Research Article Preliminary Study of the Influence of Red ...downloads.hindawi.com/journals/vmi/2014/429495.pdf · Preliminary Study of the Influence of Red Blood Cells Size on the

Research ArticlePreliminary Study of the Influence of Red Blood Cells Size onthe Determinism of the Breed in Cattle

Nezar Adili1 Mohamed Melizi1 Hadj Belabbas2 and Abdelhamid Achouri3

1 Department of Veterinary Medicine University of El-Hadj Lakhdar 05000 Batna Algeria2 Department of Microbiology and Biochemistry University of Mohamed BOUDIAF 28000 Mrsquosila Algeria3 Department of Veterinary Medicine University of Mohamed Cherif Messaadia41000 Souk Ahras Algeria

Correspondence should be addressed to Nezar Adili nezaradiliyahoofr

Received 8 November 2013 Revised 28 December 2013 Accepted 29 December 2013 Published 6 February 2014

Academic Editor Kazim Sahin

Copyright copy 2014 Nezar Adili 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

This study was carried out on five cattle groups local cross PrimrsquoHolstein Montbeliard and Brown of the Alps in order tostudy the influence of breed on erythrocytes diameter For each breed blood samples were taken from 15 adult females by jugularvenipuncture blood smears were made on slides immediately after the blood collection and stained according to the method ofMay-Grundwald Giemsa Morphometric study was realized using the OPTIKA Pro Vision software The statistical analysis wasassessed by using the descriptive boxplots test and ANOVAThe size of red blood cells is greater in the imported Brown of the Alpsbreed (532plusmn 019) and also in our local breed (523plusmn 010) whereas they were smaller in the Montbeliard breed (479plusmn 021) Thisinvestigation allowed us to show that from a drop of blood we can have an idea of the bovine breeds taking into account the sizeof the erythrocytes

1 Introduction

The mature red blood cell (RBC) of the adult bovine is bico-ncave in shape [1 2] has a width of 5-6120583m and has minimalcentral pallor and relatively long lifespan of approximately130 days [3] Red blood cell shape is relatively uniformpolychromatophils are generally absent from the blood ofadult cattle [1 3] Age sex exercise and emotional stateare variables to be considered when establishing referencesvalues in domestic cattle [4ndash10] Bulls have appreciablygreater red blood cells counts than cows breed differenceshave been reported for beef cattle which have greater redblood cells values than dairy cattle breeds lactating cows haveconsistently lower red blood cells values than nonlactatingcows [1 3 9] altitude polyglobulia is well documented andextensively studied [9 11] while few references were foundon blood cell morphometry This study was realized onlocal imported and crossed cattle in order to investigatethe influence of breed on red blood cell size its aims is todevelop criteria selection of breeds taking into account themorphometry of red blood cells

2 Materials and Methods

21 Region of the Study and Animals This study was con-ducted in the region of Batna located in the eastern part ofAlgeria 410 km from the capital city Algiers at an altitudeof about 1000m For the realization of sampling we selected15 nonpregnant adult females (3ndash5 years old) per breed inorder to eliminate the effect of age sex and pregnancy All thecattle were clinically healthy free from internal and externalparasites and belonging to the following breeds

(i) local brown of Atlas breed(ii) crossing breed (local and imported)(iii) three imported breeds PrimrsquoHolstein (Pie-Red)

Montbeliard and Brown of the Alps

22 Blood Samples and Smears After disinfecting of thesampling area blood samples were taken from the jugularvein [12] smears were confectioned on microscope slidesjust after venipuncture without anticoagulants which may

Hindawi Publishing CorporationVeterinary Medicine InternationalVolume 2014 Article ID 429495 4 pageshttpdxdoiorg1011552014429495

2 Veterinary Medicine International

interfere and induce some cytoplasmic and morphometriccell changes and on the extreme provoke degranulationof some blood cells [13ndash15] Slides are precisely identified(order number breed) and classified in slides racks which areequipped with an information sheet including breed and ageof the animals following the order number mentioned on theslide these pieces of information are recorded immediatelyafter each sampling and smear realization

23 Blood Smears Staining Staining of blood smears wascarried out following the classical mixed panoptic stainingof Pappenheim especially by the dye of May-GrundwaldGiemsa (MGG Romanowsky type) the best and mostappropriate staining to mark the mammalians erythrocytesrespecting always the protocol cited by Ledieu [12] Gabe [16]and Houwen [17]

24 Morphometric Study For several and even until the lastyears morphometric studies of red blood cells are essentiallybased on linearmeasures of erythrocytes size Using an ocularmicrometer and an objective micrometer is the only validand recognized method to measure the size of erythrocytesThe diameter of the red blood cells is measured or estimatedroughly under an optical microscope at a magnificationimmersion (times100) [18ndash20]

In view of the exponential technological progress andbesides the tedious and monotonous use of the ocularmicrometer make the use of the instrument in question dis-appearing especially with the advent and the generalizationof high performance microscopes

In our study we used a high-performance professionaloptical microscope OPTIKA B-350 (Ver400) it is a mod-ern ergonomic binocular microscope equipped with a digi-tal camera of high resolution OPTIKAM (Ver410) enablingthe display of the microscopic image of the smear placed inthe microscope on a computer in real time The morpho-metric study of red blood cells is performed with specialOPTIKA Pro Vision software of the OPTIKA microscopemeasurement operations of this software are the digitalversion of the more traditional techniques of morphometrywith optical microscopes This is an integral and ergonomicsoftware which in addition to the functions of image captureand camera control allows efficient processing and analysisof microscopic images in an easy manner for any type ofapplication and research

Before starting the actual morphometric study it isnecessary to scan the microscopic images of red blood cellsThe taking of photos for all subjects of different breeds isan essential step it allows fixing the microscopic imagesobserved and recording so that you can manipulate themusing the software

The information concerning morphometric data of redblood cells is accurate only if a scale has been correctlyentered and well calibrated which requires scanning themicrometer scale engraved on the micrometric slide on theimmersion objective (times100)

To study the influence of breed on the RBC size incattle we measured for each female the diameter of 50

400

425

450

475

500

525

550

575

600

Loca

l

Cros

sing

Mon

tbel

iard

Brow

n of

the A

lps

Prim

998400 Hol

stein

(Pie

-Red

)

Size

of r

ed b

lood

cells

(exp

ress

ed in

120583m

)

Figure 1 Influence of breed on the diameter of red blood cells incattle

red blood cells and then we determine the average for allthe cattle breeds The morphometric study of red bloodcells was performed by always respecting the guidelines andinstructions of the manufacturer of the software

25 Statistical Analysis To compare the experimental datadescriptive statistics by boxplots were realized using theMicrosoft Office Excel 2007 Analysis of variances (ANOVA)was also performed with the MedCalc statistical software(version 127 Copyright copy 1993ndash2013 Med Clack softwarebvba) 119875 value under 005 was considered statistically signifi-cant

3 Results and Discussion

In Figure 1 we presented the influence of breed on the RBCsize Firstly no overlap was detected between the group ofthe Brown of the Alps with the groups of crossed cattlethe PrimrsquoHolstein and the Montbeliard it is the same forthe group of local cattle with the crossed PrimrsquoHolsteinand the Montbeliard groups On the other hand we foundthe presence of a slight overlap between the Brown of theAlps and local breed and also between the boxes of crossedPrimrsquoHolstein and the Montbeliard cattle We should notethat the average of the Brown of the Alps group is clearlyhigher than the other groups followed respectively by thelocal and then the crossed and the PrimrsquoHolstein whereasit is the lowest for the Montbeliard which tends to indicatea significant influence of the breed factor Statistical analysisdone with ANOVA shown in Table 1 confirms major signifi-cant differences between the five breeds (119875 lt 0 001)

According to our results it seems that the breed can affectthe diameter of erythrocytes in cattle the values obtainedare still within the range of international standards cited bydifferent authors [1 3 9]

In addition we have to note that the classical measuresof red blood cells diameter with the ocular micrometerare quite difficult and imprecise Measures were done withthe software cited above allowing performing quite correct

Veterinary Medicine International 3

Table 1 Terminal values averages standard deviation and quartiles of the diameter of red blood cells of cattle (expressed in 120583m)

Breeds (119873 = 15) Min val 1198761 M amp sd 1198763 Max valLocal 506 518 523 plusmn 010 527 550Crossing 482 496 505 plusmn 016 513 543PrimrsquoHolstein (Pie-Red) 476 483 496 plusmn 016 505 535Montbeliard 449 467 479 plusmn 021 489 520Brown of the Alps 474 526 532 plusmn 019 546 555(Min val minimal value Q1 first quartile M amp sd mean and standard deviation Q3 third quartile Max val maximal value)

comparative studies in limiting the human factor involvedin the measurements realized with the ocular micrometer bychoosing the best place In addition this new measurementmethod is very simple direct easy to make and not muchexpensive

However this investigation needs to be complemented bystudies on the influence of breed on the circumference andthe surface of red blood cells not only in cattle but also for allpets

Circumference and surface measures of red blood cellsappear to be more appropriate for comparative studiesmoreover it seems that these two parameters are more repre-sentative on the research of morphological changes in themorphometry of red blood cells

4 Conclusion

This preliminary study on the influence of breed on red bloodcell diameter in cattle realized on 75 blood smears has shownthe following

(i) The size of red blood cells is greater in the importedBrown of the Alps breed and in our local breedwhereas theMontbeliard breed cattle have smaller redblood cells

(ii) It turns out that the morphometric studies of ery-throcytes made by this type of software are moreappropriate and more accurate than measurementswith the ocular micrometer

(iii) Other studies on the measurements of the circumfer-ence and the surface of red blood cells by this typeof software seem necessary in order to investigate theexact influence of breed on red blood cell morphom-etry

Finally through this study we can conclude that froma drop of blood we can have an idea about the cattlebreeds taking into account the size or the diameter of theerythrocytes

Conflict of Interests

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

Acknowledgment

The authors would like to thank all staff members in theDepartment of Veterinary Medicine Batna University forhelping them and providing facilities

References

[1] J W Kramer ldquoNormal hematology of cattle sheep and goatsrdquoin Schalmrsquos VeterInary Hematology B F Feldman J G Zinkland N C Jain Eds pp 1075ndash1084 Lippincott Williams andWilkins Philadelphia Pa USA 5th edition 2000

[2] A M Barger ldquoErythrocyte morphologyrdquo in Schalmrsquos VeterInaryHematology D J Weiss and K J Wardrop Eds pp 144ndash151Wiley-Blackwell Ames Iowa USA 6th edition 2010

[3] D Wood and G F Quiroz-Rocha ldquoNormal hematology ofcattlerdquo in Schalmrsquos Veterinary Hematology D J Weiss and KJ Wardrop Eds pp 829ndash835 Wiley-Blackwell Ames IowaUSA 6th edition 2010

[4] W E Wingfield and M E Tumbleson ldquoHematologic param-eters as a function of age in female dairy cattle Aging andhematologic valuesrdquoThe Cornell Veterinarian vol 63 no 1 pp72ndash80 1973

[5] R Adams F B Garry B M Aldridge M D Holland and K GOdde ldquoHematologic values in newborn beef calvesrdquo AmericanJournal of Veterinary Research vol 53 no 6 pp 944ndash950 1992

[6] M C Acena S Garcıa-Belenguer M Gascon and A PurroyldquoModifications hematologiques et musculaires pendant la cor-rida chez le taureau de combatrdquo Revue de Medecine Veterinairevol 146 no 4 pp 277ndash282 1995

[7] H C Brun-Hansen A H Kampen and A Lund ldquoHematologicvalues in calves during the first 6 months of liferdquo VeterinaryClinical Pathology vol 35 no 2 pp 182ndash187 2006

[8] M Mohri K Sharifi and S Eidi ldquoHematology and serumbiochemistry of Holstein dairy calves age related changes andcomparison with blood composition in adultsrdquo Research inVeterinary Science vol 83 no 1 pp 30ndash39 2007

[9] J W George V M Lane and J N Snipes ldquoChanges in bovinehematology reference intervals from 1965 to 2001rdquo VeterinaryClinical Pathology vol 36 pp 313ndash315 2008

[10] P M Kapale D G Jagtap D M Badukale and S K SahatpureldquoHaematological constituents of blood of Gaolao cattlerdquo Veteri-nary World vol 1 no 4 pp 113ndash114 2008

[11] E Kolb Physiologie des Animaux Domestiques Vigot FreresEditeurs Paris France 1975

[12] D Ledieu ldquoPrelevements en cytologierdquo in Encyclopedie Veteri-naire Elsevier 2003


[13] W J J Bacha and L M Bacha Color Atlas of VeterinaryHistology Lippincott Williams and Wilkins Philadelphia PaUSA 2nd edition 2000

[14] J W Harvey Atlas of Veterinary Hematology Blood and BoneMarrow of Domestic Animals WB Saunders Philadelphia PaUSA 2001

[15] D B DeNicola ldquoAdvances in hematology analyzersrdquo Topics inCompanion Animal Medicine vol 26 no 2 pp 52ndash61 2011

[16] MGabeTechniquesHistologiquesMasson et Cie Editeur ParisFrance 1986

[17] B Houwen ldquoBlood film preparation and staining proceduresrdquoClinics in Laboratory Medicine vol 22 no 1 pp 1ndash14 2002

[18] G C Adams ldquoA technique for the measurement of erythrocytediametersrdquo Journal of Clinical Pathology vol 7 no 1 pp 76ndash781954

[19] J C Todd Clinical Diagnosis by Laboratory Methods WBSaunders Philadelphia Pa USA 1979

[20] N Adili MMelizi andO Bennoune ldquoThe influence of age sexand altitude on the morphometry of red blood cells in bovinesrdquoVeterinary World vol 6 no 8 pp 476ndash478 2013

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Veterinary Medicine International



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interfere and induce some cytoplasmic and morphometriccell changes and on the extreme provoke degranulationof some blood cells [13ndash15] Slides are precisely identified(order number breed) and classified in slides racks which areequipped with an information sheet including breed and ageof the animals following the order number mentioned on theslide these pieces of information are recorded immediatelyafter each sampling and smear realization

23 Blood Smears Staining Staining of blood smears wascarried out following the classical mixed panoptic stainingof Pappenheim especially by the dye of May-GrundwaldGiemsa (MGG Romanowsky type) the best and mostappropriate staining to mark the mammalians erythrocytesrespecting always the protocol cited by Ledieu [12] Gabe [16]and Houwen [17]

24 Morphometric Study For several and even until the lastyears morphometric studies of red blood cells are essentiallybased on linearmeasures of erythrocytes size Using an ocularmicrometer and an objective micrometer is the only validand recognized method to measure the size of erythrocytesThe diameter of the red blood cells is measured or estimatedroughly under an optical microscope at a magnificationimmersion (times100) [18ndash20]

In view of the exponential technological progress andbesides the tedious and monotonous use of the ocularmicrometer make the use of the instrument in question dis-appearing especially with the advent and the generalizationof high performance microscopes

In our study we used a high-performance professionaloptical microscope OPTIKA B-350 (Ver400) it is a mod-ern ergonomic binocular microscope equipped with a digi-tal camera of high resolution OPTIKAM (Ver410) enablingthe display of the microscopic image of the smear placed inthe microscope on a computer in real time The morpho-metric study of red blood cells is performed with specialOPTIKA Pro Vision software of the OPTIKA microscopemeasurement operations of this software are the digitalversion of the more traditional techniques of morphometrywith optical microscopes This is an integral and ergonomicsoftware which in addition to the functions of image captureand camera control allows efficient processing and analysisof microscopic images in an easy manner for any type ofapplication and research

Before starting the actual morphometric study it isnecessary to scan the microscopic images of red blood cellsThe taking of photos for all subjects of different breeds isan essential step it allows fixing the microscopic imagesobserved and recording so that you can manipulate themusing the software

The information concerning morphometric data of redblood cells is accurate only if a scale has been correctlyentered and well calibrated which requires scanning themicrometer scale engraved on the micrometric slide on theimmersion objective (times100)

To study the influence of breed on the RBC size incattle we measured for each female the diameter of 50

400

425

450

475

500

525

550

575

600

Loca

l

Cros

sing

Mon

tbel

iard

Brow

n of

the A

lps

Prim

998400 Hol

stein

(Pie

-Red

)

Size

of r

ed b

lood

cells

(exp

ress

ed in

120583m

)

Figure 1 Influence of breed on the diameter of red blood cells incattle

red blood cells and then we determine the average for allthe cattle breeds The morphometric study of red bloodcells was performed by always respecting the guidelines andinstructions of the manufacturer of the software

25 Statistical Analysis To compare the experimental datadescriptive statistics by boxplots were realized using theMicrosoft Office Excel 2007 Analysis of variances (ANOVA)was also performed with the MedCalc statistical software(version 127 Copyright copy 1993ndash2013 Med Clack softwarebvba) 119875 value under 005 was considered statistically signifi-cant

3 Results and Discussion

In Figure 1 we presented the influence of breed on the RBCsize Firstly no overlap was detected between the group ofthe Brown of the Alps with the groups of crossed cattlethe PrimrsquoHolstein and the Montbeliard it is the same forthe group of local cattle with the crossed PrimrsquoHolsteinand the Montbeliard groups On the other hand we foundthe presence of a slight overlap between the Brown of theAlps and local breed and also between the boxes of crossedPrimrsquoHolstein and the Montbeliard cattle We should notethat the average of the Brown of the Alps group is clearlyhigher than the other groups followed respectively by thelocal and then the crossed and the PrimrsquoHolstein whereasit is the lowest for the Montbeliard which tends to indicatea significant influence of the breed factor Statistical analysisdone with ANOVA shown in Table 1 confirms major signifi-cant differences between the five breeds (119875 lt 0 001)

According to our results it seems that the breed can affectthe diameter of erythrocytes in cattle the values obtainedare still within the range of international standards cited bydifferent authors [1 3 9]

In addition we have to note that the classical measuresof red blood cells diameter with the ocular micrometerare quite difficult and imprecise Measures were done withthe software cited above allowing performing quite correct







4 Conclusion








Acknowledgment


References





























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of












4 Conclusion








Acknowledgment


References





























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






















Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






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Research Article Preliminary Study of the Influence of Red ...downloads.hindawi.com/journals/vmi/2014/429495.pdf · Preliminary Study of the Influence of Red Blood Cells Size on the