Introduction

Erythrocytes or red blood cells (RBCs) provide vital functions of oxygen transport, carbon dioxide transport, and buffering of hydrogen ions (Harvey 2008, 2010). The mature red blood cell (RBC) of the adult bovine is biconcave in shape (Kramer 2000; Harvey 2001; Barger 2010), has minimal central pallor, and has a width of 5–6 μm and a relatively long lifespan of approximately 130 days (Wood and Quiroz-Rocha 2010). Variation in sizes of erythrocytes is known as anisocytosis (Thrall 2012) which is mild to moderate in ruminants. The shape of a RBC is relatively uniform, but poikilocytosis is not unusual in blood smears of apparently healthy calves (Okabe et al. 1996). Morphology of cells has recently been indicated as a powerful indicator of cellular function (Lobo et al. 2015). The geometry of the cell has long fascinated biologists (Thompson 1917). Quantifying morphology of cell is fundamental to the statistical study of cell populations, and can help unravel mechanisms underlying cell and tissue morphogenesis (Sánchez-Corrales et al. 2018). Since there was no previous report on comparative morphometrical analyses of RBCs of castrated male and female ND cattle (Fig. 1) of Odisha, an attempt has been taken to undertake this particular study. Since comparative study on morphometrical parameters, i.e., RBCs of non-descriptive (ND) cattle with respect to age and sex are inadequate and to study the influence of age and sex on the morphometry of red blood as well as to interpret the anemic syndromes on the basis of size especially concerning the normocytic, macrocytic, and microcytic anemias; the present study is undertaken. Various authors have studied the influence of age (Adilli et al. 2013; Dash and Mohanty 2015), sex (Adilli et al. 2013), and breed (Adilli et al. 2014; Dash and Mohanty 2015) on the morphometry of RBCs.

Fig. 1
figure 1

Castrated non-descriptive cattle

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Materials and methods

Blood samples collection

ND female and castrated male cattle each having three different age groups, that is, calves, young and adult were taken for this study. After disinfecting the sampling area, blood samples were taken from the jugular vein (Sastry 1983; Brar et al. 2002; Ledieu 2003) of each animal by a skilled veterinary expert. Dry and sterilized needles (Dispo Van Single Use Needle, Hindustan Syringes & Medical Devices Ltd., Faridabad, India) and dry syringes (Dispo Van Single Use Syringe, Hindustan Syringes & Medical Devices Ltd., Faridabad, India) were used for collection of blood (Sastry 1983) and collected in EDTA (ethylene diamine tetra acetic acid) vials. Slides were precisely identified according to their respective age and sex.

Blood smears preparation, staining, and morphometric study

In the laboratory, smears were prepared on grease-free microscopic slides, air-dried, then fixed with methanol, and stained with Giemsa stain prepared from Giemsa powder (Qualigens CAS NO.51811-82-6 Product NO. 39382, scientific India Pvt. Ltd., Mumbai, Maharashtra, India) as protocol cited by Lillie (1977) . For several and even until the last years, morphometric studies of RBCs are essentially based on linear measures of erythrocyte size. For the morphometric study of erythrocytes, the ocular micrometer and an objective micrometer was used (Dash and Mohanty 2015). The entire data (30 observations) per age group of each sex were subjected for morphometrical analyses by using an ocular micrometer that was standardized against a stage micrometer (ERMA TOKYO, Japan made) using a standard light microscope (LABOSCOPE MICROSCOPES Research microscope M.No. BD-08 B, S. No. 21320 Mfg. by B.D. INSTRUMENTATION, Ambala Cantt, India) under 40X objective. Circumference and surface area of erythrocytes were calculated by using the formula 2 πr and πr2 respectively.

Photomicrography

Photomicrography of blood cells was done by a CC130-1.3 mega pixel microscopic camera (Mfg. by Catalyst Biotech, Maharastra, India) connected to a microscope (LABOSCOPE MICROSCOPES Research microscope M. No. BD-08 B, S. No. 21320 Mfg. By B.D. INSTRUMENTATION, Ambala Cantt, India) under 40X objective.

Statistical analyses

Each parameter is expressed as mean and standard error mean for all age groups and sex, and Microsoft Office Excel 2007 was used for statistical analyses. Data analyses for comparison were done with the help of Paleontological Statistics (PAST) version 2.17 (Natural History Museum, University of Oslo) for one-way analysis of variance (ANOVA) followed by Tukey’s pair-wise comparison tests. Differences were classified as significant at p < 0.05 and highly significant at p < 0.001.

Results and discussion

The erythrocytes observed were biconcave in shape (Fig. 2) and the morphometry (length and breadth) were measured in micrometer (μm). The influence of age on the male ND cattle is shown (Table 1). Among the males, the highest mean length of RBCs was observed in adult males (6.52 ± 0.11) and the lowest was observed in male calves (5.69 ± 0.15); the highest mean breadth was observed in male adults (6.20 ± 0.10) and the lowest was observed in male calves (5.46 ± 0.15) (Fig. 3). The influence of age on the female ND cattle is shown (Table 2). Among the females, the highest mean length was observed in female calves (6.33 ± 0.14) and the lowest was observed in female youngs (6.05 ± 0.10); the highest mean breadth was observed in female adults (5.86 ± 0.12) and the lowest was observed in female calves (5.71 ± 0.10) (Fig. 4). The influence of both age and sex were shown (Table 3). Among the three age groups of both males and females, the highest mean length of RBCs was observed in adult males (6.52 ± 0.11) and the lowest was observed in male calves (5.69 ± 0.15); the highest mean breadth was observed in adult males (6.20 ± 0.10) and the lowest was observed in male calves (5.46 ± 0.15) (Fig. 5). For both the mean length and breadth, no significant difference was there among the different age groups of female cattle. A highly significant difference (p < 0.001) was observed between the mean length of RBCs of the male calves with young castrated males and adult males. For the mean breadth of RBCs, a highly significant difference (p < 0.01) was there between the male calves and male adults. A highly significant difference (p < 0.01) was there between the mean length of RBCs of the male calves with the mean length of the female youngs .

Fig. 2
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Red blood cells

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Table 1 Effect of age on morphometry of RBCs of male ND cattle
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Fig. 3
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Effect of age on the morphometry of RBCs of male ND cattle

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Table 2 Effect of age on morphometry of RBCs of female ND cattle
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Fig. 4
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Effect of age on the morphometry of RBCs of female ND cattle

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Table 3 Effect of age and sex on the morphometry of RBCs of ND cattle
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Fig. 5
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Effect of both age and sex on the morphometry of RBCs of ND cattle

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The surface area was measured in μm2. The influence of age on the male ND cattle is shown (Table 4). Among the males, the highest mean circumference of RBCs was observed in adult males (20.48 ± 0.37) and the lowest was observed in the male calves (17.89 ± 0.47); the highest mean surface area was observed in the male calves (32.81 ± 1.13) and the lowest was observed in the male calves (26.01 ± 1.25). The influence of age on the female ND is shown (Table 5). Among the females, the highest mean circumference of RBCs was observed in the female calves (19.89 ± 0.44) and the lowest was observed in the female youngs (19.00 ± 0.33); the highest mean surface area was observed in the female calves (31.95 ± 1.66) and the lowest was observed in the female youngs (29.02 ± 0.92). The influence of both age and sex on the mean circumference and surface area were shown (Table 6). Among the three age groups of both males and females, the highest mean circumference of RBCs was observed in adult males (20.48 ± 0.37) and the lowest was observed in male the calves (17.89 ± 0.47); the highest mean surface area was observed in the young males (32.81 ± 1.13) and the lowest was observed in the male calf (26.01 ± 1.25). For both the mean circumference and surface area, no significant difference was there among the different age groups of female cattle. Highly significant differences (p < 0.001) were observed between the mean circumference and surface of RBCs of the male youngs, male adults, and female calves (Figs. 6 and 7).

Table 4 Effect of age on circumference and surface area of RBCs of male ND cattle
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Table 5 Effect of age on circumference and surface area of RBCs of female ND cattle
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Table 6 Effect of age and sex on circumference and surface area of RBCs of ND cattle
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Fig. 6
figure 6

Effect of both age and sex on the circumference of RBCs of ND cattle

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Fig. 7
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Effect of both age and sex on the surface area of RBCs of ND cattle

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A process which stops the functions of the testes leading to sterilization is known as castration (Carlson 1996). Several non-genetic factors including castration, i.e., removal of testis affecting hematological parameters of farm animals have been observed (Svoboda et al. 2005; Fisher et al. 2001). The castrated animals showed clinical macrocytic anemia, i.e., increased MCV which will make them be more susceptible to severe anemia than intact goats which might prove fatal thereafter (Olaifa 2018). Castration has been shown to elicit physiological stress, inflammatory reactions (indicated by acute-phase proteins), behavior associated with pain, suppression of immune function, and a reduction in performance (Molony et al. 1995; Fisher et al. 1996, 1997; Ahmed and Ahmed 2011) to varying degrees. Various authors have studied the effects of surgical castration on serum enzymes and plasma proteins (Robertson et al. 1994; Oyeyemi et al. 2000; Mohammad et al. 2008)

Anisocytosis are seen in different age groups. According to some authors (Harvey et al. 1984; Brun-Hansen et al. 2006; Aoki and Ishii 2012), age can be considered when establishing the reference values in domestic animals. According to Schlam and Carlson (1982), Harvey et al. (1984), Meinkoth and Clinkenbeard (2000), and Harvey (2008), fetal erythrocytes are larger than those of adults. During gestation and at birth, the erythron compartment increases; at birth, 9 % of the RBCs are reticulocytes (McGrath and Palis 2008). Fetal calf RBCs are less fragile and larger than adult bovine RBCs (Harvey et al. 1984). The increasing of erythrocyte diameter with an increase in age in castrated male ND cattle observed in our study could be interpreted by the persistence of RBCs after parturition formed during embryonic life and decreasing of the diameter or length by the stem cell adaptation to new conditions of life after parturition (Adilli et al. 2013). Age (Harvey et al. 1984; Brun-Hansen et al. 2006; Aoki and Ishii 2012; Dash and Mohanty 2015; Dash et al. 2015), sex (Shaikat et al. 2013), breed (Dash and Mohanty 2015; Dash et al. 2015), exercise (Aceňa et al. 1995; Zobra et al. 2011), pregnancy and lactation (Masoni et al. 1985; Roy et al. 2010; Mariella et al. 2014), and emotional states are variables to be taken in account when establishing reference values in domestic animals. Age, sex, exercise, and emotional states are variables to be considered when establishing reference values in domestic cattle (Adams et al. 1992; Aceňa et al. 1995; Brun-Hansen et al. 2006; Mohri et al. 2007; George et al. 2008; Kapale et al. 2008). The breed can affect the diameter of erythrocytes in cattle (Adilli et al. 2014).

Conclusion

Age and sex have a profound effect on the morphometry of red blood cells of non-descriptive cattle, and this study can provide a baseline reference to which further studies may be compared. By this study, interpretation of anemic syndromes in non-descriptive cattle can be well understood which will helpful for a veterinarian for proper diagnosis and interpretation of diseases as blood plays a vital role for the regulation of the physiological condition of an organism.