Hematological and Biochemical Parameters of ...

Hematological and Biochemical Parameters of Captive Andean Condors Author(s): Daniela Doussang and Cristina Palma Lucila Moreno Brayan Zambrano Eduardo Pavéz Fabiola Cerda Daniel González-Acuña Source: Journal of Raptor Research, 52(1):72-81. Published By: The Raptor Research Foundation https://doi.org/10.3356/JRR-16-14.1 URL: http://www.bioone.org/doi/full/10.3356/JRR-16-14.1

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J. Raptor Res. 52(1):72–81 Ó 2018 The Raptor Research Foundation, Inc.

HEMATOLOGICAL AND BIOCHEMICAL PARAMETERS OF CAPTIVE ANDEAN CONDORS DANIELA DOUSSANG AND CRISTINA PALMA Universidad de Concepcioń, Facultad de Ciencias Veterinarias, Casilla 537, Chillań, Biob´ıo, Postal code 3810024, Chile

LUCILA MORENO Universidad de Concepcioń, Facultad de Ciencias Naturales y Oceanogra´ficas, Concepcioń, Biob´ıo, Postal code 4030000, Chile

BRAYAN ZAMBRANO Universidad Andre´s Bello, Facultad de Ecolog´ıa y Recursos Naturales, Casilla 444, Santiago, Postal code 8370146 Chile

EDUARDO PAVE´Z Bioame´rica Consultores, Chile

FABIOLA CERDA Facultad de Ciencias de la Salud y los Alimentos, Universidad del Biob´ıo, Chile ˜ 1 DANIEL GONZA´LEZ-ACUNA

Universidad de Concepcioń, Facultad de Ciencias Veterinarias, Casilla 537, Chillań, Biob´ıo, Postal code 3810024, Chile ABSTRACT.—Hematological and biochemical parameters are used to assess the health of birds and to support conservation plans for threatened species, such as the Andean Condor (Vultur gryphus). We studied 22 captive Andean Condors from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos of Chile to determine reference values. We measured packed cell volume, total plasma solids, and total and differential leukocyte counts; we analyzed aspartate aminotransferase, gamma-glutamyl transpeptidase, lactate dehydrogenase, alkaline phosphatase, creatine kinase, creatinine, calcium, inorganic phosphorus, and uric acid. We found no blood parasites and no significant differences between sexes (P . 0.05). Our results establish a baseline for hematological and serum biochemistry parameters for the Andean Condor. KEY WORDS: Andean Condor; Vultur gryphus; biochemistry; captivity; hematology; parameter; reference values.

´ ´ PARAMETROS HEMATOLOGICOS Y BIOQUÍMICOS DE VULTUR GRYPHUS EN CAUTIVIDAD RESUMEN.—Los para´metros hematolo´gicos y bioqu´ımicos se usan para evaluar la salud de las aves y para apoyar los planes de conservacioń de especies amenazadas como Vultur gryphus. Se estudiaron 22 individuos de Vultur gryphus en cautividad mantenidos en el Centro de Rehabilitacioń de Aves Rapaces de la Unioń de Ornito´logos de Chile, para determinar valores de referencia. Medimos el volumen celular, los so´lidos plasma´ticos totales y el recuento total y diferencial de leucocitos. Analizamos aspartato aminotransferasa, gamma-glutamil transpeptidasa, lactato deshidrogenasa, fosfatasa alcalina, creatina quinasa, creatinina, calcio, fo´sforo inorgańico y aćido urico. No encontramos para´sitos sangu´ıneos ni diferencias significativas ´ entre sexos (P . 0.05). Los resultados obtenidos en este estudio establecen una referencia para los para´metros hematolo´gicos y de bioqu´ımica se´rica para esta especie. [Traduccioń del equipo editorial] 1

Email address: [email protected]

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HEMATOLOGY OF CAPTIVE ANDEAN CONDORS

The Andean Condor (Vultur gryphus) is a monotypic species. It is distributed in the Andes Mountains, from Venezuela to Cape Horn (Mart´ınez and Gonza´lez 2005). It is an uncommon, nearly threatened species (BirdLife International 2012), and has been protected in Chile since 1929 (Law No. 4601 of 1929 and Law No. 40 of 1972, Republic of Chile). The status of populations is difficult to determine, but it is probably declining (BirdLife International 2012). Serum biochemistry and hematology are the basis of medical diagnosis of diseases (Harr 2002). Clinical parameters of Andean Condors have been measured in captive (Balasch et al. 1976, Gee et al. 1981, Toro et al. 1997) and wild populations (Toro et al. 1997), but these studies lack some important parameters. In this study, we report baseline hematologic and blood chemistry values for Andean Condors, which could be used to assess the health of individuals in captivity. METHODS

Sample Collection. In January 2015, we studied 22 captive Andean Condor (10 adult females, seven adult males, and five immature birds) from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos de Chile, city of Talagante (33840.0 0 S, 70856.0 0 W). Adult birds were 5–29 yr old, and immatures 1–4 yr old. The Andean Condors were fed either chicken (Gallus gallus domesticus) or dayold chicks once a day, and water was provided ad libitum. We collected 4 ml of blood from the metatarsal vein of each condor using sterile syringes with 21-gauge needles, and transferred the samples to Vacutainert tubes with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant (Becton, Dickinson and Company, Franklin Lakes, NJ U.S.A.), and plain Vacutainert tubes (Becton, Dickinson and Company, Plymouth, U.K.) for hematology and biochemistry analyses, respectively. We collected blood samples between 1200 and 1500 H to avoid variations in the blood parameters due to the circadian rhythm (Garc´ıa-Rodr´ıguez et al. 1987). We stored samples at 48C, and analyzed them within 12 hr of collection at the Clinical Laboratory of the Facultad de Ciencias Veterinarias, Universidad de Concepcioń. We used serum for biochemical analyses and whole blood for hematological analysis. A veterinarian physically evaluated all Andean Condors included in this study and considered all to be clinically healthy. We determined age and sex of each bird by plumage (Mart´ınez and Gonza´lez

73

2005). In this study, conditions such as habitat, diet, and veterinary management were the same for all individuals sampled. Sample Analysis. We measured packed cell volume (PCV; %) from the microhematocrit tubes (Hirschmann Laborgeräte GmbH & CO. KG, Eberstadt, Germany) after centrifuging for 4 min at 14,490 3 g in a microhematocrit centrifuge (Campbell and Coles 1986; Digital micro centrifuge, KHT-410E, Taipei, Taiwan, Republic of China). We determined total plasma solids (TPS; g/L) using Goldberg’s refractometer (Golberg TS Meter Clinical Refractometer, Reichert Analytical Instrument, New York, NY U.S.A.). We calculated white blood cell (WBC; x109/L) using a Neubauer chamber (Brand GmbH & Co. KG, Wertheim, Germany) and a modified version of the Rees and Ecker solution (Lucas and Jamroz 1961). We stained blood smears with Diff-Quickt (Romanowski stain variant, Quick Panoptic, Qu´ımica Cl´ınica Anal´ıtica, Barcelona, Spain). We performed a differential white blood cell count by counting 200 leukocytes by oil immersion microscopy (Merck KGaA, Darmstadt, Germany) at 10003 magnification (Microscopy Zeiss Primo Star, Carl Zeiss Inc., Oberkochen, Germany). We determined the presence of blood parasites in the stained smear by oil immersion at 10003 magnification (Merino et al. 1997; Microscopy Zeiss Primo Star, Carl Zeiss Inc., Oberkochen, Germany). We centrifuged the blood samples at 2500 3 g for 10 min (Centrifuge DSC 200A-2, Already Enterprise Inc., Taipei, Taiwan, Republic of China) and obtained serum for blood chemistry analyses. We determined gamma-glutamyl transpeptidase (GGT; U/L), aspartate aminotransferase (AST; U/L), lactate dehydrogenase (LDH; U/L), alkaline phosphatase (AP; U/L), creatine kinase (CK: U/L), creatinine (CRE; lmol/L), calcium (Ca; mmol/L), inorganic phosphorus (Phos; mmol/L), and uric acid (UA: lmol/L) using a commercial test (DiaSys Diagnostic Systems GmbH, Alte Strasse 9, Holzheim, Germany). The AST, LDH, Phos, and CK were read at 340 nm wavelength, GGT and AP at 405 nm, CRE at 546 nm, Ca at 570 nm, and UA at 520 nm in a spectrophotometer (Wiener lab, MetroLAB 2300 Plus, UV-Vis Metrolab S.A., Buenos Aires, Argentina). Statistical Analysis. We tested normality with the Anderson-Darling test (RefValAdv, National Veterinary School, Toulouse, France). Many analytes were not normally distributed, and data were transformed

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Table 1. Hematological parameters of Andean Condor juveniles (n ¼ 5), from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos de Chile, Talagante, Chile. ANALYTE

MEAN

SD

MINIMUM

MAXIMUM

PCV (%) TPSb (g/L) WBCc (3109/L) Heterophil (3109/L) Lymphocytes (3109/L) Monocyte (3109/L) Eosinophils (3109/L) Basophils (3109/L)

46.2 44.0 8.9 ND 0.6 0.9 0.0 0.0

4.1 4.0 1.4 1.1 0.2 0.3 0.0 0.1

42.0 38.0 6.6 5.6 0.1 0.5 0.0 0.0

51.0 48.0 10.1 8.0 1.6 1.2 0.1 0.2

a

a b c

PCV ¼ packed cell volume. TPS ¼ total plasma solids. WBC ¼ white blood cell count.

to approximate a normal distribution using the BoxCox transformation. This transformation is a family of transformations expressed by the equation: Y ðkÞ ¼ ðY k 1Þ=kðfor k „ 0Þ Y ðkÞ ¼ loge ðY Þðfor k ¼ 0Þ where k is the number that maximizes the loglikelihood function: n v vX loge y L ¼ loge ðST2 Þ þ ðk 1Þ 2 n i¼1

where m is the degrees of freedom, n is the sample size, and ST2 is the variance of the transformed values of Y (Gotelli and Ellison 2004). We assessed outliers using a strict Dixon’s test and excluded data identified as outliers and suspect outliers from subsequent calculation RefValAdv. However, after analysis of the residuals from the transformed data, the heterogeneity of the variance was not reduced with any transformation; therefore, we treated those variables as ordinal. We determined nonparametric reference intervals using a bootstrap method. We determined reference intervals and 90% confidence intervals of the limits using the RefValAdv. We used MANOVA on the normal data to detect differences between sexes with JMPt7.0.1 (SAS Institute). The sample size for immatures (n ¼ 5) was too small to assess whether values differed from adults. All statistical findings were considered significant at P 0.05. Data are expressed as mean 6 standard deviation (SD), minimum and maximum.

RESULTS

Hematology and blood chemistry values of Andean Condors did not differ between males and females (P ¼ 0.9883; Tables 1–4). We did not find blood parasites in the blood smears. DISCUSSION

This study established baseline reference intervals for common serum biochemical and hematological parameters in healthy captive Andean Condors and evaluated sex-related differences. Many of hematological and biochemical ranges recorded in this study were within the values given in literature for Andean Condors (Table 5). However, there are some hematological parameters recorded in this study with variations outside the ranges observed for other raptors. These variations could be attributed to characteristics of the species, nutritional status, environmental variables, conditions of captivity, age, sex, social structure, health status, and techniques used for sample analysis (Clark et al. 2009). With respect to this issue, other studies indicate some parameters are affected by the use of EDTA. For example, plasma protein and PCV values were significantly lower in samples with lithium heparin than in those with K2EDTA, whereas lymphocyte numbers were significantly higher in lithium heparin samples than in K2EDTA samples (SanchezMigallon et al. 2008). We recommend using the same anticoagulant to follow trends within the same individual, especially when considering plasma protein concentration, PCV, and lymphocyte count (Sanchez-Migallon et al. 2008). However, EDTA has little effect on cell morphology and produces fewer staining artifacts. It is the anticoagulant of choice in hematology to conserve samples for short periods of

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Table 2. Hematological parameters of Andean Condor adults (seven males and 10 females), from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos de Chile, Talagante, Chile. ANALYTE a

PCV (%) TPSb (g/L) WBCc (3109/L) Heterophil (3109/L) Lymphocytes (3109/L) Monocyte (3109/L) Eosinophils (3109/L) Basophils (3109/L) a b c

SEX

MEAN

SD

MINIMUM

MAXIMUM

Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female

47.3 46.6 46.0 45.6 7.0 7.0 5.6 5.9 0.6 0.5 0.7 0.5 0.0 0.0 0.1 0.1

2.9 2.9 2.8 4.8 2.4 1.4 1.7 1.1 0.5 0.4 0.3 0.3 0.0 0.0 0.1 0.1

41.0 41.0 42.0 38.0 3.1 5.2 2.7 4.5 0.1 0.1 0.2 0.2 0.0 0.0 0.0 0.0

49.0 51.0 50.0 50.0 10.4 9.8 7.8 7.5 1.5 1.2 1.0 1.0 0.1 0.1 0.3 0.1

PCV ¼ packed cell volume. TPS ¼ total plasma solids. WBC ¼ white blood cell count.

time. However, in very small samples, EDTA causes hemolysis (Beynon and Cooper 1999), which did not occur in our study because the blood samples were fairly large (4 ml). Mean values for PCV were within the normal range (35–55%) reported for bird species in general (Campbell 1994), but higher than those described

for captive Andean Condors by Balasch et al. (1976) and Gee et al. (1981; Table 5). The season in which samples were collected could explain these differences. Balasch et al. (1976) collected samples in winter, Gee et al. (1981) collected samples in autumn, and we collected samples in summer. The hydration status and water availability are important

Table 3. Biochemical parameters of Andean Condor juveniles (n ¼ 5), from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos de Chile, Talagante, Chile. ANALYTE

MEAN

SD

MINIMUM

MAXIMUM

Ca (mmol/L) Phosb (mmol/L) UAc (lmol/L) CKd (U/L) CREe (lmol/L) LDHf (U/L) GGTg (U/L) APh (U/L) ASTi (U/L)

3.1 1.6 452.4 509.8 25.1 456.8 6.1 291.3 11.4

0.2 0.6 181.7 97.0 12.7 78.3 2.8 155.5 3.1

2.8 0.9 281.7 403.7 9.4 378.8 2.5 143.9 7.6

3.4 2.4 761.9 617.8 42.0 546.0 9.8 536.2 16.0

a

a

Ca ¼ calcium. Phos ¼ inorganic phosphorus. UA ¼ uric acid. d CK ¼ creatine kinase. e CRE ¼ creatinine. f LDH ¼ lactate deshidrogenase. g GGT ¼ gamma-glutamyl transpeptidase. h AP ¼ alkaline phosphatase. i AST ¼ aspartate aminotransferase. b c

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Table 4. Biochemical parameters of Andean Condor adults from the Rehabilitation Center for Raptors of the Unioń de Ornito´logos de Chile, Talagante, Chile. ANALYTE a

Ca (mmol/L) Phosb (mmol/L) UAc (lmol/L) CKd (U/L) CREe (lmol/L) LDHf (U/L) GGTg (U/L) APh (U/L) ASTi (U/L)

SEX

n

MEAN

SD

MINIMUM

MAXIMUM

Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female

6 8 7 9 7 9 7 9 7 10 7 10 6 9 7 10 7 9

2.7 2.8 1.5 1.4 305.1 309.0 362.5 346.2 28.3 31.6 361.0 356.7 4.9 5.9 100.2 150.0 6.9 6.9

0.2 0.1 0.5 0.4 103.6 66.6 66.6 75.6 6.4 15.5 93.3 71.8 2.3 2.3 29.5 87.8 2.2 1.7

2.4 2.6 0.9 1.0 180.8 226.5 231.4 243.7 18.9 14.3 249.9 256.1 2.2 2.0 66.8 81.8 3.6 4.1

2.9 2.9 2.3 2.0 449.0 445.8 447.7 501.5 36.2 62.8 530.3 478.2 7.1 10.1 153.6 359.8 9.6 9.4

a

Ca ¼ calcium. Phos ¼ inorganic phosphorus. c UA ¼ uric acid. d CK ¼ creatine kinase. e CRE ¼ creatinine. f LDH ¼ lactate deshidrogenase. g GGT ¼ gamma-glutamyl transpeptidase. h AP ¼ alkaline phosphatase. i AST ¼ aspartate aminotransferase. b

factors to consider when interpreting PCV values, as high levels of PCV may indicate dehydration (Smith and Bush 1978). Although our values were higher, they were within the normal ranges reported for other species of free-living vultures (Coleman et al. 1988, Hernańdez and Margalida 2010). PCV values tend to be higher in males than females (Campbell 2015). Our results were consistent with these findings; males appeared to have higher mean values than females, but differences were not statistically significant and the sample size was small. Differences may result from higher estrogen levels in females, which depresses erythropoiesis, whereas in males, it is stimulated by androgens and thyroxine (Campbell 2015). Values of TPS were within the normal range described for birds (30 to 50 g/L; Gee et al. 1981, Ferrer et al. 1987, Coleman et al. 1988), and within the ranges previously described for the species by Toro et al. (1997) in captive (Table 5) and free-living condors. However, the mean value was higher than that determined for Andean Condors (Balasch et al.

1976, Gee et al. 1981; Table 5) and for other species of vultures (Balasch et al. 1976, Coleman et al. 1988, Polo et al. 1992, Dell’Omo and Cavallina 1996, Dobado-Berr´ıos and Tella 1998, Dujowich et al. 2005, Hernańdez and Margalida 2010). Moreover, variations in TPS could be attributed to environmental conditions at the time of sampling. TPS increased with the rise in environmental temperature, because plasma water was used to dissipate heat by evaporation (Dawson and Bortolotti 1997). As with PCV, the sampling season could affect these values. Unfortunately Toro et al. (1997) did not report the sample collection season. False elevations will occur if samples are hemolyzed (Samour 2006). Otherwise, the method used to measure proteins also can contribute to observed variations. Some authors demonstrated that total proteins measured by refractometry were higher than those provided by the spectrophotometric method (Biuret methods), because substances other than proteins contribute to the total solids of the refractive index (Caprita et al. 2013). Lumeij and Maclean (1996) indicated that

41.0 38.0 3.1 2.7 0.1 0.2 0.0 0.0 2.4 0.9 128.0 231.4 14.3 249.9 2.0 66.8 3.6

2.7 (0.1) 1.4 (0.4) 307.3 (81.6) 353.3 (69.9) 30.2 (12.4) 358.5 (78.5) 5.5 (2.3) 129.5 (72.8) 6.9 (1.9)

b

MIN

46.9 (2.8) 45.8 (4.0) 7.0 (1.8) 5.8 (1.3) 0.6 (0.4) 0.6 (0.3) 0.0 (0.0) 0.1 (0.1)

MEAN (6SD)

PCV ¼ packed cell volume. TPS ¼ total plasma solids. c WBC ¼ white blood cell count. d Ca ¼ calcium. e Phos ¼ inorganic phosphorus. f UA ¼ uric acid. g CK ¼ creatine kinase. h CRE ¼ creatinine. i LDH ¼ lactate deshidrogenase. j GGT ¼ gamma-glutamyl transpeptidase. k AP ¼ alkaline phosphatase. l AST ¼ aspartate aminotransferase.

a

Hematological parameters PCVa (%) TPSb (g/L) WBCc (3109/L) Heterophil (3109/L) Lymphocytes (3109/L) Monocyte (3109/L) Eosinophils (3109/L) Basophils (3109/L) Biochemical parameters Cad (mmol/L) Phose (mmol/L) UAf (lmol/L) CKg (U/L) CREh (lmol/L) LDHi (U/L) GGTj (U/L) APk (U/L) ASTl (U/L)

ANALYTE

THIS STUDY (n ¼ 17)

2.9 2.3 449.0 501.5 62.8 530.3 10.1 359.8 9.6

51.0 50.0 10.4 7.8 1.5 1.0 0.1 0.3

MAX

-

43.7 (6.7) 29.8 (7.8) -

MEAN (6SD)

BALASCH ET AL. 1976 (n ¼ 2)

2.2 (0.1) 0.7 (0.3) 529.4 (142.8) 70.7 (8.8) 274 (56) 2.0 (1) 69.0 (51) -

42 (3) 37 (2) -

MEAN (6SD)

2.1 0.2 267.7 53.0 192 1 36 -

39 35 -

MIN

GEE ET AL. 1981 (n ¼ 9)

2.4 1.0 672.1 88.4 360 3 193 -

48 42 -

MAX

2.7 (0.4) 1.3 (0.5) 143 (48.5) 277.5 (107.5) 13 (4.0)

43 (10) -

MEAN (6SD)

1.4 0.8 77 121 6

26 -

MIN

TORO ET AL. 1997 (n ¼ 12)

2.7 2.4 242 414 19

62 -

MAX

Table 5. Hematological and biochemical parameters obtained in our study and in other studies of captive Andean Condor (Vultur gryphus) adults. The symbol – indicates that the authors did not report this value.

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DOUSSANG ET AL.

plasma protein concentration measured by a refractometer may be considered only a rough estimate of total protein concentration obtained by the Biuret method. Nevertheless, other authors demonstrated that there was no difference and recommended refractometry because it is faster and less expensive than other methods (Pessoa et al. 2017). This study reported a wide range in WBC; nevertheless, these results coincided with reference intervals reported previously in other species of vultures (Polo et al. 1992, Dujowich et al. 2005, Naidoo et al. 2008, Hernańdez and Margalida 2010) and raptors (Cooper 2002). The WBC estimate method may lack accuracy and precision depending on the quality of the stained blood film (Hernańdez et al. 1990). In our study, the same person performed cell counts using the same procedure. Our results of leukogram indicated that heterophils were the most numerous leukocyte in the peripheral blood of Andean Condors. The stress of handling can cause an increase in the number of heterophils. This response is not immediate, because although corticosteroid levels change rapidly in response to stress, the absolute number of leukocytes changes more slowly (30–60 min after capture; Cirule et al. 2012). We believe that this was not the case due to the short time of handling the birds. Calcium values were higher than those determined in other studies of Andean Condors (Gee et al. 1981, Toro et al. 1997; Table 5), and for other vulture species (Polo et al. 1992, Dell’Omo and Cavallina 1996, Dobado-Berr´ıos and Tella 1998, Dujowich et al. 2005, Hernańdez and Margalida 2010). The higher levels of calcium reported in our study could be correlated with protein levels, because there is a positive correlation between plasma proteins and calcium (Lumeij et al. 1993). Calcium concentrations depend on the reproductive cycle, sex, and possibly breeding season (Harr 2006), but samples in this study were not from the breeding season. Increased calcium concentrations have been reported with dietary excesses of Vitamin D, osteolytic bone tumors, and hyperparathyroidism (Harr 2006). Furthermore, dehydration can cause hyperkalemia (Hochleithner 1994). Mean values obtained for inorganic phosphorus were similar to those determined in female and male of Andean Condors by Toro et al. (1997) in captive vultures and Gee et al. (1981) in females (1.9 mmol/ L) but they obtained higher mean values in males (2.8 mmol/L; Table 5). Similar values were obtained in other vultures (Dell’Omo and Cavallina 1996,

VOL. 52, NO. 1

Dobado-Berr´ıos and Tella 1998, Villegas et al. 2002, Dujowich et al. 2005). False elevations will occur if samples are hemolyzed, because the phosphate concentration in erythrocytes is higher than that of plasma (Hochleithner 1994). In this study the samples were not hemolyzed. The mean value obtained for UA was lower than the mean values in Andean Condors determined by Gee et al. (1981; Table 5). Nevertheless, levels of UA were similar to those reported in other vultures (Polo et al. 1992, Dujowich, et al. 2005, Hernańdez and Margalida 2010). Uric acid concentrations in serum is used to detect renal disease in birds (Hochleithner 1994), but other factors such as postprandial state and dehydration can increase the values obtained (Harr 2006). The mean value obtained for CK (390.6 U/L) was higher than those described for Andean Condors (Toro et al. 1997; Table 5) and California Condors (Gymnogyps californianus; Dujowich et al. 2005), but similar mean values for CK were measured in other studies of vultures (Polo et al. 1992, Naidoo et al. 2008). High CK activity is associated with muscular trauma in vulture species (Naidoo et al. 2008). In this study no individual had traumatic injuries. Mean values obtained for LDH and GGT were higher than those determined for Andean Condors by Gee et al. (1981; Table 5). The range of LDH recorded for raptors is wide (100–750 U/L) and higher values may indicate liver disease (Cooper 2002). Differences in methodologies for measuring GGT may account for the marked differences in reference values (Harr 2006). Enzyme activity in normal birds typically falls below the sensitivity range of most analyzers (Hochleithner 1994), which may be the case in this study. Values outside the range (0–6.0 U/L) can be observed when there is liver disorder (Cooper 2002). The mean value obtained for AP was higher than that determined by Gee et al. (1981) but lower than that obtained by Toro et al. (1997) for Andean Condors (Table 5). Compared to others species of vultures, the mean value was higher (Polo et al. 1992, Dobado-Berr´ıos and Tella 1998, Villegas et al. 2002, Dujowich et al. 2005, Hernańdez and Margalida 2010). Large ranges have been described in birds of prey (25–500 U/L) and high values may indicate osteoblastic activity (Cooper 2002). AST was lower than the mean described by Toro et al. (1997) for captive Andean Condors (Table 5) but within the ranges and similar to other species of vultures (Polo et al. 1992, Dobado-Berr´ıos and Tella

MARCH 2018


1998, Villegas et al. 2002, Dujowich et al. 2005, Naidoo et al. 2008, Hernańdez and Margalida 2010). Damage in skeletal muscle and high levels of protein in the diet can influence the values of AST (Polo et al. 1992, Dobado-Berr´ıos and Tella 1998). TPS, PCV, calcium, and UA increase due to dehydration (Hochleithner 1994); therefore, when these parameters are altered, the conditions of captivity and sampling station should be considered. We took our samples in summer; this could explain the difference in our results compared to other studies. We found no significant differences between males and females in parameters examined (Table 2, 4). Results were similar to that described in wild Bearded Vultures (Gypaetus barbatus; Hernańdez and Margalida 2010) and Andean Condors (Toro et al. 1997); in both studies, males and females did not differ. Moreover, Gee et al. (1981) found significant differences in the values of serum AST and CA between males and females of Andean Condors, but the sample size was small (n ¼ 9). Also Dujowich et al. (2005) found that chloride, cholesterol, and TPS concentrations were higher in males than females among California Condors. The effect of the sex of the birds on the hematological characteristics of some species and the hematological values may vary depending on reproductive status of birds. Clark et al. (2009), for example, found that ovulating hens have significantly higher calcium levels than nonreproductive females (Hochleithner 1994). Age may be an important factor affecting hematological parameters. We could not compare adults and immatures because the sample size for immatures was very small (n ¼ 5). Wild Bearded Vultures exhibited significant age-related differences in biochemical parameters (urea, uric acid, triglycerides, total serum protein, inorganic phosphorus, magnesium concentrations, AST, CK, LDH, AP, amylase, and lipase activities). Additionally, significant agerelated differences occurred in hematological parameters, PCV, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, fibrinogen level, and heterophils, lymphocytes, and eosinophils (Herna´ ndez and Margalida 2010). Some differences observed among studies may be attributed to the conditions of captivity and characteristics of each individual. The hematological and biochemical parameters obtained in this study could be compared to those found in wild condors, but the technique of extraction, analysis, and sampling

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season can produce variation. As a result, MacedaVeiga et al. (2015) recommend standard protocols for extraction and analysis of samples, and suggest storing results in a public database to facilitate comparative studies and meta-analysis. Hematological studies are currently being used to evaluate the health of ecosystems. Any of the biochemical parameters analyzed could indicate tissue damage (Polo-Cavia et al. 2013) that could be associated with environmental contamination. For example, blood plasma activity is described as a biomarker sensitive to the exposure of insecticides such as organophosphorus, carbamate, paraoxonmethyl, carbofuran, and carbaryl, which cause inhibition of cholinesterase in plasma or serum (Oropesa et al. 2013). Hematological parameters may be useful to monitor contamination events, because the blood redistributes contaminants to the tissues. Bioaccumulation of toxins may occur in the liver and cause liver damage, which when associated with other antecedents may be markers of environmental contamination (Pe´rez et al. 2008). The hematological and biochemical values published in this study can assist the veterinarian laboratory and clinical assessment of condors kept in captivity for conservation programs. ACKNOWLEDGMENTS The authors thank the Centro de Rehabilitacioń de Aves Rapaces de la Unioń de Ornito´logos de Chile (and personally Jürgen Rottmann and Francisca Izquierdo). We also thank Daniel Bengtsson, Karen Ardiles, Sofia Gonza´lez, and Hakan Johansson for their help in gathering samples. Thanks to the Chilean Livestock and Agricultural Services (SAG) for granting official permit No. 815, issued on 21 March 2014.

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