100mLThe mean packed cell volume (PCV) was more in females(5155 plusmn 095) than in males (50625 plusmn 110) (Table 1)The mean corpuscular volume (MCV) mean corpuscularhaemoglobin (MCH) and mean corpuscular haemoglobinconcentration (MCHC) of the males were observed to be85131 plusmn 2712 120583

3 10005 plusmn 366 pg and 1174 plusmn 017respectively In females the mean values of MCV MCH andMCHC were 82241 plusmn 1331 1205833 9671 plusmn 690 pg and 1175 plusmn073 respectively However the differences between themeans of the corpuscular valueswere not significant (Table 1)In Polypedates maculatus percentage of hemoglobin MCVandMCHC showed significant differences between male andfemale frogs [8]

34WBCCount Differential Leukocyte Count andNeutrophilto Lymphocyte Ratio More WBCs were observed in femalesthan in males (Table 1) As mean WBC count was 1212 times103

plusmn 033mm3 in males and 1213 times 103 plusmn 069mm3 in

females the difference was not significant (Table 1) Howeverpercentage of lymphocytes monocytes eosinophils andbasophils remained higher in males than in females But thepercentage of neutrophils was higher in females than inmales(Table 1) The difference was significant only for neutrophils(119905 = 3535 df = 6 119875 = 0012)

The neutrophil to lymphocyte (NL) ratio in females(048 plusmn 002) was more than in males (042 plusmn 003) (Table 1)and the difference was found to be significant (119905 = 3328df = 6 119875 = 0058) According to Davis [36] the averagereference range of amphibian NL ratios falls within 001 to067 and the NL ratio of the present study falls within thisrange There exists a close link between leucocyte profilesand glucocorticoid levels Specifically these hormones act toincrease the percentage of neutrophils while decreasing thepercentage of lymphocytes This phenomenon is seen in allthe five vertebrate taxa namely pisces amphibia reptiliaaves and mammals in response to either natural stressorsor exogenous administration of stress hormones [37] Forecologists therefore high ratio of neutrophil to lymphocytesin blood samples reliably indicates high glucocorticoid levelsFurthermore this close relationship between stress hormonesand NL ratio needs to be highlighted more prominently inhematological assessments of stress for a better interpretationof results [37]

35 Serum Biochemical Parameters The levels of bloodserum protein cholesterol glucose urea and uric acid werefound to be higher in females than in males However theserum creatinine value was more in males than in females(Table 2) The difference was found to be significant for thelevels of glucose (119905 = 370 df = 6 119875 = 0010) urea (119905 = 1404df = 6 119875 = 00001) and creatinine (119905 = 332 df = 6119875 = 0016) No significant differences were observed for thelevel of protein cholesterol and uric acid in the blood withinthe sexes

4 Conclusion

As the study represents the first hematological serum bio-chemical investigation of the Rhacophorid frog Polypedatesteraiensis the values can serve as general reference valuesfor future investigations involving this species and otheranuran species This study would be helpful to understand


the relevance of the data described earlier for the tadpoles ofthis species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank DST Government of Indiafor the financial assistance under PURSE grant to the PGDepartment of Zoology Utkal University Madhusmita Daswould like to thankUGC for aResearch Fellowship (RFSMS)

References

[1] P Artacho M Soto-Gamboa C Verdugo and R F NespololdquoUsing haematological parameters to infer the health and nutri-tional status of an endangered black-necked swan populationrdquoComparative Biochemistry and Physiology A vol 147 no 4 pp1060ndash1066 2007

[2] J H Sarasola J J Negro and A Travaini ldquoNutritional condi-tion and serum biochemistry for free-living Swainsonrsquos Hawkswintering in central Argentinardquo Comparative Biochemistry andPhysiology A vol 137 no 4 pp 697ndash701 2004

[3] D A Seaman C G Guglielmo and T D Williams ldquoEffects ofphysiological state mass change and diet on plasma metaboliteprofiles in the western sandpiper Calidris maurirdquo Journal ofExperimental Biology vol 208 no 4 pp 761ndash769 2005

[4] S H Newman J F Piatt and J White ldquoHematological andplasma biochemical reference ranges of Alaskan seabirds theirecological significance and clinical importancerdquo Waterbirdsvol 20 no 3 pp 492ndash504 1997

[5] C Carey ldquoHow physiological methods and concepts can beuseful in conservation biologyrdquo Integrative and ComparativeBiology vol 45 no 1 pp 4ndash11 2005

[6] P E Seiser L K Duffy A D Mcguire D D Roby G H Goletand M A Litzow ldquoComparison of pigeon guillemot Cepphuscolumba blood parameters from oiled and unoiled areas ofAlaska eight years after the Exxon Valdez oil spillrdquo MarinePollution Bulletin vol 40 no 2 pp 152ndash164 2000

[7] M Kuramoto ldquoRelationships between number size and shapeof red blood cells in amphibiansrdquoComparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[8] B B Mahapatra M Das S K Dutta and P K MahapatraldquoHematology of Indian rhacophorid tree frog Polypedates mac-ulatus Gray 1833 (Anura Rhacophoridae)rdquo Comparative Clini-cal Pathology vol 21 no 4 pp 453ndash460 2012

[9] S K Dutta M V Nair P P Mohapatra and A K Mahap-atra Amphibians and Reptiles of Similipal Biosphere ReservesRegional Plant Resource Centre 2009

[10] M Das and P K Mahapatra ldquoBlood cell profiles of the tadpolesof the Duboisrsquos tree frog Polypedates teraiensis Dubois 1986(Anura Rhacophoridae)rdquo The Scientific World Journal vol2012 Article ID 701746 11 pages 2012

[11] K Wright ldquoAmphibian medicine and captive husbandryrdquo inAmphibian Medicine and Captive Husbandry K Wright and BWhitaker Eds pp 128ndash146 Kreiger Publishing Malabar FlaUSA 2001

[12] R J Turner ldquoAmphibiansrdquo in Vertebrate Blood Cells A FRawley and N A Ratcliff Eds pp 129ndash209 Cambridge Uni-versity Press Cambridge UK 1998

[13] J J Heatley and M Johnson ldquoClinical technique amphib-ian hematology a practitionerrsquos guiderdquo Journal of Exotic PetMedicine vol 18 no 1 pp 14ndash19 2009

[14] S K Arserim and A Mermer ldquoHematology of the Uludag frogRana macrocnemis Boulenger 1885 in Uludag National park(Bursa Turkey)rdquo E U Journal of Fisheries and Aquatic Sciencesvol 25 no 1 pp 39ndash46 2008

[15] J A Coppo N B Mussart S A Fioranelli and P A ZeinstegerldquoBlood and urine physiological values in captive bullfrog Ranacatesbeiana (Anura Ranidae)rdquoAnalecta Veterinaria vol 25 pp15ndash17 2005

[16] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[17] H L Rosenthal M L Pfluke and S Buscaglia ldquoA stable ironreagent for determination of cholesterolrdquo The Journal of Labo-ratory and Clinical Medicine vol 50 no 2 pp 318ndash322 1957

[18] H Szarski and G Czopek ldquoErythrocyte diameter in someamphibians and reptilesrdquo Bulletin de lrsquoAcademie Polonaise desSciences Serie des Sciences Biologiques vol 14 pp 437ndash4431966

[19] F J Vernberg ldquoHematological studies on salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 pp 129ndash133 1955

[20] A K Davis J R Milanovich J L DeVore and J C Maerz ldquoAninvestigation of factors influencing erythrocyte morphology ofred-backed salamanders (Plethodon cinereus)rdquo Animal Biologyvol 59 no 2 pp 201ndash209 2009

[21] G K Snyder and B A Sheafor ldquoRed blood cells centerpiecein the evolution of the vertebrate circulatory systemrdquo TheAmerican Zoologist vol 39 no 2 pp 189ndash198 1999

[22] F A Hartman and M A Lessler ldquoErythrocyte measurementsin birdsrdquo Auk vol 80 pp 467ndash473 1963

[23] M K Atatur H Arikan and E I Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23no 2 pp 111ndash114 1999

[24] G Ruiz M Rosenmann and A Veloso ldquoAltitudinal distribu-tion and blood values in the toad Bufo spinulosus wiegmannrdquoComparative Biochemistry and Physiology A vol 94 no 4 pp643ndash646 1989

[25] T Pages V I Peinado and G Viscor ldquoSeasonal changes inhematology and blood chemistry of the freshwater turtleMau-remys caspica leprosardquo Comparative Biochemistry and Physiol-ogy A vol 103 no 2 pp 275ndash278 1992

[26] G Ruiz M Rosenmann and A Cortes ldquoThermal acclimationand seasonal variations of erythrocyte size in theAndeanmousePhyllotis xanthopygus rupestrisrdquo Comparative Biochemistry andPhysiology A vol 139 no 4 pp 405ndash409 2004

[27] T R Gregory ldquoThe bigger the C-value the larger the cellgenome size and red blood cell size in vertebratesrdquo Blood CellsMolecules and Diseases vol 27 no 5 pp 830ndash843 2001

[28] H M Smith ldquoCell size and metabolic activity in AmphibiardquoBiologial Bulletin vol 48 no 5 pp 347ndash378 1925

[29] M A Monnickendam and M Balls ldquoThe relationship betweencell sizes respiration rates and survival of amphibian tissuesin long-term organ culturesrdquo Comparative Biochemistry andPhysiology A vol 44 no 3 pp 871ndash880 1973

[30] G RuizM Rosenmann andAVeloso ldquoRespiratory and hema-tological adaptations to high altitude in Telmatobius frogs from


the chilean andesrdquo Comparative Biochemistry and Physiology Avol 76 no 1 pp 109ndash113 1983

[31] M Stock and W R Grobe ldquoErythrocyte size and ploidy deter-mination in green toads (Bufo viridis complex) from MiddleAsiardquo Alytes vol 15 pp 72ndash90 1997

[32] T Schroer andH Greven ldquoVerbreitung Populationsstrukturenund Ploidiegrade von Wasserfroschen in Westfalenrdquo Zeitschiftfur Feldherpetology vol 5 pp 1ndash14 1998

[33] A LMartino andU Sinsch ldquoSpeciation by polyploidy inOdon-tophrynus americanusrdquo Journal of Zoology vol 257 no 1 pp67ndash81 2002

[34] S D Rosset D Baldo C Lanzone and N G Basso ldquoReviewof the geographic distribution of diploid and tetraploid pop-ulations of the Odontophrynus americanus species complex(Anura Leptodactylidae)rdquo Journal of Herpetology vol 40 no4 pp 465ndash477 2006

[35] S Llacuna A Gorriz M Riera and J Nadal ldquoEffects ofair pollution on hematological parameters in passerine birdsrdquoArchives of Environmental Contamination and Toxicology vol31 no 1 pp 148ndash152 1996

[36] A K Davis ldquoThe Wildlife Leukocytes Webpage the ecologistrsquossource for information about leukocytes of wildlife speciesrdquo2009 httpwildlifehematologyugaedu

[37] A K Davis D L Maney and J C Maerz ldquoThe use of leukocyteprofiles tomeasure stress in vertebrates a review for ecologistsrdquoFunctional Ecology vol 22 no 5 pp 760ndash772 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

Figure 1 (a) Adult Polypedates teraiensis (b) elliptical RBCwith centrally placed nucleus (c) elliptical RBCwith eccentrically placed nucleus(d) circular RBC with centrally placed nucleus (e) spindle-shaped RBC (f) teardrop-shaped cell (g) dividing erythrocytes (h) destructionof erythrocytesrsquo membrane (i) large lymphocyte (j) small lymphocyte (k) monocyte (l) eosinophil (m) tetralobed neutrophil (n) trilobedneutrophil (o) basophil and (p) cluster of thrombocytes (scale bar = 10120583m (b)ndash(p))

grams) and snout to vent length (SVL) was measured (incm) Blood was collected in the morning hours to avoiddiurnal variation Collected blood was transferred from thesyringe to a penicillin vial and kept undisturbed for clottingAfter following retraction of clot the supernatant serumwas pipetted into an eppendorf tube The serum was thenused for all biochemical investigations For other hemato-logical investigations the collected blood was transferredfrom the syringe to a penicillin vial containing pinch ofethylene diamine tetra acetic acid (EDTA) an anticoagulantThe blood was mixed well without frothing Hematologicalinvestigations included morphology and morphometry oferythrocytesmorphometry of leucocytes packed cell volume(PCV) hemoglobin content (Hb) mean corpuscular volume(MCV) mean corpuscular hemoglobin (MCH) mean cor-puscular hemoglobin concentration (MCHC) erythrocyte or

red blood cell (RBC) count leukocyte or white blood cell(WBC) count differential leukocyte count and neutrophilto lymphocyte (NL) ratio Serum biochemical parametersincluded estimation of total protein cholesterol glucoseurea uric acid and creatinine content

For morphology and morphometry of blood cells bloodsmears were prepared using push slide technique The driedblood smears were stained with Giemsarsquos stain and observedunder light microscope (Hund H500 WETZLAR) Differenttypes of blood cells present in the smear were identifiedfollowing Turner [12] and Heatley and Johnson [13] Slideswere viewed in zigzag pattern covering all parts of the bloodsmear and leukocytes were counted in each field of view until100 cells were counted Blood cells were photographed withthe help of a Canon EOS 450 122 Mega pixel camera (EF-S 18-55 1S Kit) and measured using an ocular micrometer

































4 Conclusion






Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































4 Conclusion






Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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