Hindawi Publishing CorporationInternational Journal of ZoologyVolume 2013 Article ID 716183 14 pageshttpdxdoiorg1011552013716183

Research ArticleBlood Cell Profile of the Developing Tadpoles and Adults of theOrnate Frog Microhyla ornata (Anura Microhylidae)

Jutshina Hota Madhusmita Das and Pravati Kumari Mahapatra

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

Correspondence should be addressed to Pravati Kumari Mahapatra mahap pkyahoocom

Received 31 May 2013 Revised 30 July 2013 Accepted 5 August 2013

Academic Editor Roger P Croll

Copyright copy 2013 Jutshina Hota et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Metamorphosis happens to be an important event in the lifetime of amphibians Our study offers a record of blood cell profile oflaboratory reared tadpoles during development and metamorphosis (Gosner stage 26 to 46) and adults of Microhyla ornata Thelarval erythrocytes were observed to be circular oval and elliptical in shape However other variations were distinct during theprometamorphic and metamorphic stages Crenulated erythrocytes showed a pattern of appearance and the crenulations variedfrom minute serrations to highly spiked projections Correlations between the morphometric values of erythrocytes during thelarval development were also determined The leukocyte profile of the tadpoles showed a high percentage of lymphocytes duringlarval development while the percentage of monocytes eosinophils and neutrophils remained high during metamorphosis andwere positively correlated with the developing stages Blood thrombocytes of the tadpoles were small and were found in clustersElliptical erythrocytes were the most common in the adult frogs However few erythrocytes were also circular in shape In adultsthe percentage of lymphocytes was found to be more in comparison with the other leucocytes and neutrophils showed variouspolymorphic forms Thrombocytes were nucleated and spindle shaped

1 Introduction

Metamorphosis in amphibians is often a time of dramaticdevelopmental change affecting nearly the entire organismand has been a subject of investigation in several directions[1 2] During this time a large proportion of the animalrsquosstructure changes the larva and adults are unrecognizableas being the same individual Interestingly these two phaseshave opposing effects on tissues as in the first phase thereis growth and development of tissues and increase in bodysize and in the second phase massive tissue reconstructionand breakdown leading to a reduction in overall bodysize occurs [3] This brings about changes morphologicallyphysiologically and behaviorally to prepare for a new modeof existence

Herpetologists are becoming increasingly aware of theimportance of hematological parameters for evaluating thewelfare of their study animals [4] Interpretation of thehematological parameters offers valuable pieces of infor-mation concerning the health status of the organism [4]The erythrocyte size has also been described to be used in

ploidy determination [5] The effects of the phenomenonof tissue growth and lysis on the relative distribution ofwhite blood cells in circulation have been considered as animportant biological phenomenon since the 1920s [3] and agrowing number of ecologists are turning to the enumerationof white blood cells from blood smears to assess stressin animals [4] Recent interest in counts of leucocytes inamphibians for environmental monitoring emphasizes theneed to understand how white blood cells naturally varythroughout larval life [6ndash9] There also exists considerablevariation in cell morphology in amphibians due to variationin metabolism [10ndash12]

Since normal hematology of many species is poorlyunderstood and reference values are scarce [13] thepresent study aims to investigate the blood cell profile ofthe laboratory reared tadpoles and adult frogs of Microhylaornata (Dumeril and Bibron 1841) inhabiting NandankananBhubaneswar (20∘2410158401010158401015840N 85∘4810158401310158401015840E) Odisha IndiaMicrohyla ornata is a microhylid frog belonging to classanura A considerable work on M ornata has been donewith respect to life history [14] red blood cell sizes of adults

2 International Journal of Zoology

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

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

(i) (j)

Figure 1 Adult frog and developmental stages of the ornate frog Microhyla ornata (a) Adult M ornata (b) premetamorphic tadpole(Gosner stage 28) (c) premetamorphic tadpole (Gosner stage 31) (d) prometamorphic tadpole (Gosner stage 36) (e) prometamorphictadpole (Gosner stage 39) (f) prometamorphic tadpole (Gosner stage 40) (g) metamorphic stage (Gosner stage 42) (h) metamorphic stage(Gosner stage 43) (i) metamorphic stage (Gosner stage 45) and (j) metamorphic stage (Gosner stage 46) Scale bar = 5mm

[12] toxicities of some heavy metals [15] morphologicaland acoustic comparisons between M ornata M fissipesand M okinavensis [16] taxonomic relationships usingmtDNA analysis [17] and changes in polyamine contents[18] However hematological study of both tadpoles andadult frogs remains unexplored in this species in IndiaStudy of hematological parameters during premetamorphicprometamorphic and metamorphic stages is crucial to gainproper understanding of changes occurring in the bloodcells during metamorphosis as change in mode of life bringsabout several new challenges This paper provides the recordof blood cell profile during development and metamorphosisin the tadpoles and also adult frogs ofM ornata

2 Materials and Methods

21 Collection of Egg Nests and Adult Frogs Egg nests andadult frogs of Microhyla ornata were collected from theirnatural habitat The egg nests were kept in plastic tubs con-taining conditioned 20mm deep tap water (tap water storedand aerated for 72 hours)The tadpoles were reared followingstandardized procedure [19] and were fed with yolk of boiledegg of Gallus gallus domesticus (breed White Leghorn) adlibitum The adult frogs (Figure 1(a)) after collection weremaintained in the terrarium for acclimatization to laboratoryconditions All procedureswere approved by theAnimal CareReview Committee at Utkal University

22 Tadpoles Investigated For the study tadpoles fromGosner stages 26 to 46 [20] were considered which werecomparable to the Taylor andKollros [21] stages I to XXV Tennumbers of tadpoles for each stage were selected for investi-gationThe tadpoles were divided into three subgroups basedon developmental periods that are premetamorphic (stages25 to 35) (Figures 1(b) and 1(c)) prometamorphic (stages36 to 41) (Figures 1(d) 1(e) and 1(f)) and metamorphic(stages 42 to 46) (Figures 1(g) 1(h) 1(i) and 1(j)) accordingto McDiarmid and Altig [22] Prior to drawing blood forperipheral smear snout to tail tip length (STL) of tadpolesand snout to vent length of metamorphosed froglets weremeasured

23 Preparation of Blood Smears Procedures described byDas and Mahapatra [23] were followed Blood samples foradult frogs were successfully collected following standardizedmethods [24] without harming the animals in the morninghours Tadpoles were anesthetized by exposing them to 03MS-222 (Tricaine Methane Sulphonate) solution The bloodof tadpoles from stage 26 to 44 was obtained from tailamputation through the middle of the tail For stages 45 and46 blood was collected from the heart using a 26 gaugesyringe needle Blood smears were prepared using push slidetechniqueThedried blood smearswere stainedwithGiemsarsquosstain or Leishmanrsquos stain and were observed under lightmicroscope (Hund H500)

International Journal of Zoology 3

24 Identification and Counting of Blood Cells Erythrocytesand their variations were identified following Hadji-Azimiet al [25] Sood [26] and Thrall [27] The leucocyteswere identified as lymphocytes monocytes eosinophils neu-trophils and basophils following Hadji-Azimi et al [25] andThrall [27] Slides were viewed in zigzag pattern coveringall parts of the blood smear and all leucocytes were countedin each field of view until 100 cells were counted Per bloodsmear 150 fields of views were randomly selected to assesserythrocytes Only field of views with even distribution oferythrocytes was used For the morphometric analysis oferythrocytes fifty cells per blood smears were measuredThesize of erythrocytes and their nuclei was measured by anocular micrometer which was standardized against a stagemicrometer (ERMA Japan) For morphometric analysisthe formula of Arserim and Mermer [28] was followedPhotographs of the leucocytes and erythrocytes were takenwith the help of a Canon EOS 450 122-megapixel camera(EF-S 18ndash55 1S Kit) connected to Hund 500 WETZLARmicroscope

25 Statistical Analysis The relationship between develop-mental stages of tadpoles and blood cell profiles was assessedby drawing scatter plots The correlation coefficient ldquo119903rdquo wascalculated in each case by Karl Pearsonrsquos method [29]

3 Results

31 Blood Cells Profile of the Tadpoles of M ornata

311 Morphology of Erythrocytes The erythrocytes in allstages of the tadpoles were found to be circular (Figure 2(a))oval (Figure 2(b)) and elliptical in shape (Figure 2(c)) Nucleiof these three types of erythrocytes were round in shapeand placed mostly in the center However few erythrocyteshad eccentric nuclei pushed to the periphery (Figure 2(d))In tadpoles of stages 37 to 42 the erythrocytes were eitherunusually larger in size or smaller than the normal erythro-cytes (Figures 2(e) and 2(f)) Poikilocytosis (20ndash30) wasobserved during different developmental stages (stages 26 to33) Few erythrocytes (01ndash05) lacked distinct membrane(Figure 2(g)) and some (05ndash1) showed irregular shapes(Figures 2(h) 2(i) and 2(j)) Several tear drop forms (5ndash7)(also called dacrocytes) with tapering or slightly blunt ends(Figure 2(k)) and comma-shaped cells (Figure 2(l)) werenoticed in tadpoles between stages 38 and 42 Some erythro-cytes (1-2) were found to lack nuclei (Figure 2(m)) whilein others (05ndash1) the nuclei were indistinct (Figure 2(n))Several smaller dark bodies surrounding regular erythrocyteswere found in tadpoles of stages 40ndash42 (Figure 2(o)) Inseveral smears (stages 40 to 42) the erythrocytes werefound to have vacuole like structures where the nuclei werepushed to the extreme periphery (Figure 2(p)) Dark patcheswere found inside some intact erythrocytes (Figure 2(q)) Inother such cells the membrane appeared to be disintegrated(Figure 2(r))

Of all the variations in the shape of the erythrocytesthe most remarkable was the presence of several kinds ofcrenulated erythrocytes that had strong resemblance withechinocytes and acanthocytes as reported in mammalianperipheral smears The crenulations showed a pattern ofappearance as they were found in the peripheral smear of tad-poles between Gosner stages 37 and 45 Initially crenulationsappeared as fine serrations in the erythrocytes of tadpolesof Gosner stages 37ndash40 resembling echinocytes (Figures 3(a)and 3(b)) Subsequently highly crenulated forms resemblingacanthocytes appeared on erythrocytes towards the climaxstage (Gosner stages 41 to 45) (Figures 3(c) 3(d) and3(e)) However the maximum number of such elaboratelyspiked cells was seen in stage 44 tadpoles These cells wereabsent in the froglets of stage 46 The crenulated cells hadapproximately 8ndash10 spiny projections distributed uniformlyall over (Figures 3(c) and 3(d)) while few had developed3-4 projections only at one side (Figure 3(e)) Moreoverseveral degenerating erythrocytes (Figure 3(f)) were recordedin the blood smears of the tadpoles of stages 42 to 44 Inthese stages many erythrocytes were in the state of division(Figures 3(g) 3(h) 3(i) and 3(j)) Aggregation of erythrocyteswas evident throughout developmental stages of the tadpoles(Figure 3(k))

312 Morphology of Leucocytes Leucocytes were of fivedifferent types that is lymphocytes monocytes eosinophilsneutrophils and basophils The lymphocytes both large andsmall (Figures 4(a) and 4(b) resp) were round in shapeTheir nuclei were also rounded and occupied almost theentire cell leaving a narrow rim of light violet cytoplasmtowards the periphery Some lymphocytes showed irregularmembrane (Figure 4(c)) Monocytes had eccentrically placedindented nuclei (Figure 4(d)) Eosinophils showed bilobednuclei where the connections between the lobes were dis-tinct (Figure 4(e)) Neutrophils (Figure 4(f)) with trilobedand tetralobed nuclei were also observed Neutrophils withtrilobed nuclei were themost common in tadpoles Basophilswere identified by the presence of dark violet stained granulesover the nuclei as well as entire cells (Figure 4(g))

313 Blood Thrombocytes The blood thrombocytes weresmall in size and were found in clusters of 8 to 25 cells(Figure 4(h))

314 Morphometry of Erythrocytes The length (L) of ery-throcytes ranged from 1530 plusmn 294 120583m (Gosner stage 42)to 1999 plusmn 260 120583m (Gosner stage 44) while the breadth(B) of the erythrocytes ranged from 1530 plusmn 224 120583m and1530 plusmn 364 120583m (Gosner stages 36 and 43 resp) to 1893 plusmn211 120583m (Gosner stage 30) respectively (Table 1) Similarlythe length (1198711015840) and breadth (1198611015840) of the nuclei ranged from742 plusmn 150 120583m (Gosner stage 42) to 1060 plusmn 179 120583m (Gosnerstage 27) and 742 plusmn 150 120583m (Gosner stages 41 and 43) to1030 plusmn 148 120583m (Gosner stage 30) respectively (Table 1)Moreover area occupied by the erythrocytes (A) ranged from18206 plusmn 2290 120583m2 (Gosner stage 43) to 28200 plusmn 4194 120583m2(Gosner stage 30) while the area of nuclei of the erythrocytes

4 International Journal of Zoology

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

(a) (b) (c)

(m) (n) (o)

(g)(e) (f)(d)

(p) (q) (r)

Figure 2 Erythrocytes and its variations in the tadpoles of the ornate frogMicrohyla ornata (a) Circular erythrocytes (b) oval erythrocyte(c) elliptical erythrocytes (d) erythrocyte with eccentrically positioned nucleus (e) large sized erythrocyte (f) small sized erythrocyte and(g) erythrocyte lacking a distinct membrane (stained in Leishmanrsquos stain) ((h) (i) (j)) Irregular shaped erythrocytes (k) tear drop shapederythrocyte (stained in Leishmanrsquos stain) (l) comma shaped erythrocyte (stained in Leishmanrsquos stain) (m) erythrocyte lacking nucleus(n) erythrocyte lacking a distinct nucleus (o) small dark bodies surrounding regular erythrocytes (p) large erythrocyte with a vacuolatedstructure and nucleus pushed towards the periphery (q) dark patches in intact erythrocytes and (r) Disintegrated erythrocyte with darkpatches Scale bar = 10 120583m

(1198601015840) ranged from 4505 plusmn 1286 120583m2 (Gosner stage 42) to8397 plusmn 2328 120583m2 (Gosner stage 30) The ratio of the areaof the nuclei of erythrocytes to the area of the erythrocytes(1198601015840A) ranged from 021 plusmn 003 (Gosner stage 40) to 032 plusmn003 (Gosner stage 28) The length to breadth (LB) ratio ofthe erythrocytes ranged between 083 plusmn 011 (Gosner stage45) and 108 plusmn 011 (Gosner stage 27 and Gosner stage 40)while the length to breadth ratio of the nuclei (11987110158401198611015840) ranged

from 094 plusmn 006 (Gosner stage 32) to 116 plusmn 006 (Gosnerstage 43) in the tadpoles of different developmental stages

315 Differential Leucocyte Count Lymphocytes were themost abundant cells amongst all leucocytes (Table 2) Therewas a surge in their percentage from 4809 plusmn 12 (stage46) to 9588 plusmn 16 (stage 26) The percentage of monocytesranged from 0 to 1706 plusmn 09 The highest percentage of

International Journal of Zoology 5

(g) (h) (i)

(j) (k)

(a) (b) (c)

(d) (e) (f)

Figure 3 Different types of erythrocytes in the tadpoles of the ornate frogMicrohyla ornata (a) Early stage of crenulated erythrocytes and(b) erythrocyte with increased size and distinct serrations (resembling echinocytes) ((c) (d)) Erythrocytes (resembling acanthocytes) withdistinct spikes uniformly around the cell (e) erythrocyte (resembling acanthocytes) having spikes on only one side and (f) degeneratingerythrocyte and ((g) (h) (i) (j)) Dividing erythrocytes (k) Aggregation of erythrocytes Scale bar = 10 120583m

monocytes was found in stage 42 tadpoles Interestinglytheir percentage remained high during metamorphic stagesin comparison with the earlier stages The percentage ofeosinophils ranged from 0 to 1613 plusmn 03 and the highestpercentage was observed during stage 46 However theirnumber was low during the early developmental stagesThe neutrophils remained the second abundant leucocytesafter lymphocytes Their percentage ranged between 0 and1966 plusmn 09 No neutrophils were observed during stage29 while they were high during Gosner stage 46 (Table 2)

The highest percentage of basophils was recorded duringstage 46 tadpoles Their overall percentage was low in theperipheral blood smears of the tadpoles as their percentagefluctuated between 0 and 605 plusmn 02

32 Blood Cells Profile of Adult Microhyla ornata

321 Morphology of Erythrocytes The erythrocytes of adultM ornata were elliptical in shape (Figure 5(a)) while only

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 International Journal of Zoology

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

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

(i) (j)

Figure 1 Adult frog and developmental stages of the ornate frog Microhyla ornata (a) Adult M ornata (b) premetamorphic tadpole(Gosner stage 28) (c) premetamorphic tadpole (Gosner stage 31) (d) prometamorphic tadpole (Gosner stage 36) (e) prometamorphictadpole (Gosner stage 39) (f) prometamorphic tadpole (Gosner stage 40) (g) metamorphic stage (Gosner stage 42) (h) metamorphic stage(Gosner stage 43) (i) metamorphic stage (Gosner stage 45) and (j) metamorphic stage (Gosner stage 46) Scale bar = 5mm

[12] toxicities of some heavy metals [15] morphologicaland acoustic comparisons between M ornata M fissipesand M okinavensis [16] taxonomic relationships usingmtDNA analysis [17] and changes in polyamine contents[18] However hematological study of both tadpoles andadult frogs remains unexplored in this species in IndiaStudy of hematological parameters during premetamorphicprometamorphic and metamorphic stages is crucial to gainproper understanding of changes occurring in the bloodcells during metamorphosis as change in mode of life bringsabout several new challenges This paper provides the recordof blood cell profile during development and metamorphosisin the tadpoles and also adult frogs ofM ornata

2 Materials and Methods

21 Collection of Egg Nests and Adult Frogs Egg nests andadult frogs of Microhyla ornata were collected from theirnatural habitat The egg nests were kept in plastic tubs con-taining conditioned 20mm deep tap water (tap water storedand aerated for 72 hours)The tadpoles were reared followingstandardized procedure [19] and were fed with yolk of boiledegg of Gallus gallus domesticus (breed White Leghorn) adlibitum The adult frogs (Figure 1(a)) after collection weremaintained in the terrarium for acclimatization to laboratoryconditions All procedureswere approved by theAnimal CareReview Committee at Utkal University

22 Tadpoles Investigated For the study tadpoles fromGosner stages 26 to 46 [20] were considered which werecomparable to the Taylor andKollros [21] stages I to XXV Tennumbers of tadpoles for each stage were selected for investi-gationThe tadpoles were divided into three subgroups basedon developmental periods that are premetamorphic (stages25 to 35) (Figures 1(b) and 1(c)) prometamorphic (stages36 to 41) (Figures 1(d) 1(e) and 1(f)) and metamorphic(stages 42 to 46) (Figures 1(g) 1(h) 1(i) and 1(j)) accordingto McDiarmid and Altig [22] Prior to drawing blood forperipheral smear snout to tail tip length (STL) of tadpolesand snout to vent length of metamorphosed froglets weremeasured

23 Preparation of Blood Smears Procedures described byDas and Mahapatra [23] were followed Blood samples foradult frogs were successfully collected following standardizedmethods [24] without harming the animals in the morninghours Tadpoles were anesthetized by exposing them to 03MS-222 (Tricaine Methane Sulphonate) solution The bloodof tadpoles from stage 26 to 44 was obtained from tailamputation through the middle of the tail For stages 45 and46 blood was collected from the heart using a 26 gaugesyringe needle Blood smears were prepared using push slidetechniqueThedried blood smearswere stainedwithGiemsarsquosstain or Leishmanrsquos stain and were observed under lightmicroscope (Hund H500)

International Journal of Zoology 3

24 Identification and Counting of Blood Cells Erythrocytesand their variations were identified following Hadji-Azimiet al [25] Sood [26] and Thrall [27] The leucocyteswere identified as lymphocytes monocytes eosinophils neu-trophils and basophils following Hadji-Azimi et al [25] andThrall [27] Slides were viewed in zigzag pattern coveringall parts of the blood smear and all leucocytes were countedin each field of view until 100 cells were counted Per bloodsmear 150 fields of views were randomly selected to assesserythrocytes Only field of views with even distribution oferythrocytes was used For the morphometric analysis oferythrocytes fifty cells per blood smears were measuredThesize of erythrocytes and their nuclei was measured by anocular micrometer which was standardized against a stagemicrometer (ERMA Japan) For morphometric analysisthe formula of Arserim and Mermer [28] was followedPhotographs of the leucocytes and erythrocytes were takenwith the help of a Canon EOS 450 122-megapixel camera(EF-S 18ndash55 1S Kit) connected to Hund 500 WETZLARmicroscope

25 Statistical Analysis The relationship between develop-mental stages of tadpoles and blood cell profiles was assessedby drawing scatter plots The correlation coefficient ldquo119903rdquo wascalculated in each case by Karl Pearsonrsquos method [29]

3 Results

31 Blood Cells Profile of the Tadpoles of M ornata

311 Morphology of Erythrocytes The erythrocytes in allstages of the tadpoles were found to be circular (Figure 2(a))oval (Figure 2(b)) and elliptical in shape (Figure 2(c)) Nucleiof these three types of erythrocytes were round in shapeand placed mostly in the center However few erythrocyteshad eccentric nuclei pushed to the periphery (Figure 2(d))In tadpoles of stages 37 to 42 the erythrocytes were eitherunusually larger in size or smaller than the normal erythro-cytes (Figures 2(e) and 2(f)) Poikilocytosis (20ndash30) wasobserved during different developmental stages (stages 26 to33) Few erythrocytes (01ndash05) lacked distinct membrane(Figure 2(g)) and some (05ndash1) showed irregular shapes(Figures 2(h) 2(i) and 2(j)) Several tear drop forms (5ndash7)(also called dacrocytes) with tapering or slightly blunt ends(Figure 2(k)) and comma-shaped cells (Figure 2(l)) werenoticed in tadpoles between stages 38 and 42 Some erythro-cytes (1-2) were found to lack nuclei (Figure 2(m)) whilein others (05ndash1) the nuclei were indistinct (Figure 2(n))Several smaller dark bodies surrounding regular erythrocyteswere found in tadpoles of stages 40ndash42 (Figure 2(o)) Inseveral smears (stages 40 to 42) the erythrocytes werefound to have vacuole like structures where the nuclei werepushed to the extreme periphery (Figure 2(p)) Dark patcheswere found inside some intact erythrocytes (Figure 2(q)) Inother such cells the membrane appeared to be disintegrated(Figure 2(r))

Of all the variations in the shape of the erythrocytesthe most remarkable was the presence of several kinds ofcrenulated erythrocytes that had strong resemblance withechinocytes and acanthocytes as reported in mammalianperipheral smears The crenulations showed a pattern ofappearance as they were found in the peripheral smear of tad-poles between Gosner stages 37 and 45 Initially crenulationsappeared as fine serrations in the erythrocytes of tadpolesof Gosner stages 37ndash40 resembling echinocytes (Figures 3(a)and 3(b)) Subsequently highly crenulated forms resemblingacanthocytes appeared on erythrocytes towards the climaxstage (Gosner stages 41 to 45) (Figures 3(c) 3(d) and3(e)) However the maximum number of such elaboratelyspiked cells was seen in stage 44 tadpoles These cells wereabsent in the froglets of stage 46 The crenulated cells hadapproximately 8ndash10 spiny projections distributed uniformlyall over (Figures 3(c) and 3(d)) while few had developed3-4 projections only at one side (Figure 3(e)) Moreoverseveral degenerating erythrocytes (Figure 3(f)) were recordedin the blood smears of the tadpoles of stages 42 to 44 Inthese stages many erythrocytes were in the state of division(Figures 3(g) 3(h) 3(i) and 3(j)) Aggregation of erythrocyteswas evident throughout developmental stages of the tadpoles(Figure 3(k))

312 Morphology of Leucocytes Leucocytes were of fivedifferent types that is lymphocytes monocytes eosinophilsneutrophils and basophils The lymphocytes both large andsmall (Figures 4(a) and 4(b) resp) were round in shapeTheir nuclei were also rounded and occupied almost theentire cell leaving a narrow rim of light violet cytoplasmtowards the periphery Some lymphocytes showed irregularmembrane (Figure 4(c)) Monocytes had eccentrically placedindented nuclei (Figure 4(d)) Eosinophils showed bilobednuclei where the connections between the lobes were dis-tinct (Figure 4(e)) Neutrophils (Figure 4(f)) with trilobedand tetralobed nuclei were also observed Neutrophils withtrilobed nuclei were themost common in tadpoles Basophilswere identified by the presence of dark violet stained granulesover the nuclei as well as entire cells (Figure 4(g))

313 Blood Thrombocytes The blood thrombocytes weresmall in size and were found in clusters of 8 to 25 cells(Figure 4(h))

314 Morphometry of Erythrocytes The length (L) of ery-throcytes ranged from 1530 plusmn 294 120583m (Gosner stage 42)to 1999 plusmn 260 120583m (Gosner stage 44) while the breadth(B) of the erythrocytes ranged from 1530 plusmn 224 120583m and1530 plusmn 364 120583m (Gosner stages 36 and 43 resp) to 1893 plusmn211 120583m (Gosner stage 30) respectively (Table 1) Similarlythe length (1198711015840) and breadth (1198611015840) of the nuclei ranged from742 plusmn 150 120583m (Gosner stage 42) to 1060 plusmn 179 120583m (Gosnerstage 27) and 742 plusmn 150 120583m (Gosner stages 41 and 43) to1030 plusmn 148 120583m (Gosner stage 30) respectively (Table 1)Moreover area occupied by the erythrocytes (A) ranged from18206 plusmn 2290 120583m2 (Gosner stage 43) to 28200 plusmn 4194 120583m2(Gosner stage 30) while the area of nuclei of the erythrocytes

4 International Journal of Zoology

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

(a) (b) (c)

(m) (n) (o)

(g)(e) (f)(d)

(p) (q) (r)

Figure 2 Erythrocytes and its variations in the tadpoles of the ornate frogMicrohyla ornata (a) Circular erythrocytes (b) oval erythrocyte(c) elliptical erythrocytes (d) erythrocyte with eccentrically positioned nucleus (e) large sized erythrocyte (f) small sized erythrocyte and(g) erythrocyte lacking a distinct membrane (stained in Leishmanrsquos stain) ((h) (i) (j)) Irregular shaped erythrocytes (k) tear drop shapederythrocyte (stained in Leishmanrsquos stain) (l) comma shaped erythrocyte (stained in Leishmanrsquos stain) (m) erythrocyte lacking nucleus(n) erythrocyte lacking a distinct nucleus (o) small dark bodies surrounding regular erythrocytes (p) large erythrocyte with a vacuolatedstructure and nucleus pushed towards the periphery (q) dark patches in intact erythrocytes and (r) Disintegrated erythrocyte with darkpatches Scale bar = 10 120583m

(1198601015840) ranged from 4505 plusmn 1286 120583m2 (Gosner stage 42) to8397 plusmn 2328 120583m2 (Gosner stage 30) The ratio of the areaof the nuclei of erythrocytes to the area of the erythrocytes(1198601015840A) ranged from 021 plusmn 003 (Gosner stage 40) to 032 plusmn003 (Gosner stage 28) The length to breadth (LB) ratio ofthe erythrocytes ranged between 083 plusmn 011 (Gosner stage45) and 108 plusmn 011 (Gosner stage 27 and Gosner stage 40)while the length to breadth ratio of the nuclei (11987110158401198611015840) ranged

from 094 plusmn 006 (Gosner stage 32) to 116 plusmn 006 (Gosnerstage 43) in the tadpoles of different developmental stages

315 Differential Leucocyte Count Lymphocytes were themost abundant cells amongst all leucocytes (Table 2) Therewas a surge in their percentage from 4809 plusmn 12 (stage46) to 9588 plusmn 16 (stage 26) The percentage of monocytesranged from 0 to 1706 plusmn 09 The highest percentage of

International Journal of Zoology 5

(g) (h) (i)

(j) (k)

(a) (b) (c)

(d) (e) (f)

Figure 3 Different types of erythrocytes in the tadpoles of the ornate frogMicrohyla ornata (a) Early stage of crenulated erythrocytes and(b) erythrocyte with increased size and distinct serrations (resembling echinocytes) ((c) (d)) Erythrocytes (resembling acanthocytes) withdistinct spikes uniformly around the cell (e) erythrocyte (resembling acanthocytes) having spikes on only one side and (f) degeneratingerythrocyte and ((g) (h) (i) (j)) Dividing erythrocytes (k) Aggregation of erythrocytes Scale bar = 10 120583m

monocytes was found in stage 42 tadpoles Interestinglytheir percentage remained high during metamorphic stagesin comparison with the earlier stages The percentage ofeosinophils ranged from 0 to 1613 plusmn 03 and the highestpercentage was observed during stage 46 However theirnumber was low during the early developmental stagesThe neutrophils remained the second abundant leucocytesafter lymphocytes Their percentage ranged between 0 and1966 plusmn 09 No neutrophils were observed during stage29 while they were high during Gosner stage 46 (Table 2)

The highest percentage of basophils was recorded duringstage 46 tadpoles Their overall percentage was low in theperipheral blood smears of the tadpoles as their percentagefluctuated between 0 and 605 plusmn 02

32 Blood Cells Profile of Adult Microhyla ornata

321 Morphology of Erythrocytes The erythrocytes of adultM ornata were elliptical in shape (Figure 5(a)) while only

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Zoology 3

24 Identification and Counting of Blood Cells Erythrocytesand their variations were identified following Hadji-Azimiet al [25] Sood [26] and Thrall [27] The leucocyteswere identified as lymphocytes monocytes eosinophils neu-trophils and basophils following Hadji-Azimi et al [25] andThrall [27] Slides were viewed in zigzag pattern coveringall parts of the blood smear and all leucocytes were countedin each field of view until 100 cells were counted Per bloodsmear 150 fields of views were randomly selected to assesserythrocytes Only field of views with even distribution oferythrocytes was used For the morphometric analysis oferythrocytes fifty cells per blood smears were measuredThesize of erythrocytes and their nuclei was measured by anocular micrometer which was standardized against a stagemicrometer (ERMA Japan) For morphometric analysisthe formula of Arserim and Mermer [28] was followedPhotographs of the leucocytes and erythrocytes were takenwith the help of a Canon EOS 450 122-megapixel camera(EF-S 18ndash55 1S Kit) connected to Hund 500 WETZLARmicroscope

25 Statistical Analysis The relationship between develop-mental stages of tadpoles and blood cell profiles was assessedby drawing scatter plots The correlation coefficient ldquo119903rdquo wascalculated in each case by Karl Pearsonrsquos method [29]

3 Results

31 Blood Cells Profile of the Tadpoles of M ornata

311 Morphology of Erythrocytes The erythrocytes in allstages of the tadpoles were found to be circular (Figure 2(a))oval (Figure 2(b)) and elliptical in shape (Figure 2(c)) Nucleiof these three types of erythrocytes were round in shapeand placed mostly in the center However few erythrocyteshad eccentric nuclei pushed to the periphery (Figure 2(d))In tadpoles of stages 37 to 42 the erythrocytes were eitherunusually larger in size or smaller than the normal erythro-cytes (Figures 2(e) and 2(f)) Poikilocytosis (20ndash30) wasobserved during different developmental stages (stages 26 to33) Few erythrocytes (01ndash05) lacked distinct membrane(Figure 2(g)) and some (05ndash1) showed irregular shapes(Figures 2(h) 2(i) and 2(j)) Several tear drop forms (5ndash7)(also called dacrocytes) with tapering or slightly blunt ends(Figure 2(k)) and comma-shaped cells (Figure 2(l)) werenoticed in tadpoles between stages 38 and 42 Some erythro-cytes (1-2) were found to lack nuclei (Figure 2(m)) whilein others (05ndash1) the nuclei were indistinct (Figure 2(n))Several smaller dark bodies surrounding regular erythrocyteswere found in tadpoles of stages 40ndash42 (Figure 2(o)) Inseveral smears (stages 40 to 42) the erythrocytes werefound to have vacuole like structures where the nuclei werepushed to the extreme periphery (Figure 2(p)) Dark patcheswere found inside some intact erythrocytes (Figure 2(q)) Inother such cells the membrane appeared to be disintegrated(Figure 2(r))

Of all the variations in the shape of the erythrocytesthe most remarkable was the presence of several kinds ofcrenulated erythrocytes that had strong resemblance withechinocytes and acanthocytes as reported in mammalianperipheral smears The crenulations showed a pattern ofappearance as they were found in the peripheral smear of tad-poles between Gosner stages 37 and 45 Initially crenulationsappeared as fine serrations in the erythrocytes of tadpolesof Gosner stages 37ndash40 resembling echinocytes (Figures 3(a)and 3(b)) Subsequently highly crenulated forms resemblingacanthocytes appeared on erythrocytes towards the climaxstage (Gosner stages 41 to 45) (Figures 3(c) 3(d) and3(e)) However the maximum number of such elaboratelyspiked cells was seen in stage 44 tadpoles These cells wereabsent in the froglets of stage 46 The crenulated cells hadapproximately 8ndash10 spiny projections distributed uniformlyall over (Figures 3(c) and 3(d)) while few had developed3-4 projections only at one side (Figure 3(e)) Moreoverseveral degenerating erythrocytes (Figure 3(f)) were recordedin the blood smears of the tadpoles of stages 42 to 44 Inthese stages many erythrocytes were in the state of division(Figures 3(g) 3(h) 3(i) and 3(j)) Aggregation of erythrocyteswas evident throughout developmental stages of the tadpoles(Figure 3(k))

312 Morphology of Leucocytes Leucocytes were of fivedifferent types that is lymphocytes monocytes eosinophilsneutrophils and basophils The lymphocytes both large andsmall (Figures 4(a) and 4(b) resp) were round in shapeTheir nuclei were also rounded and occupied almost theentire cell leaving a narrow rim of light violet cytoplasmtowards the periphery Some lymphocytes showed irregularmembrane (Figure 4(c)) Monocytes had eccentrically placedindented nuclei (Figure 4(d)) Eosinophils showed bilobednuclei where the connections between the lobes were dis-tinct (Figure 4(e)) Neutrophils (Figure 4(f)) with trilobedand tetralobed nuclei were also observed Neutrophils withtrilobed nuclei were themost common in tadpoles Basophilswere identified by the presence of dark violet stained granulesover the nuclei as well as entire cells (Figure 4(g))

313 Blood Thrombocytes The blood thrombocytes weresmall in size and were found in clusters of 8 to 25 cells(Figure 4(h))

314 Morphometry of Erythrocytes The length (L) of ery-throcytes ranged from 1530 plusmn 294 120583m (Gosner stage 42)to 1999 plusmn 260 120583m (Gosner stage 44) while the breadth(B) of the erythrocytes ranged from 1530 plusmn 224 120583m and1530 plusmn 364 120583m (Gosner stages 36 and 43 resp) to 1893 plusmn211 120583m (Gosner stage 30) respectively (Table 1) Similarlythe length (1198711015840) and breadth (1198611015840) of the nuclei ranged from742 plusmn 150 120583m (Gosner stage 42) to 1060 plusmn 179 120583m (Gosnerstage 27) and 742 plusmn 150 120583m (Gosner stages 41 and 43) to1030 plusmn 148 120583m (Gosner stage 30) respectively (Table 1)Moreover area occupied by the erythrocytes (A) ranged from18206 plusmn 2290 120583m2 (Gosner stage 43) to 28200 plusmn 4194 120583m2(Gosner stage 30) while the area of nuclei of the erythrocytes

4 International Journal of Zoology

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

(a) (b) (c)

(m) (n) (o)

(g)(e) (f)(d)

(p) (q) (r)

Figure 2 Erythrocytes and its variations in the tadpoles of the ornate frogMicrohyla ornata (a) Circular erythrocytes (b) oval erythrocyte(c) elliptical erythrocytes (d) erythrocyte with eccentrically positioned nucleus (e) large sized erythrocyte (f) small sized erythrocyte and(g) erythrocyte lacking a distinct membrane (stained in Leishmanrsquos stain) ((h) (i) (j)) Irregular shaped erythrocytes (k) tear drop shapederythrocyte (stained in Leishmanrsquos stain) (l) comma shaped erythrocyte (stained in Leishmanrsquos stain) (m) erythrocyte lacking nucleus(n) erythrocyte lacking a distinct nucleus (o) small dark bodies surrounding regular erythrocytes (p) large erythrocyte with a vacuolatedstructure and nucleus pushed towards the periphery (q) dark patches in intact erythrocytes and (r) Disintegrated erythrocyte with darkpatches Scale bar = 10 120583m

(1198601015840) ranged from 4505 plusmn 1286 120583m2 (Gosner stage 42) to8397 plusmn 2328 120583m2 (Gosner stage 30) The ratio of the areaof the nuclei of erythrocytes to the area of the erythrocytes(1198601015840A) ranged from 021 plusmn 003 (Gosner stage 40) to 032 plusmn003 (Gosner stage 28) The length to breadth (LB) ratio ofthe erythrocytes ranged between 083 plusmn 011 (Gosner stage45) and 108 plusmn 011 (Gosner stage 27 and Gosner stage 40)while the length to breadth ratio of the nuclei (11987110158401198611015840) ranged

from 094 plusmn 006 (Gosner stage 32) to 116 plusmn 006 (Gosnerstage 43) in the tadpoles of different developmental stages

315 Differential Leucocyte Count Lymphocytes were themost abundant cells amongst all leucocytes (Table 2) Therewas a surge in their percentage from 4809 plusmn 12 (stage46) to 9588 plusmn 16 (stage 26) The percentage of monocytesranged from 0 to 1706 plusmn 09 The highest percentage of

International Journal of Zoology 5

(g) (h) (i)

(j) (k)

(a) (b) (c)

(d) (e) (f)

Figure 3 Different types of erythrocytes in the tadpoles of the ornate frogMicrohyla ornata (a) Early stage of crenulated erythrocytes and(b) erythrocyte with increased size and distinct serrations (resembling echinocytes) ((c) (d)) Erythrocytes (resembling acanthocytes) withdistinct spikes uniformly around the cell (e) erythrocyte (resembling acanthocytes) having spikes on only one side and (f) degeneratingerythrocyte and ((g) (h) (i) (j)) Dividing erythrocytes (k) Aggregation of erythrocytes Scale bar = 10 120583m

monocytes was found in stage 42 tadpoles Interestinglytheir percentage remained high during metamorphic stagesin comparison with the earlier stages The percentage ofeosinophils ranged from 0 to 1613 plusmn 03 and the highestpercentage was observed during stage 46 However theirnumber was low during the early developmental stagesThe neutrophils remained the second abundant leucocytesafter lymphocytes Their percentage ranged between 0 and1966 plusmn 09 No neutrophils were observed during stage29 while they were high during Gosner stage 46 (Table 2)

The highest percentage of basophils was recorded duringstage 46 tadpoles Their overall percentage was low in theperipheral blood smears of the tadpoles as their percentagefluctuated between 0 and 605 plusmn 02

32 Blood Cells Profile of Adult Microhyla ornata

321 Morphology of Erythrocytes The erythrocytes of adultM ornata were elliptical in shape (Figure 5(a)) while only

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

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International Journal of

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International Journal of

Microbiology

4 International Journal of Zoology

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

(a) (b) (c)

(m) (n) (o)

(g)(e) (f)(d)

(p) (q) (r)

Figure 2 Erythrocytes and its variations in the tadpoles of the ornate frogMicrohyla ornata (a) Circular erythrocytes (b) oval erythrocyte(c) elliptical erythrocytes (d) erythrocyte with eccentrically positioned nucleus (e) large sized erythrocyte (f) small sized erythrocyte and(g) erythrocyte lacking a distinct membrane (stained in Leishmanrsquos stain) ((h) (i) (j)) Irregular shaped erythrocytes (k) tear drop shapederythrocyte (stained in Leishmanrsquos stain) (l) comma shaped erythrocyte (stained in Leishmanrsquos stain) (m) erythrocyte lacking nucleus(n) erythrocyte lacking a distinct nucleus (o) small dark bodies surrounding regular erythrocytes (p) large erythrocyte with a vacuolatedstructure and nucleus pushed towards the periphery (q) dark patches in intact erythrocytes and (r) Disintegrated erythrocyte with darkpatches Scale bar = 10 120583m

(1198601015840) ranged from 4505 plusmn 1286 120583m2 (Gosner stage 42) to8397 plusmn 2328 120583m2 (Gosner stage 30) The ratio of the areaof the nuclei of erythrocytes to the area of the erythrocytes(1198601015840A) ranged from 021 plusmn 003 (Gosner stage 40) to 032 plusmn003 (Gosner stage 28) The length to breadth (LB) ratio ofthe erythrocytes ranged between 083 plusmn 011 (Gosner stage45) and 108 plusmn 011 (Gosner stage 27 and Gosner stage 40)while the length to breadth ratio of the nuclei (11987110158401198611015840) ranged

from 094 plusmn 006 (Gosner stage 32) to 116 plusmn 006 (Gosnerstage 43) in the tadpoles of different developmental stages

315 Differential Leucocyte Count Lymphocytes were themost abundant cells amongst all leucocytes (Table 2) Therewas a surge in their percentage from 4809 plusmn 12 (stage46) to 9588 plusmn 16 (stage 26) The percentage of monocytesranged from 0 to 1706 plusmn 09 The highest percentage of

International Journal of Zoology 5

(g) (h) (i)

(j) (k)

(a) (b) (c)

(d) (e) (f)

Figure 3 Different types of erythrocytes in the tadpoles of the ornate frogMicrohyla ornata (a) Early stage of crenulated erythrocytes and(b) erythrocyte with increased size and distinct serrations (resembling echinocytes) ((c) (d)) Erythrocytes (resembling acanthocytes) withdistinct spikes uniformly around the cell (e) erythrocyte (resembling acanthocytes) having spikes on only one side and (f) degeneratingerythrocyte and ((g) (h) (i) (j)) Dividing erythrocytes (k) Aggregation of erythrocytes Scale bar = 10 120583m

monocytes was found in stage 42 tadpoles Interestinglytheir percentage remained high during metamorphic stagesin comparison with the earlier stages The percentage ofeosinophils ranged from 0 to 1613 plusmn 03 and the highestpercentage was observed during stage 46 However theirnumber was low during the early developmental stagesThe neutrophils remained the second abundant leucocytesafter lymphocytes Their percentage ranged between 0 and1966 plusmn 09 No neutrophils were observed during stage29 while they were high during Gosner stage 46 (Table 2)

The highest percentage of basophils was recorded duringstage 46 tadpoles Their overall percentage was low in theperipheral blood smears of the tadpoles as their percentagefluctuated between 0 and 605 plusmn 02

32 Blood Cells Profile of Adult Microhyla ornata

321 Morphology of Erythrocytes The erythrocytes of adultM ornata were elliptical in shape (Figure 5(a)) while only

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Zoology 5

(g) (h) (i)

(j) (k)

(a) (b) (c)

(d) (e) (f)

Figure 3 Different types of erythrocytes in the tadpoles of the ornate frogMicrohyla ornata (a) Early stage of crenulated erythrocytes and(b) erythrocyte with increased size and distinct serrations (resembling echinocytes) ((c) (d)) Erythrocytes (resembling acanthocytes) withdistinct spikes uniformly around the cell (e) erythrocyte (resembling acanthocytes) having spikes on only one side and (f) degeneratingerythrocyte and ((g) (h) (i) (j)) Dividing erythrocytes (k) Aggregation of erythrocytes Scale bar = 10 120583m

monocytes was found in stage 42 tadpoles Interestinglytheir percentage remained high during metamorphic stagesin comparison with the earlier stages The percentage ofeosinophils ranged from 0 to 1613 plusmn 03 and the highestpercentage was observed during stage 46 However theirnumber was low during the early developmental stagesThe neutrophils remained the second abundant leucocytesafter lymphocytes Their percentage ranged between 0 and1966 plusmn 09 No neutrophils were observed during stage29 while they were high during Gosner stage 46 (Table 2)

The highest percentage of basophils was recorded duringstage 46 tadpoles Their overall percentage was low in theperipheral blood smears of the tadpoles as their percentagefluctuated between 0 and 605 plusmn 02

32 Blood Cells Profile of Adult Microhyla ornata

321 Morphology of Erythrocytes The erythrocytes of adultM ornata were elliptical in shape (Figure 5(a)) while only

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 International Journal of Zoology

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

(e) (f) (g)

(h)

Figure 4 Leucocytes and thrombocytes in the tadpoles of the ornate frog Microhyla ornata (a) Large lymphocyte (b) small lymphocyte(c) lymphocyte with irregular margin (stained in Leishmanrsquos stain) (d) monocyte (stained in Leishmanrsquos stain) (e) eosinophil (f) neutrophil(g) basophil and (h) thrombocytes Scale bar = 10 120583m

few were found to be circular (Figure 5(b)) with centrallyplaced nuclei Only a few tear drop shaped cells (Figure 5(c))and comma shaped cells (Figure 5(d)) were found and othervariations in shapes of erythrocytes were not recorded at all

322 Morphology of Leucocytes Both granulocytes andagranulocytes were found in the peripheral blood smearof adult frogs The lymphocytes were of two types large(Figure 5(e)) and small (Figure 5(f)) Their nuclei were largecentrally placed and occupied majority of the area leavinga narrow rim of cytoplasm Monocytes (Figure 5(g)) hadeccentrically placed indented nuclei Eosinophils were iden-tified with the bilobed nuclei (Figure 5(h)) Neutrophils hadmultilobed nuclei Usually 3 or 4 lobeswere recorded (Figures5(i) and 5(j) resp) However few cells had hypersegmentednuclei (Figures 5(k) and 5(l)) Several neutrophils had ring-like nucleus in which all lobes joined or folded to give a ring-like appearance (Figure 5(m)) Band neutrophils were also

recorded (Figure 5(n)) Basophils (Figure 5(o)) were darklystained and smaller cells with heavy granule deposition

323Thrombocytes Thrombocytes were nucleated and spin-dle shaped (Figure 5(p))

324 Morphometry of Erythrocytes The surface area coveredby erythrocytes in adult males was found to be 25439 plusmn4082 120583m2 while it was recorded to be 23829 plusmn 2975 120583m2 inadult females (Table 1) Therefore the surface area occupiedby erythrocytes was more in males than females The meanlength and breadth of erythrocytes were found to be 2211 plusmn216 120583m and 1318plusmn220 120583m in adult males In adult femalestheir values were found to be 2196 plusmn 211 120583m and 1393 plusmn222 120583m respectively Similarly the mean length and breadthof nuclei of erythrocytes in adult males were recorded tobe 1227 plusmn 116 120583m and 681 plusmn 162 120583m respectively while

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

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International Journal of

Microbiology

International Journal of Zoology 7

Table1Morph

ometry

oferythrocytes

durin

gthelarvald

evelo

pmentand

inthea

dults

oftheo

rnatefrogMicrohylaornata

Stages

STL

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Gosner

Taylor

and

Kollros

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)26

I15plusmn001605plusmn2221727plusmn289092plusmn01121845plusmn4811909plusmn165939plusmn132096plusmn0066810plusmn2006031plusmn003

27II

15plusmn001924plusmn1731772plusmn198108plusmn01126746plusmn36321060plusmn179939plusmn163112plusmn0067858plusmn2162029plusmn003

28III

17plusmn001711plusmn2461651plusmn178103plusmn01122167plusmn3581939plusmn132969plusmn181096plusmn0067136plusmn1658032plusmn003

29IV

17plusmn0041666plusmn2241681plusmn150099plusmn01121950plusmn3170939plusmn132924plusmn066101plusmn0066806plusmn1032031plusmn003

30V

17plusmn0041908plusmn2551893plusmn211100plusmn01128200plusmn41941030plusmn2001030plusmn14100plusmn0068397plusmn2328029plusmn003

31VI

17plusmn0121878plusmn1541818plusmn135103plusmn01126780plusmn2614924plusmn116863plusmn173107plusmn0066235plusmn1353023plusmn003

32VII

17plusmn0091545plusmn3691560plusmn307099plusmn01118278plusmn2976772plusmn150818plusmn168094plusmn0064866plusmn1040026plusmn003

33VIII

18plusmn0091757plusmn1811636plusmn176107plusmn01122458plusmn2316984plusmn131924plusmn066106plusmn0067136plusmn992031plusmn003

34IX

19plusmn0041665plusmn1511757plusmn181097plusmn01122996plusmn3173939plusmn090939plusmn090100plusmn0066990plusmn1513030plusmn003

35X

18plusmn001575plusmn1541560plusmn198100plusmn01119248plusmn2642772plusmn150802plusmn144096plusmn0064865plusmn1319025plusmn003

36XI

20plusmn001575plusmn1541530plusmn224102plusmn01119319plusmn3363924plusmn066833plusmn131110plusmn0066054plusmn1102031plusmn003

37XII

25plusmn001665plusmn1511590plusmn131104plusmn01120868plusmn3002909plusmn135802plusmn144113plusmn0065766plusmn1494027plusmn003

38XIII

24plusmn0161636plusmn1761621plusmn173100plusmn01120761plusmn2797893plusmn150818plusmn138109plusmn0065766plusmn1494027plusmn003

39XIV

24plusmn0181666plusmn2241666plusmn151100plusmn01121630plusmn2243878plusmn163818plusmn138107plusmn0065694plusmn1593026plusmn003

40XV

-XVII22plusmn021802plusmn1781666plusmn179108plusmn01123574plusmn3312833plusmn131772plusmn178107plusmn0065153plusmn1694021plusmn003

41XV

III-

XIX

23plusmn0041787plusmn1631651plusmn202108plusmn01123103plusmn3033818plusmn138742plusmn150110plusmn0064793plusmn1353027plusmn003

42XX

22plusmn001530plusmn2941726plusmn396088plusmn01119540plusmn4244742plusmn150772plusmn150096plusmn0064505plusmn1286022plusmn003

43XXI

14plusmn0041590plusmn3811530plusmn364103plusmn01118206plusmn2290863plusmn111742plusmn150116plusmn0065081plusmn13560290plusmn003

44XXII

12plusmn001999plusmn2601378plusmn310145plusmn01121127plusmn2424893plusmn066818plusmn138109plusmn0065766plusmn1138027plusmn003

45XXIII-

XXIV

07plusmn0081545plusmn2691848plusmn269083plusmn01121956plusmn2144848plusmn121878plusmn090096plusmn0065838plusmn989026plusmn003

46XXV

06plusmn001605plusmn1381621plusmn173099plusmn01120402plusmn2436818plusmn138759plusmn151107plusmn0064865plusmn1286023plusmn003

Correlationcoeffi

cient(119903)

minus0208

minus0348

+012

4minus0438

minus0591

minus0758

+0296

minus0713

minus0591

Sex(no

ofadult

frogs

used)

Average

body

weight(g)

plusmnSD

Average

SVL

(cm)plusmn

SD

Size

ofcell

Size

ofnu

clei

A1015840AplusmnSD

Leng

th(Lplusmn

SD)(120583m)

Breadth(B

plusmnSD

)(120583m)

LBplusmnSD

Area(

AplusmnSD

)(120583m2

)

Leng

th(1198711015840

plusmnSD

)(120583m)

Breadth

(1198611015840

plusmnSD

)(120583m)

L1015840B1015840

plusmnSD

Area

(1198601015840

plusmnSD

)(120583m2

)

Males

(7)

1012plusmn

084

18plusmn031

2211plusmn216

1318plusmn220

173plusmn034

25439plusmn40

82

1227plusmn116

681plusmn16

218

6plusmn032

6596plusmn1813

0265plusmn008

Females

(7)

1186plusmn

073

19plusmn012

2196plusmn211

1393plusmn222

162plusmn032

23829plusmn2975

1242plusmn16

3772plusmn17

816

6plusmn031

7643plusmn2351

0319plusmn008

STLsnou

ttotailtip

leng

thSVLsnou

ttovent

leng

thSDstand

arddeviation120583mm

icrometer

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 International Journal of Zoology

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

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

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

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

Figure 5 Blood cells of the adult ornate frog Microhyla ornata (a) Elliptical erythrocytes (b) circular erythrocyte (c) tear drop shapederythrocyte (d) comma shaped erythrocyte (e) large lymphocyte (f) small lymphocyte (g) monocyte (h) eosinophil (i) neutrophil with3 lobed nuclei (j) neutrophil with 4 lobed nuclei and ((k) (l)) neutrophils with hypersegmented nuclei (m) neutrophil with folded nucleiforming ring (n) band neutrophil (o) basophil and (p) thrombocyte Scale bar = 10 120583m

their values in adult females were found to 1242 plusmn 163 120583mand 772 plusmn 178 120583m respectively The ratio between the areaof nuclei of the erythrocytes to the area of the erythrocytes(1198601015840A) was found to be 026 plusmn 008 in male frogs while it was031 plusmn 008 in the females The length to breadth ratio of theerythrocytes (LB) was found to be 173 plusmn 034 and 162 plusmn032 in the male and female frogs respectively Similarly thelength to breadth ratio of nuclei of the erythrocytes (11987110158401198611015840) inadult males and females was recorded to be 186 plusmn 032 and166 plusmn 031 respectively

325 Differential Leucocyte Count The mean lymphocytesand monocytes percentage in adult males were found to be5042 plusmn 52 and 624 plusmn 12 respectively In adult femalesthe percentage was found to be 5128 plusmn 63 and 528 plusmn 18respectively In adult males mean eosinophil percentage was1021 plusmn 19 while it was 932 plusmn 19 in adult females (Table 2)Mean neutrophil percentage in adult males was 3041 plusmn 68and in adult females it was found to be 3121plusmn102Themeanbasophil percentage in adult males was 272plusmn10while it was291 plusmn 07 in adult females In the adult frogs percentage of

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Zoology 9

Table 2 Percentage of leucocytes during larval development and in the adults of the ornate frogMicrohyla ornata

Stages Lymphocytes plusmn SD Monocytes plusmn SD Eosinophils plusmn SD Neutrophils plusmn SD Basophils plusmn SDGosner stage Taylor and Kollros stage26 I 9588 plusmn 16 117 plusmn 16 000 plusmn 00 295 plusmn 00 000 plusmn 00

27 II 9286 plusmn 25 428 plusmn 15 000 plusmn 00 286 plusmn 15 000 plusmn 00

28 III 8787 plusmn 21 305 plusmn 21 060 plusmn 13 363 plusmn 13 485 plusmn 16

29 IV 8778 plusmn 24 112 plusmn 24 555 plusmn 39 000 plusmn 00 555 plusmn 00

30 V 9144 plusmn 10 428 plusmn 10 144 plusmn 00 142 plusmn 00 142 plusmn 10

31 VI 8775 plusmn 35 318 plusmn 01 000 plusmn 00 585 plusmn 27 322 plusmn 22

32 VII 9317 plusmn 05 106 plusmn 00 126 plusmn 04 323 plusmn 00 128 plusmn 04

33 VIII 8710 plusmn 22 000 plusmn 00 000 plusmn 00 839 plusmn 17 451 plusmn 17

34 IX 8709 plusmn 04 230 plusmn 01 125 plusmn 17 53 plusmn 20 406 plusmn 00

35 X 8585 plusmn 16 356 plusmn 08 059 plusmn 07 529 plusmn 17 471 plusmn 17

36 XI 9443 plusmn 07 187 plusmn 07 143 plusmn 00 227 plusmn 03 000 plusmn 00

37 XII 8645 plusmn 06 217 plusmn 04 233 plusmn 05 223 plusmn 08 582 plusmn 13

38 XIII 9371 plusmn 14 000 plusmn 00 000 plusmn 00 234 plusmn 05 395 plusmn 08

39 XIV 8213 plusmn 11 421 plusmn 01 776 plusmn 11 276 plusmn 07 314 plusmn 02

40 XV-XVII 8867 plusmn 13 382 plusmn 12 186 plusmn 01 377 plusmn 00 188 plusmn 00

41 XVIII-XIX 8238 plusmn 13 392 plusmn 00 382 plusmn 02 784 plusmn 00 204 plusmn 15

42 XX 6109 plusmn 07 1706 plusmn 09 1508 plusmn 02 524 plusmn 03 153 plusmn 04

43 XXI 5880 plusmn 11 1108 plusmn 06 1477 plusmn 01 1209 plusmn 05 326 plusmn 08

44 XXII 5726 plusmn 01 943 plusmn 03 1506 plusmn 02 1619 plusmn 01 206 plusmn 03

45 XXIII-XXIV 5806 plusmn 01 917 plusmn 01 1284 plusmn 01 1501 plusmn 03 492 plusmn 04

46 XXV 5809 plusmn 12 1007 plusmn 01 1613 plusmn 03 1966 plusmn 09 605 plusmn 02

Correlation coefficient (119903) minus0801 +0654 +0779 +0719 +0249

Adult Frogs Males 5042 plusmn 52 624 plusmn 12 1021 plusmn 19 3041 plusmn 68 272 plusmn 10

Females 5128 plusmn 63 528 plusmn 18 932 plusmn 19 3121 plusmn 102 291 plusmn 07

SD standard deviation

monocytes and eosinophils was higher inmales than femalesbut percentages of lymphocytes basophils and neutrophilswere higher in females than males (Table 2)

33 Statistical Analysis In the tadpoles a negative correlationwas observed between different stages with respect to length(119903 = minus0208) breadth (119903 = minus0348) and area (119903 = minus0438) ofthe erythrocytes (Figure 6) Similar negative correlation wasobserved for length (119903 = minus0591) breadth (119903 = minus0758) andarea (119903 = minus0713) of nuclei of the erythrocytes (Figure 7)However the aspect ratio of the erythrocytes (119871119861) and theirnuclei (11987110158401198611015840) were positively correlated 119903 = +0124 inFigure 6(c) and 119903 = +0296 in Figure 7(c) respectivelyThe 1198601015840A ratio was found to be negatively correlated (119903 =minus0591) (Figure 7) The monocytes (119903 = +0654) eosinophils(119903 = +0779) neutrophils (119903 = +0719) and basophils (119903 =+0249) showed a positive correlation whereas lymphocytes(119903 = minus0801) showed a negative correlation with differentstages during development (Figure 8)

4 Discussion

The erythrocytes of the tadpoles ofM ornata were observedto be round oval or elliptical in shape (Figures 2(a) 2(b)and 2(c)) This observation is similar to previous studies

on larval amphibians which suggest presence of two generalforms of erythrocytes larval and adult forms [30ndash32] Thelarval form is large and elongated while the adult formis smaller and rounder Nucleus a characteristic featureof amphibian erythrocytes was found to be placed at thecenter of the erythrocytes in almost all peripheral smearsHowever few variationswere also recordedwhere nuclei wereeither eccentrically positioned or pushed to the periphery(Figure 2(d)) In tadpoles of stages 37 to 42 several erythro-cytes were noticed to be either unusually larger in size orsmaller than the normal erythrocytes (Figures 2(e) and 2(f))Few erythrocytes lacked a distinct membrane (Figure 2(g))while several others had irregular appearance (Figures 2(h)2(i) and 2(j)) Poikilocytosis was noticed in the peripheralblood smears of the tadpoles during the early stages inpatches only Abnormal shape of the erythrocytes includedseveral tear drop forms (Figure 2(k)) and comma-shapederythrocytes (Figure 2(l)) Some erythrocytes were found tolack nuclei (Figure 2(m)) while in others the nuclei wereindistinct (Figure 2(n)) Similar large sized erythrocytes anderythrocytes lacking nucleus (senile erythrocytes) have beenreported in tadpoles of Polypedates teraiensis [23] Such senileerythrocytes have also been reported during metamorphosisin other anurans [33] Several smaller dark bodies sur-rounding regular erythrocytes (Figure 2(o)) were observedin the blood smears of the tadpoles of stages 40 to 42 In

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 International Journal of Zoology

0

5

10

15

20

25

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Leng

th o

f ery

thro

cyte

s (120583

m)

r = minus0208y = minus0046x + 1864

(a)

02468

101214161820

26 28 30 32 34 36 38 40 42 44 46Gosner stages

r = minus0348y = minus0067x + 1903

Brea

dth

of er

ythr

ocyt

es (120583

m)

(b)

002040608

1121416

26 28 30 32 34 36 38 40 42 44 46

Leng

thb

read

th o

f ery

thro

cyte

s

Gosner stages

y = 0002x + 0944

r = +0124

(c)

0

50

100

150

200

250

300

26 28 30 32 34 36 38 40 42 44 46Gosner stages

Are

a of e

ryth

rocy

tes (120583

m2) y = minus1725x + 2805

r = minus0438

(d)

Figure 6 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

these stages few erythrocytes appeared large in size havingvacuolated structures that pushed the nuclei to the peripherymaking them look inconspicuous (Figure 2(p)) Moreoverdark patches were found inside some intact erythrocytes(Figure 2(q)) In other such cells the membrane appearedto be disintegrated (Figure 2(r)) Interestingly a remarkabledegree of crenulated erythrocytes was also noticed (Figures3(a) 3(b) 3(c) 3(d) and 3(e)) towards the late prometa-morphic and metamorphic stages Crenulations have beenpreviously reported in red blood cells of Rana pipiens afterforelimb emergence [30] These cells resembling echinocytesand acanthocytes of mammalian red blood cells have alsobeen reported earlier in thyroid-treated tadpoles of Ranacatesbeianawhere cells were irregular withmany cytoplasmicprojections [34] and in tadpoles of P teraiensis [23] Such cellshave been described to be present during anemic conditions[34 35] and the ectothermic animals are capable towithstandanemic conditions for a long period without mortality [2336] Erythrocytes which had undergone degeneration werealso observed (Figure 3(f))

The size and shape of erythrocyte give an indication ofthe surface available for the exchange of gases in respiratoryfunctions [37] Morphometric measurement of the erythro-cytes (Table 1) confirmed a negative correlation existingbetween the developmental stages with respect to the sizeof the erythrocytes and their nuclei However their aspectratio (lengthbreadth) was found to be positive A gradualdecline in the area occupied by the erythrocytes was observed

in the tadpoles with progress in development as a negativecorrelation existed between the developmental stages withrespect to the area of the erythrocytes and their nucleiThe smallest erythrocytes were present in the metamor-phosed froglets Broyles [38] Duellman and Trueb [39] havedescribed replacement of larger larval erythrocytes by smalleradult erythrocytes duringmetamorphosis in several anuransSeveral cells were found to be in different stages of divisionas nuclear division was distinct (Figures 3(g) 3(h) 3(i) and3(j)) An earlier report of increase in erythropoietic activityduring metamorphosis has also been reported in R cates-beiana [40] Another interesting observationwas aggregationof several erythrocytes during different developmental stages(Figure 3(k))

Several trendswith respect to the percentage of leucocytesduring different developmental stages appeared consistentwith the earlier observations [3 6 7 23] Lymphocyteswere found to be most abundant during the growth phaseof larval development Their percentage decreased towardsend of metamorphosis (Table 2) Davis has reported 70of lymphocytes in the tadpoles of stages 30 to 33 in Rcatesbeiana which decreased with onset of metamorphosisThe trend inmonocyte which is a phagocytic cell remained atpar with the earlier observations of Davis [3] Their numberincreased significantly during the metamorphic stages Thehigher number of monocytes has been correlated with theincreased cellular debris left over from the tissue lysis during

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Zoology 11

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0077x + 1170

r = minus0591

Leng

th o

f the

nuc

lei o

fer

ythr

ocyt

es (120583

m)

(a)

0

2

4

6

8

10

12

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus0100x + 1214

r = minus0758

Brea

dth

of th

e nuc

lei o

fer

ythr

ocyt

es (120583

m)

(b)

0

02

04

06

08

1

12

14

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

r = +0296

y = 0003x + 093

Leng

thb

read

th o

f nuc

lei o

fer

ythr

ocyt

es

(c)

0102030405060708090

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Are

a of n

ucle

i of e

ryth

rocy

tes (120583

m2)

r = minus0713y = minus1246x + 1050

(d)

0

005

01

015

02

025

03

035

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Are

a of n

ucle

i of e

ryth

rocy

tes

area

of

eryt

hroc

ytes

Gosner stages

y = minus0003x + 0392

r = minus0591

(e)

Figure 7 Correlation between different morphometric values of erythrocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

remodeling of larval structures [3] and a positive correlationwas observed with developmental stages

However an unusual spike of neutrophils was observedduring the metamorphic climax in the present study unlikethe earlier reports on Rana catesbeiana [3] and P teraien-sis [23] where these cells were least abundant during themetamorphic period Neutrophils remained significantlypositive with respect to the developmental stages The rise inthe level of neutrophil in this species is suggested to be relatedto physiological condition Eosinophils remained positively

correlated with the developmental stages of the tadpolesSince these cells are known to produce a variety of chemicalsubstances [41] and respond to tissue injury [42] an elevationin the level of eosinophils in the present study is suggestedto act to modulate the process of lysis of tissue during meta-morphosis The percentage of basophils fluctuated duringdevelopment However a positive correlation was observedbetween percentage of basophils and developmental stagesbut the correlation was not significant Increase in thebasophil level has been reported in tadpoles of R catesbeiana

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 International Journal of Zoology

0

20

40

60

80

100

120

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

y = minus1905x + 1499

r = minus0801

Lym

phoc

ytes

()

(a)

02468

1012141618

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Mon

ocyt

es (

)

y = 0456x minus 1184

r = +0654

(b)

0

5

10

15

20

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Eosin

ophi

ls (

)

minus5

y = 0757x minus 2240

r = +0779

(c)

0

5

10

15

20

25

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Neu

troph

ils (

)

y = 0612x minus 1589

r = +0719

(d)

0

1

2

3

4

5

6

7

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46Gosner stages

Baso

phils

()

y = 0078x + 0237

r = +0249

(e)

Figure 8 Correlation between the percentages of different types of leucocytes with respect to the developmental stages of the tadpoles of theornate frogMicrohyla ornata

[3] and Polypedates teraiensis [23]Thrombocytes were foundto be in clusters or stacked with each other during differentdevelopmental stages

The erythrocytes of adult Microhyla ornata were ellip-soidal in shape with centrally placed nuclei and only afew rounded erythrocytes were observed The characteristicshape of anuran erythrocyte has been reported to be ellip-soidal [43] Tok et al [44] have reported morphologicallysimilar erythrocytes among various species of anurans Ellip-soidal erythrocytes have also been reported in the bloodsmear of Polypedates maculatus [23] Erythrocytes being themost important carrier of oxygen and carbon dioxide it hasbeen suggested that an elliptical body is more efficient thana spherical one of the same volume as far as greater rate of

exchange is concerned [37] Only a few tear drop shapedcells and comma-shaped cells were found in the peripheralsmear However other variations in shapes of erythrocyteswere not recorded in the adults as found in their larvalcounterparts In the present study the surface area coveredby erythrocytes was found to be greater in males than infemales (Table 1) Similar reports of higher surface area oferythrocytes in males (24315 plusmn 7841120583m2) in comparisonwith females (21058 plusmn 38279120583m2) have been reported inadult frogs of Pmaculatus [23] In the present study themeanbreadth of the erythrocytes and their nuclei was found to beslightly higher in females than of males (Table 1) Arserimand Mermer [28] have reported larger erythrocyte lengthand breadth in cases of females (2303120583m and 1459 120583m

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Zoology 13

resp) than males (2232 120583m and 1365 120583m resp) in Ranamacrocnemis The size of erythrocytes of adult M ornatawas found to be higher in comparison with the erythrocyteslength (154 120583m plusmn 104) and breadth (1147 120583m plusmn 082) ofthe balloon frogGlyphogloossus molossus belonging to familymicrohylidae [45]

Leucocyte differential in the adult frogs showed per-centage of monocytes and eosinophils to be slightly higherin males than females but percentages of lymphocytesbasophils and neutrophils were higher in females Davis andDurso [4] have reported lymphocytes and neutrophils tobe the most commonly seen cell types within amphibiansThey have also reported the average of the white blood cellsfor anurans as 526 for lymphocytes 291 for neutrophils70 for eosinophils 75 for basophils and 40 for monocytesComparable percentages of lymphocytes and neutrophilswere observed in the present study But the percentages ofbasophils remained low while the percentages of eosinophilsand monocytes remained higher Thrombocytes in thepresent study were nucleated and spindle shaped Earlierreports suggest amphibian thrombocytes to be nucleated andhaving spindle appearance [13 28]

Thus our study provides the baseline information onblood cell profile of tadpoles and adults of the ornate frogMicrohyla ornata Moreover it describes the changes thattake place in shape size and number of blood cells duringmetamorphosis necessary for the aquatic tadpoles to adapt toa terrestrial environment as a froglet

Acknowledgments

The authors would like to thank the Head of the P G Depart-ment of Zoology Utkal University for providing necessaryfacilities Madhusmita Das would like to thank UGC for aResearch Fellowship (RFSMS) The authors would like tothank DST the Government of India for financial assistanceunder PURSE Grant to the P G Department of ZoologyUtkal University

References

[1] J Wojtaszek and A Adamowicz ldquoHaematology of the fire-bellied toadBombina bombina LrdquoComparative Clinical Pathol-ogy vol 12 no 3 pp 129ndash134 2003

[2] S F Gilbert Developmental Biology Sinauer Associates Sun-derland Mass USA 2003

[3] A K Davis ldquoMetamorphosis-related changes in leukocyteprofiles of larval bullfrogs (Rana catesbeiana)rdquo ComparativeClinical Pathology vol 18 no 2 pp 181ndash186 2009

[4] A K Davis and A M Durso ldquoWhite blood cell differentials ofnorthern cricket frogs (Acris c crepitans) with a compilationof published values from other amphibiansrdquoHerpetologica vol65 no 3 pp 260ndash267 2009

[5] M K Atatur H Arikan and I E Cevik ldquoErythrocyte sizes ofsome anurans from Turkeyrdquo Turkish Journal of Zoology vol 23pp 111ndash114 1999

[6] H E Jordan and C C Speidel ldquoLeukocytes in relation to themechanism of thyroid-accelerated metamorphosis in the larval

frogrdquo Proceedings of the Society for Experimental Biology andMedicine vol 20 pp 380ndash383 1922

[7] H E Jordan and C C Speidel ldquoThe behavior of the leucocytesduring coincident regeneration and thyroid-induced metamor-phosis in the frog larva with a consideration of growth factorsrdquoThe Journal of Experimental Medicine vol 40 pp 1ndash11 1924

[8] J M Kiesecker ldquoSynergism between trematode infection andpesticide exposure a link to amphibian limb deformities innaturerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 99 no 15 pp 9900ndash9904 2002

[9] S Barni E Boncompagni A Grosso et al ldquoEvaluation ofRana snk esculenta blood cell response to chemical stressors inthe environment during the larval and adult phasesrdquo AquaticToxicology vol 81 no 1 pp 45ndash54 2007

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

[11] F J Vernberg ldquoHematological studies in salamanders in rela-tion to their ecologyrdquo Herpetologica vol 11 no 2 pp 129ndash1331955

[12] K Mitsuru ldquoRelationships between number size and shape ofred blood cells in amphibiansrdquo Comparative Biochemistry andPhysiology A vol 69 no 4 pp 771ndash775 1981

[13] M C Allender and M M Fry ldquoAmphibian hematologyrdquoVeterinary Clinics of North America vol 11 no 3 pp 463ndash4802008

[14] P Mohanty-Hejmadi S K Dutta and I Khan ldquoLife history ofthe Indian frogs IIIThe ornate frogMicrohyla ornatardquo Journalof the Zoological Society of India vol 32 no 1-2 pp 43ndash48 1980

[15] J Rao andM N Madhyastha ldquoToxicities of some heavy metalsto the tadpoles of frogMicrohyla ornata (Dumeril amp Bibron)rdquoToxicology Letters vol 36 no 2 pp 205ndash208 1987

[16] M Kuramoto and S H Joshy ldquoMorphological and acousticcomparisons ofMicrohyla ornataM fissipes andM okinaven-sis (Anura Microhylidae)rdquo Current Herpetology vol 25 no 1pp 15ndash27 2006

[17] M Matsui H Ito T Shimada et al ldquoTaxonomic relationshipswithin the pan-oriental narrow-mouth toadMicrohyla ornataasrevealed by mtDNA analysis (Amphibia Anura Microhyli-dae)rdquo Zoological Science vol 22 no 4 pp 489ndash495 2005

[18] K Joseph and T G Baby ldquoChanges in polyamine contentsduring development of the frogMicrohyla ornatardquoDevelopmentGrowth and Differentiation vol 32 no 3 pp 329ndash334 1990

[19] P Mohanty-Hejmadi ldquoCare and management of amphibianembryosrdquo Prakruti Utkal University Journal vol 11 pp 81ndash871977

[20] K L Gosner ldquoA simplified table for staging anuran embryos andlarvaerdquo Herpetologica vol 16 pp 183ndash190 1960

[21] A C Taylor and J J Kollros ldquoStages in the normal developmentof Rana pipiens larvaerdquoTheAnatomical Record vol 94 no 1 pp7ndash23 1946

[22] W McDiarmid and R Altig Tadpoles The Biology of AnuranLarvae The University of Chicaga Press Chicago Ill USA2000

[23] 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

[24] K NWright ldquoAmphibian hematologyrdquo inAmphibianMedicineand Captive Husbandry K N Wright and B R Whitaker Edspp 129ndash146 Krieger Malabar Fla USA 1st edition 2001

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 International Journal of Zoology

[25] I Hadji-Azimi V Coosemans and C Canicatti ldquoAtlas ofadult Xenopus laevis laevis hematologyrdquo Developmental andComparative Immunology vol 11 no 4 pp 807ndash874 1987

[26] R Sood Haematology for Students and Practitioners JaypeeBrothers New Delhi India 1996

[27] M A Thrall ldquoHematology of amphibiansrdquo in VeterinaryHematology and Clinical Chemistry Test and Clinical TestPresentations M A Thrall D C Baker and E D Lassen EdsLippincott Williams and Wilkins Philadelphia Pa USA 2004

[28] S K Arserim and A Mermer ldquoHematology of the Uludagfrog Rana macrocnemis Boulenger 1885 in Uludag NationalPark (Bursa Turkey)rdquo European Union Journal of Fisheries andAquatic Sciences vol 25 no 1 pp 39ndash46 2008

[29] R G D Steel and J N Torrie Principles and Procedures ofStatistics McGraw Hill London UK 1980

[30] J G Hollyfield ldquoErythrocyte replacement at metamorphosis inthe frog Rana pipiensrdquo Journal of Morphology vol 119 no 1 pp1ndash6 1966

[31] J Benbassat ldquoErythroid cell development during naturalamphibian metamorphosisrdquo Developmental Biology vol 21 no4 pp 557ndash583 1970

[32] R H Broyles G M Johnson P B Maples and G R KindellldquoTwo erythropoietic microenvironments and two larval red celllines in bullfrog tadpolesrdquo Developmental Biology vol 81 no 2pp 299ndash314 1981

[33] C C Speidel ldquoBile pigment production and erythrocytedestruction in thyroid-treated amphibian larvaerdquo The Journalof Experimental Medicine vol 63 pp 703ndash712 1926

[34] G L Vankin E M Brandt and W DeWitt ldquoUltrastructuralstudies on red blood cells from thyroxin-treated Rana cates-beiana tadpolesrdquo The Journal of Cell Biology vol 47 no 3 pp767ndash772 1970

[35] College of American Pathologists ldquoBlood cell identificationrdquoHematology and Clinical Microscopy Glossary pp 3ndash21 2010

[36] M E Feder and W W Burggren Environmental Physiology ofthe Amphibians University of Chicago Press 1st edition 1992

[37] F A Hartman andMA Lessler ldquoErythrocytemeasurements infishes amphibians and reptilesrdquoThe Biological Bulletin vol 126no 1 pp 83ndash88 1964

[38] R H Broyles ldquoChanges in the blood during amphibian meta-morphosisrdquo in Metamorphosis a Problem in DevelopmentalBiology L I Gilbert and E Frieden Eds pp 461ndash490 PlenumPress New York NY USA 2nd edition 1981

[39] W L Duelleman and L Trueb Biology of Amphibians McGrawHill New York NY USA 1986

[40] G M Maniatis and V M Ingram ldquoErythropoiesis duringamphibianmetamorphosis I site of maturation of erythrocytesin Rana catesbeianardquo Journal of Cell Biology vol 49 no 2 pp372ndash379 1971

[41] M E Rothenberg and S P Hogan ldquoThe eosinophilrdquo AnnualReview of Immunology vol 24 pp 147ndash174 2006

[42] D J Adamko S O Odemuyiwa D Vethanayagam and RMoqbel ldquoThe rise of the phoenix the expanding role of theeosinophil in health and diseaserdquo Allergy vol 60 no 1 pp 13ndash22 2005

[43] B B Mahapatra M Das S K Dutta and P K Mahapa-tra ldquoHematology of Indian rhacophorid tree frog Polypedatesmaculatus Gray 1833 (Anura Rhacophoridae)rdquo ComparativeClinical Pathology vol 21 no 4 pp 453ndash460 2012

[44] C V Tok M Tosunoglu D Ayaz K Cicek and C GulldquoHematology of the lycian salamander lyciasalamandra fazilaerdquoNorth-Western Journal of Zoology vol 5 no 2 pp 321ndash329 2009

[45] S PonsenN-ANarkkong S Pamok K Sappaso andWAeng-wanich ldquoHematological values and morphological observationof blood cells in balloon frogGlyphogloossus molossusrdquo Journalof Microscopy Society of Thailand vol 22 pp 71ndash75 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology