Preliminary study of the influence of red blood cells morphometry on the species determinism of domestic animals

Aim: This survey was realized on cattle, sheep, goats, horses, and dogs, in order to study the influence of three morphometric parameters: the diameter, the circumference and the surface of red blood cells on the determinism of these species. Materials and Methods: For each species, blood samples were taken from 15 adult female by jugular venipuncture with confection of blood smears on microscope slides immediately after blood collection and stained according to the method of May-Grundwald Giemsa. Morphometric study was realized using the software OPTIKA Pro Vision. To better describe the results, the statistical analysis was assessed by using the descriptive Boxplots test, ANOVA, and the Student's t-test. Results: The morphometric parameters of red blood cells are biggest in dogs followed by horses, cattle, and sheep, while goats have the lowest ones. Conclusion: This investigation allowed us to show that from a drop of blood we can have an idea about the animal species taking into account the diameter, the circumference, and the surface of erythrocytes.


Introduction
with discoid red blood cells [14,15].Polychromasia and reticulocytes are generally absent from the blood Erythrocytes or red blood cells (RBCs) provide of normal adult sheep and goat [13].vital functions of oxygen transport, carbon dioxide The equine red blood cell measure 5-6µm in transport, and buffering of hydrogen ions [1,2].The diameter that has mild to no central pallor, and have a typical RBCs shape for multiple veterinary species is lifespan of 140-155 days.Polychromatophilic RBCs the disc or biconcave disc (discoid) resulting in a high are rarely detected in the peripheral blood of horses surface area to volume ration making the red blood because equine reticulocytes mature in the bone cells deformable.Central pallor can be observed to marrow even when erythropoiesis is increased [16,17].variable degrees in these species on examination of a Rouleaux formation is common in horses because peripheral blood smear [3,4].RBCs have no nuclei and horses have decreased red membrane charge compared no organelles, and thereby no ability to synthesize to other species.Howell-Jolly bodies can be seen occaproteins.The full complement of functional proteins sionally in equine peripheral blood films in health [17].must be present by the time the reticulocyte matures The canine erythrocyte measure approximately [5].
7µm in diameter and have a lifespan of 110-115 days The mature red blood cell of the adult bovine has a [18].The biconcave shape is more pronounced in the width of 5-6 µm, and has minimal central pallor and dog than in other domestic animals, giving their red relatively lifespan of approximately 130-150 days [6, cells a clearly visible central pallor when viewed in the 7].Anisocytosis is mild to moderate in bovine.monolayer of well-made blood film [19].Polychromatophils are generally absent from the blood Age [20,21,22], sex [23], breed, exercise [24, 25], of normal adult cattle [8,9].
pregnancy and lactation [26,27,28]; emotional state Ovine red blood cells are some of the smallest of are variables to be considered when establishing refemammalian RBCs, has a width of 3,2-5µm and lifespan rences values in domestic animals; altitude polyglobulia of 70-150 days [10,11], and do not aggregate or deform (increased RBCs count at high altitude) is well as readily as erythrocytes of other species [12,13].
documented and extensively studied [7, 29] ; while few Caprine red blood cells are generally discoid with references were found on red blood cell morphometry.2,5-3,9µm of width and lifespan of 125 days; with an This study was realized on cattle, sheep, goats, exception in the Angora breed, which frequently have a horses and dogs; the purpose is to investigate the high percentage of fusiform-shaped RBCs mixed in influence of morphometric parameters of red blood cells on the determinism of the species, in order to develop criteria selection of domestic animals, taking into account the erythrocytes morphometry.

Materials and Methods
professional optical microscope: OPTIKA B-350 (Ver.4.0.0) ; it is a binocular, modern and ergonomic Ethical approval: Adequate measures were taken to microscope equipped with a digital camera of high minimize pain or discomfort in accordance with the resolution OPTIKAM (Ver.4.1.0)enabling the display International Animal Ethics Committee.The study was of the microscopic image of the smear placed in the approved by the committee framed for the research by microscope on a computer in real time.the university authority.During the study visits, the The study of morphometric parameters of red researchers introduced themselves and explained the TM blood cells is performed with special OPTIKA (Italy) objective and methodology of the study to all animal Pro Vision software of the OPTIKA microscope; breeders.
measurement operations of this software are the digital Study area and animals: This study was conducted in version of the more traditional techniques of morphothe region of Batna, located in the eastern part of metry with optical microscopes.This is an integral Algeria, 410km from the capital city Algiers at an software, which in addition to the functions of image altitude of about 1000m.
capture and camera control, it allows efficient For the realization of sampling we selected 15 non processing and analysis of microscopic images in an pregnant adult females per species, in order to easy manner for any type of application and research.eliminate the effect of age, sex and pregnancy.All the Before starting the actual morphometric study, it animals were clinically healthy, free from internal and is necessary to scan the microscopic images of red external parasites, and belonging to the following local blood cells.The taking of photos for all subjects of breeds: Brown of atlas for cattle, Ouled Djellal in different species is an essential step; it allows fixing the sheep, Arbia for goats, Arab thoroughbred in horses microscopic images observed and recording so that we and Sloughi for dogs.can manipulate them using the software.The information concerning morphometric data of red Blood samples and smears: After disinfecting of the blood cells is accurate only if a scale has been correctly sampling area, blood samples were taken from the entered and well calibrated, which requires scanning jugular vein [30]; smears were confectioned on microthe micrometer scale engraved on the micrometric scope slides just after venipuncture without anticoaguslide on the immersion objective (x100).lants which may interfere and induce some cytoplasmic To study the influence of RBCs morphometry on and morphometric cell changes and on the extreme the determinism of the species of pets, we measured the provoke degranulation of some blood cells [31,32,33].
diameter of red blood cells, which is still estimated in Slides are precisely identified (order number, species) relation to the shape of the cell, and so we developed and classified in slides racks which are equipped with two new parameters that have not previously treated, an information sheet including specie and age of the which are the circumference and the surface of erythroanimals following the order number mentioned on the cytes.slide; these information are recorded immediately after The morphometric study of red blood cells was each sampling and smear realization.
performed by always respecting the guidelines and Blood smears staining: Staining of blood smears was instructions of the manufacturer of the software.We carried out following the classical mixed Panoptic measured for each female, the diameter, the circumstaining of Pappenheim, especially by the dye of Mayference and the area of 50 red blood cells, and then we Gründwald Giemsa (M.G.G, Romanowsky type) the determine the average for all the animal species.best and most appropriate staining to mark the Statistical analysis: To compare the experimental data, mammalians erythrocytes, respecting always the a descriptive statistics by boxplots was realized using protocol cited by the authors [30,34].
the Microsoft Office Excel 2010.Analysis of variances Morphometric study: For several and even until the last (ANOVA) and Student T test, were also performed years, morphometric studies of red blood cells are with the MedCalc statistical software (version 12.7, essentially based on linear measures of erythrocytes Copyright © 1993-2013 Med Clack software bvba), P size.Using an ocular micrometer and an objective value under 0,05 was considered statistically significant.micrometer is the only valid and recognized method to

measure the size of erythrocytes. The diameter of the
The Figure-1 and Table-1 shows the influence of red blood cells is measured or estimated roughly under the diameter of RBCs on the determinism of the species an optical microscope at a magnification immersion in domestic animals.We find that the mean of the group (x100) [35,36,37].
of dogs is the highest, followed by that of horses, cattle In view of the tedious and the monotonous use of and sheep respectively; while the average in goats is the ocular micrometer, other difficulties in non-linear lowest; the different boxes do not overlap which structures measures make the use of the instrument in suggests significant differences between species.As question disappearing, especially with the exponential well as the statistical analysis by ANOVA and Student's technological progress, the advent and the generali-T test (comparison of means of different species two by zation of high performance microscopes. two) confirms the presence of very highly significant In our study, we used a high-performance difference (p < 0,001).phometric parameters of red blood cells have an The results presented in the Figure-2 and in the influence on the determinism of the species of Table-2, show the influence of the circumference of red domestic animals.For the diameters of erythrocytes, blood cells on the determinism of the species of the values obtained in the present study are still within domestic animals; like for the erythrocyte diameter, the range of international standards cited by the circumference of red blood cells was also higher in different authors [9, 13,17,19].dogs while the lowest value was recorded in goats, the Concerning the circumference and the surface of different groups do not overlap ; it should be noted that red blood cells, we tried to propose reference values all the differences are highly significant and at p < given by intervals (Table-4), since there is practically 0,001 (using ANOVA and Student test).
no studies and publications that provide detailed Changes relative to the influence of the surface of information on these two parameters.red blood cells on the determinism of the species are In addition, we should note that the classical shown in Figure-3 and also the Table-3; there are linear and rectilinear measures of the diameter of red differences between species with always an advantage blood cells, with the ocular micrometer are quite for dogs (no overlap between boxes).Using analytical difficult and imprecise.The measures of circumference ANOVA and Student's T test, it turns out that these and surface using the evoked software are more differences are also very highly statistically significant appropriate for comparative studies especially in front with always p < 0,001.
of varying sizes cells.Measures done with this type of software, allowing performing quite correct compa-

Discussion
rative studies in limiting the human factor involved in According to our results, it seems that the morthe measurements realized with the ocular micrometer

quartileFigure- 1 .
Figure-1.Influence of red blood cells diameter on the determinism of the species of pets.

Figure- 2 .
Figure-2.Influence of red blood cells circumference on the determinism of the species of pets.

Table - 1
. Terminal values, averages, standard deviation and quartiles of the diameter of red blood cells of pets (expressed in µm).

Table - 2
. Terminal values, averages, standard deviation and quartiles of the circumference of red blood cells of pets (expressed in µm).

Table - 3
. Terminal values, averages, standard deviation and quartiles of the surface of red blood cells of pets (expressed in µm ).