Hematological and serum biochemical parameters of the captive long‐legged buzzard (Buteo rufinus) in Iran

Abstract Background Although normal haematological and serum biochemical values for both pet and wild birds have been published, little information is available on the haematological and serum biochemical values in long‐legged buzzards (Buteo rufinus). Objectives This is the first study that aimed to define reference values of haematological, biochemical parameters, and protein electrophoretic fractions of long‐legged buzzards in Iran. Methods Blood samples were collected from 30 clinically healthy adult long‐legged buzzards of both sexes. Hematological, biochemical parameters, and protein electrophoretic fractions were measured. The mean and standard deviations were calculated. Results Mean values for red blood cells, packed cell volume, haemoglobin, and white blood cells were 2.72 ± 0.60 ×106/μl, 39.10 ± 3.70%, 13.45 ± 1.30 g/dl, and 3.92 ± 1.39 ×103/μl, respectively. Mean values for biochemistry parameters were total protein 4.46 ± 1.27 g/dl, albumin 1.78 ± 0.55 g/dl, creatinine 0.54 ± 0.22 mg/dl, uric acid 7.81 ± 2.89 mg/dl, calcium 9.63 ± 2.22 mg/dl, phosphorus 4.31 ± 1.00 mg/dl, glucose 398.87 ± 96.90 mg/dl, blood urea nitrogen 10.46 ± 3.85 mg/dl, alkaline phosphatase 127.01 ± 1.46 IU/L, aspartate aminotransferase 262.22 ± 116.30 IU/L, and alanine aminotransferase 56.63 ± 27.85 IU/L. Mean values for serum protein fractions included pre‐albumin, albumin, α‐1 globulin, α‐2 globulin, β‐ globulin, and ϒ‐globulin was 0.20 ± 0.09, 2.35 ± 0.67, 0.28 ± 0.13, 0.32 ± 0.07, 0.62 ± 0.24, and 0.68 ± 0.53 g/dl, respectively. Conclusion The reference data presented in this study can be used as health assessment values for veterinary laboratories and clinicians when developing release criteria for rehabilitated long‐legged buzzards.


Birds
The present study was performed on 30 clinically healthy adult longlegged buzzards. These long-legged buzzards were housed at the Pardisan Wildlife Rehabilitation Center in Tehran.
The birds were checked by an expert veterinarian when they first arrived in the rehabilitation centre. If there was a sign or symptom indicating a potential injury, the birds were placed in recovery cages until they had fully recovered. Accordingly, all the sample collections in the present study were from apparently healthy birds which had already been treated for several weeks to months and were completely ready for successful release at the time of blood sampling. The birds were fed either chickens or rats, and management conditions such as cage situation, diet, and water supply were the same for all birds. These birds were previously found by members of the public and brought to the wildlife rehabilitation centre, and their exact age could not be specified.
However, only adults were sampled based on a dark trailing edge to the wing as characteristic of adult long-legged buzzards (Rodriguez et al., 2013

Laboratory analysis
Blood samples were collected from the brachial vein using a 24gauge needle. Samples were divided into vacuumed sterile tubes with ethylene diamine tetraacetic acid as anticoagulant and serum collection tubes with clot activator for haematology and biochemistry analyses, respectively. Total erythrocyte count, total leucocyte count, haemoglobin concentration, and packed cell volume (PCV) values were assessed using avian haematological techniques (Thrall et al., 2012). To differentiate leucocyte counts, 100 leucocytes were counted on blood smears stained with Giemsa stain (Voigt & Swist, 2011).

Data analysis
The mean and standard deviations (SD) were calculated. Statistical analysis was performed using SPSS software version 16(SPSS Inc., Chicago, USA).

RESULTS
The mean ± SD of haematological and biochemical profiles and serum protein electrophoresis results for 30 long-legged buzzards are listed in Tables 1, 2,   Patterns for the distribution of proteins determined by serum protein electrophoresis are shown in Figures 5 and 6. Serum proteins were separated into pre-albumin, albumin, and α1-, α2-, β-, and γ-globulins.
The β-globulin fraction contained β1and β2-globulins in two cases, but it had a single peak in 28 other long-legged buzzards.

DISCUSSION
To our knowledge, this is the first report on haematology, biochemistry, and serum protein electrophoresis from long-legged buzzards (B. rufinus) in Iran. There is a large number of published studies discussing the reference haematological and biochemical values of wild raptors (García-Montijano et al., 2002;Gelli et al., 2009;Heatley & Russell, 2020;Hernandez et al., 1990;Le Souëf et al., 2013;Nazifi et al., 2008;Samour, 2016;Spagnolo et al., 2006). These data could be valuable clinically to evaluate the health of raptors in rehabilitation centres before release (Black et al., 2011). Some of these values could be considered as a beneficial predictive indicator in rehabilitation or veterinary centres (Heatley & Russell, 2020;Molina-López et al., 2015).
In determining reference values for analysis of haematological and biochemical parameters of birds and other exotic animals, the sample size should be between 20 and 40, and less than 20 samples are not suitable for reference values (Cray, 2015). Therefore, in this study, blood samples were taken from 30 apparently healthy long-legged buzzards kept in Pardisan Wildlife Rehabilitation Center located in Tehran.
The birds studied were all adults, and it was impossible to determine their sex. Therefore, the effect of age and sex on the measured parameters was not considered.
The number and type of leucocytes could vary among the raptor species. Except for some of the owl species, heterophil is the most common cell of other raptors (Joseph, 1999). Total leucocyte count in the long-legged buzzard was lower than common buzzard (Buteo buteo) (Hernandez et al., 1990;Spagnolo et al., 2006), Spanish imperial eagle (Aquila adalberti) (García-Montijano et al., 2002), red-tailed hawk (Buteo jamaicensis), Cooper's hawk (Accipiter cooperii) (Black et al., 2011), and golden eagle (Aquila chrysaetos) (Nazifi et al., 2008). This could be due to the clinical health of the birds or subclinical or unknown diseases in other species. The heterophil was the most numerous leucocytes found in the blood of long-legged buzzards, and lymphocytes were the next most abundant leukocytes. No basophil was observed in blood smears of the long-legged buzzard. The percentage of leukocytes in the longlegged buzzard was similar to the common buzzard (B. buteo) reported by Hernandez et al. (1990).
The hemogram of raptors could be altered by blood parasites, stress response, and infectious diseases (Joseph, 1999). Total red blood cell count and haemoglobin concentration in the long-legged buzzard were similar to the results published in common buzzard (B. buteo) (Hernandez et al., 1990;Spagnolo et al., 2006) and Spanish imperial eagle (A. adalberti) (García-Montijano et al., 2002) but were higher than those reported in golden eagle (A. chrysaetos) (Nazifi et al., 2008).
PCV has been shown as a good predictive indicator with clinical significance in the rehabilitation medicine of birds of prey (Molina-López et al., 2015). Raptors with a PCV <35% are considered anaemic, and raptors with a PCV >45% are considered dehydrated (Joseph, 1999).
Reported serum calcium levels do not show wide variations among the raptors species (Joseph, 1999). However, the concentration of serum calcium in long-legged buzzards was lower than that in F I G U R E 5 Electrophoretogram of cellulose acetate electrophoresis of serum proteins of adult long-legged buzzards (pre-albumin, albumin, and α1-, α2-, β-, and γ globulins) F I G U R E 6 Electrophoretogram of cellulose acetate electrophoresis of serum proteins of adult long-legged buzzards (pre-albumin, albumin, and α1-, α2-, β1-, and β2-, and γ-globulins) common buzzard (B. buteo) (Gelli et al., 2009;Hernandez et al., 1990;Spagnolo et al., 2006). In most of bird species, the total plasma calcium concentration of less than 8.0 mg/dl is considered hypocal-  (Harrison et al., 1994;Kaneko et al., 2008;Thrall et al., 2012). Serum phosphorus concentration of long-legged buzzards was similar to common buzzard (B. buteo) in one report (Hernandez et al., 1990) and lower than common buzzard (B. buteo) in another report (Spagnolo et al., 2006). These differences were probably dietary phosphorus status.
Creatinine levels in long-legged buzzards varied from other buzzard species (Gelli et al., 2009;Hernandez et al., 1990;Spagnolo et al., 2006). In this study, creatinine levels in long-legged buzzards were in agreement with the findings of Spagnolo et al. (2006). Blood urea nitrogen in the long-legged buzzard was higher than in common buzzard (B. buteo) (Spagnoloet al., 2006) and golden eagle (A. chrysaetos) (Nazifi et al., 2008). High plasma urea levels can be associated with conditions that reduce the flow of urine, such as dehydration or bilateral ureteral obstruction (Harrison et al., 1994). The birds in our study were in normal hydration status based on clinical examination, PCV, total protein, and albumin concentrations. Carnivorous birds have higher levels of uric acid in comparison with other birds, and slightly higher plasma urea levels in this study were probably due to the high protein diet. Greater levels than 20 mg/dl could be caused by kidney disease, shock, starvation, or during the training period of raptors in rehabilitation centres (Joseph, 1999). Serum uric acid concentration in the long-legged buzzard was similar to common buzzard (B. buteo) (Gelli et al., 2009;Hernandez et al., 1990;Spagnolo et al., 2006).