Morphometric analysis on erythrocytes of various livestock being reared in the Cholistan desert

ABSTRACT The present study aimed to assess the influence of physiological exposure to erythrocyte indices; the morphometric parameters of the size of erythrocytes, as well as synergistic effects on the erythrocyte spectrum changes in various species being reared in different strip-wasteland conditions. We collected samples from 400 animals from the jugular veins and made 2000 blood smears, sample blood was taken by an expert veterinary doctor and transferred into an EDTA vacutainer. Blood smears were stained through the Pappenheim procedure and slides were photomicrographed under 100x objective. The parameters of morphometry of erythrocytes were largest in cattle after buffalo, sheep, while goats have the lowest one. The influence of age was significant p<0.05. The influence of sex was not significant in Cattle p<0.01, values of a parameter of erythrocytes were higher in young than in adults in sheep, goats, cattle, and buffalo. The size of erythrocytes in males was higher than females in sheep, goat, and buffalo but in cattle values of female erythrocytes were higher than in males. Further, gender, altitude, and age have a profound influence on the morphometric attributes of erythrocytes. It will be helpful in interpreting etiopathogenetic conditions in human beings and other animals.


INTRODUCTION
Erythrocytes deliver oxygen to the body's organs, extract outside carbon dioxide, and buffer H + ions (Dash, 2020). Erythrocytes have no organelles, no nuclei, and have no ability to make proteins (Thamer et al., 2016). Erythrocytes are the largest biological component in circulating blood. The number of erythrocytes in circulation is roughly 05 million per cubic millimeter of blood (Samson Adewoyin et al., 2019). An increase in erythrocyte concentration was observed for species exposed to direct sunlight. Erythrocytes are concave on both sides enucleate that transport oxygen/carbon dioxide between lungs and body tissues, their high hemoglobin concentration adds to their O 2 capacity (Menon and Ghaffari, 2021).
The breeds of goat (Capra aegagrus hircus), sheep (Ovis aries), cattle (Bos taurus indicus), and buffalo (Bubalus bubalis) are one of the most domesticated livestock that are extensively found and commonly raised in a huge desert situated in the Bahawalpur region of Punjab, Pakistan, situated on the southwest side of the province and located at a longitude of 57 to 60 east and latitude of 27 to 40 north (Type et al., 2021). It is at a level of 112 meters above sea level (Ali et al., 2009). It has a vast significance in culture, economy, and agriculture from the era of the Neolithic agricultural insurgency (Naderi et al., 2008).
Erythrocytes of Angora breed goat are discoid with the 2.5 to 3.9 µm of width, which normally has a greater fraction of fusiform shaped erythrocytes which is assorted in discoid erythrocytes (Kramer, 2000). Erythrocytes of ovine have a lifespan of 70-150 days having a width of 3.2-5µm (Thamer et al., 2016). A Caprine erythrocyte has a lifespan of 125 days and a width of 2.5-3.9µm, which is commonly discoid (Mariella et al., 2014). A breed of cattle zebu or humped breed is of Indian origin like other zebu cattle found in Pakistan. The literature was reviewed with respect to the morphometry of erythrocytes and the influences with respect to age, sex, and species. Various authors have studied the influence of age (Adili et al., 2013;Mohanty, 2015), sex (Adili et al., 2013), and breed Mohanty, 2015) on the morphometry of erythrocytes. Gender, age, altitude, physiological state, seasonal and diurnal change, feeding level, breed, and temperature of animals all contribute to variations in blood parameters (Mbassa and Poulsen, 1993).
This work is based on the hypothesis that the selected areas cause specific changes in the surface roughness, stiffness, and morphology (area (a), length (l), width (w), perimeter (p), height (h), ferret (f), roundness (r), and solidity (s)) of erythrocytes. We deployed optical microscopy (OM) to establish specific morphometric features and alterations in the male, female, young, and adult modules of erythrocytes from different altitudes and their dependence on the cell size. We found considerable differences in the gender, altitude, and age distribution of the size of erythrocytes derived from discussed species in the three studied regions. It is envisaged that the present study will assist to give information about the anemic syndrome and species type with the drop of blood measuring the parameters of the size of the erythrocytes.

MATERIALS AND METHODS
The experimental animals comprised Cholistani breeds of goat (beetal), sheep (kajli), cattle (zebu), and buffalo (Nili-Ravi) belonging to private or government livestock farms or nomadic pastoralists having the age between 1-6 months of young, and 14-50 months for adult; for goat and sheep, 1-10 month for young, and 30-60 for adult cattle and buffalo, respectively, with 4 groups of each species that are male, female, young, and adult groups. A total of 400 blood samples with 1 of each species with 5 blood smears from each sample, a total of 2000 blood smears, livestock were randomly taken from three different regions A Khairprur Tamewali (KPT), B Derawar (DWR), and C Yazman (YZN) of Cholistan.
The climate of this area is a hot, dry subtropical, continental type with low and irregular rainfall (100-150 mm), high temperature (50 0 C), annual evapotranspiration rate (300cm), low relative humidity, high rate of disappearance, and intense summer winds. The region of KPT, DWR, and YZN is one of the driest and hottest areas in the Cholistan, situated at 130m, 106m, and 115m, respectively, above sea level with a mean annual temperature of 28.33 0 C.
Blood sampling was done aseptically through a disposable syringe from the jugular vein of all these animals. About 5ml of blood samples were taken from each animal. Samples of blood were venipuncture from the jugular vein (Ledieu, 2003) by a veterinary doctor. Before the venipuncture, hair on the site of puncture was trimmed off and the skin was disinfected according to the perspective of the standard veterinary procedure. The storage capacity for blood samples was at +4 0 C and applied procedures in the laboratory on the very same day of the collection of the blood (Žaja et al., 2019). The blood sample was shifted into a screw-capped tube which has 0.5ml of 1% EDTA (ethylene diamine tetra-acetic acid) solution as an anticoagulant. A mixture of classical panoptic staining of the Pappenheim procedure was carried out instead of the dye of May-Grunwald-Giemsa (Infectious, 2001) (Romanowsky type) stain field A which is methylene blue and Azure1 dissolved in phosphate buffer solution. While another one is a stain field B, which is Eosin Y in buffer solution was used, which is the best and very suitable procedure of staining to spot the erythrocytes of mammals in the laboratory (Ledieu, 2003). A high-performance optical microscope, named "OPTIKA 4083.B3" was used. Microscopic pictures were selected and then it was opened in ImageJ and a set scale was done and results were analyzed. The statistical analysis was examined by using the student's ttest for the subject of gender and age, while ANOVA was used for altitude. To better describe the results, we use Python for the graph, module name ". matplotlib" (Ucuzal et al., 2019). Table 1 showed that a slight difference is present in the three different regional sites in the area and perimeter of erythrocytes, a significant difference is present in shape parameters (roundness and solidity) between the KPT and DWR but there was no difference between the values of solidity in DWR and YAZ. It seems that the altitude can affect the parameters of the size of erythrocytes in sheep, goats, buffalo, and cattle. Climatic conditions like temperature, pressure, moisture, etc., could cause a change in blood morphology. authors. These results showed that there is a significant difference (p<0.001) present in the values of area, perimeter, and round parameters between the male and female in sheep erythrocytes, while in cattle, values of parameters of erythrocytes are lower in females than in males, however, the values of parameters of the size of erythrocytes in male buffalos are higher than females. Variations in the influence of the age of species were observed very keenly (Table. 3). It was more obvious that the values of area and perimeter in young are higher than that in adults, similarly, the other parameters of erythrocytes of goats are also bigger in young than in adults. It indicates that the effect of age on caprine (male and female) size of erythrocytes is larger in young goats than in adults (p<0.001). Two characteristic components were attained with the help of Principal Component Analysis (PCA) and Cluster Analysis (CA) of erythrocytes morphometry with an Eigen number greater than one (1). Variations on the influence of the age of sheep were studied by the Mann-Whitney test, which showed a highly significant difference (p<0.001) was present between young and adult sheep. The influence of the age of cattle is higher in adults as compared to the young; a significant difference of p<0.01 lies between the different parameters of young and adults. These results determine that the parameters of erythrocytes have remained higher in DWR as compared to the other two regions. Regarding variations on the effect of the age of buffaloes of erythrocytes, results showed a lack of any significant differences (P>0.01) between adult and young buffaloes. Hypoxia occurs at high altitudes. The increase in the size of a erythrocyte causes hypoxia, which leads to the reduction in an erythropoiesis time period, the erythrocyte size is higher in the DWR region followed by YZN and KPT regions. As DWR is so hot, the dry region, water, and Arq. Bras. Med. Vet. Zootec., v.75, n.3, p.365-375, 2023 vegetation depend on rainfall which is very low. In the case of buffalo, the values of erythrocyte parameters remained higher in KPT as compared to the other two regions (Table. 4). Table 4. Morphometric parameters (area, length, width, perimeter, height, ferret, roundness, and solidity) of size of erythrocytes of species (100 blood smears from every specie with 500 measured erythrocytes, 5 from each blood smear) distributed randomly in three distinct regions (KPT, DWR, YZN) with respect to age (young, adult) and gender (male, female) of livestock. (All results expressed in microns). (N=400) (Sample smear=2000) The statistical values of the various parameters of morphometry of size and shape of erythrocytes in livestock are shown in Table 5. The parameters of shape are roundness and solidity. All the values of all the parameters of size and shape were significant. These values are according to (Adili et al., 2016) and remained in international standards. Avg= ( Table 6 showed that the values of morphometric parameters of shape of erythrocytes i.e., round are higher (0.930) in cattle followed by buffalo (0.914), then sheep (0.901) and goat respectively, the lowest was seen in the goat (0.893).

RESULTS
Similarly, the values of solidity are equal in buffalo and cattle (1.003), while the value of solidity was lower but equal in goats and sheep, that is (1.002). All values were expressed in microns.  Table 7 showed the typical shapes of erythrocytes for livestock are a biconcave disc or discoid. However, the change in the form of erythrocytes occurs in different morphological and physiological conditions of the hematological disturbances or anemia (Žaja et al., 2019).

DISCUSSION
According to (Adili et al., 2013), the erythrocytes of young are greater than those of the erythrocytes of adults. The size of the cell decreases from proerythroblast to erythrocyte during erythropoiesis. The increase in erythrocyte size seen in our observations could be explained by hypoxia, which causes a reduction in erythropoiesis time by passing through several stages of maturation, resulting in large erythrocytes. According to this study, the circumference, area, and diameter of erythrocytes appeared to have a significant influence on the species of the determination of domestic animals. Moreover, ovine erythrocytes are slightly more minor than many mammal's erythrocytes, they have a width of (3.2-5) m, a diameter of (4.5) m, and a life cycle of (70-150) days. Further, they do not collect or lose shaped as early as erythrocytes of other species. A very small difference in values of the cited morphometrical parameters related to the study (Adili et al., 2016(Adili et al., , 2017, is expected to be correlated to the various breeds of sheep. The values of (round 0.901) m and (solidity 1.002) m are nearly equal to those of (Žaja et al., 2019), although sheep breeds are different. The value of area obtained in this study (9.670) m does not relate with the results described by , their results of the area of erythrocytes were (15_19) m. Both rams and ewes were nearly equal, which is astonishing. In sheep results of the influence of age exactly matches to (Adili et al., 2013), that the parameters of erythrocytes in young are bigger than that of adults.
In cattle the results were conversely different, the erythrocytic size has no significant difference between the young and adult within the females, while a significant difference (p<0.001) was present in young and adult males; the results match with (Dash, 2020). According to (Adili et al., 2013), size of the erythrocyte is larger in males as compared to females, our results were the same as (Adili et al., 2013), so it may be considered a bovine property or linked trait in all these species of animals. As DWR is a hot, dry region, water and vegetation depends on rainfall which is very low. Area of erythrocytes (16.424) m, perimeter (14.344) m, height (4.571) m, length (4.275) m, and width (4.552) m was in cattle's erythrocytes. However, age affects the size of erythrocytes in other animals. The size of erythrocyte of young in buffalo is higher than adults. The stage at which erythrocytes are discharged into the circulation from the bone marrow influence their size (Manwani and Bieker, 2008). The cellular oxygen level regulates erythropoiesis and erythrocyte mass in normal animals. From pro-erythroblast to erythrocyte, cell size decreases during erythropoiesis (Manwani and Bieker, 2008). For the diameter of erythrocytes, it's vibrant to see that the results are consistent and always correlate to worldwide reference values cited by (Meinkoth, 2000) and (Rizzi and Clinkenbeard, 2010).
Furthermore, as there are almost no studies and published work on buffalo at all on all these parameters of the size of erythrocytes were found, detailed information on morphometric parameters of erythrocytes of buffalo, cattle, sheep, and goats. We further tried to establish a reference value for all those given in Table 6 by obtaining statistically interpretable results. In livestock of Cholistan (Goat, Sheep, Buffalo, and Cattle), various forms of erythrocytes were observed. They were either biconcave with no central pallor in buffalo or in other shapes. In the case of cattle, biconcave and crenated (echinocytes) types of erythrocytes were found. Echinocytes or crenated types of erythrocytes have evenly spaced spicules. Acanthocytes or spur cells of erythrocytes with irregular shapes in different sizes of spicules were found in goats. Dacrocytes or teardrop-shaped erythrocytes were also observed in all age groups of male and female goats. Spindle-shaped erythrocytes were also found in goats. Acanthocytes or disc-shaped erythrocytes were found in sheep, as seen in the (Dash, 2020), the morphological features and breed on the morphometry of erythrocytes of female cattle.
Statistical analysis was carried out with ANOVA in Table 6, which shows major significant differences between the three regions (p ≤ 0.9). According to gained results, it looks like altitude can influence the size of erythrocytes in goats. This increase in the erythrocyte size parameters at higher sea levels shown in our results might be interpreted as hypoxia that may occur at higher sea levels. It may shorten the time it takes for erythropoiesis to occur by delaying some phases of maturation to produce larger erythrocytes. The stage at which erythrocytes are discharged into circulation from the bone marrow determines their size (Manwani and Bieker, 2008;Schalm, 1974) the cellular O 2 level regulates erythropoiesis and erythrocyte mass in normal animals (Grondin, 2010). From problast to erythrocyte, cell size decreases during erythropoiesis (Manwani and Bieker, 2008). Firstly, in (Fig. 1) an overlap was seen between the three regions. Firstly, in (Fig. 1) an overlap was seen between the three regions. The altitude of KPT remained highest, whereas the size of erythrocyte in a parameter of area was lowest. While the altitude of DWR was lowest and size of erythrocyte was remained highest as compared to others. Secondly, YZN and KPT are closely connected to each other. The pattern remained same for goat and sheep, whereas some irregularities were observed in Cattle and Buffalo. Furthermore, we must point out that traditional ocular micrometer measurements of erythrocyte size are difficult and imprecise. The abovementioned software was used to measure the trails, allowing for more exact learning of comparisons in preventing the anthropoid feature intricated in the quantities understood with the ocular micrometer by selecting the top spot. In accumulation, this novel method of measurement Arq. Bras. Med. Vet. Zootec., v.75, n.3, p.365-375, 2023 was very modest, shortest, easy to type, classy, and not very pricey.

CONCLUSION
A morphometric investigation of goat erythrocytes with a substantial impact on four hundred (400) blood sample smears based on altitude, gender and age revealed that erythrocytes are much larger in young animals than in adults. The morphometry of caprine erythrocytes was unaffected by gender, as was the case in the previous study (Adili et al., 2013). It appeared to be quite evident that the shape and size of the erythrocytes in sheep morphological characteristics are important for detecting variations in normal erythrocytic morphometry. Furthermore, it can be inferred that ovine erythrocytes may be investigated by computerguided image analysis application of erythrocytes descriptive approaches, such as cluster analysis and principal component analysis, based on morphometric size and form criteria. However, the different physiological conditions may change the distribution of erythrocytes. Age and sex have a profound effect on the morphometry of erythrocytes of zebu cattle. Extended studies of different breeds of livestock are highly recommended. According to the findings, morphometric investigations of erythrocytes performed using advanced and reliable computer-guided software were more suitable and error-free than traditional morphometric approaches.
Based on our findings, we suggest that in the future there shall be more variation in the number of animals and samples. In our experiments, we defined twelve (12) categories for the number of males, females, young, and adults. These findings may be useful in the diagnosis of anemic illnesses in animals in veterinary medicine, notably in the cases of microcytic, normocytic, and macrocytic anemia. The shape of erythrocytes is significant in diagnosing anemia and other blood diseases. Smears of good quality with adequate Romanowsky/special staining, as well as morphologists' expertise, are still extremely valuable.
Funding: This study is a part of PhD thesis of the first author and is not funded by any national or international funding agency.