Acta Veterinaria-Beograd 2014, 64 (2), 200-212 UDK: 599.723.2-111.1 DOI: 10.2478/acve-2014-0019
Research article
HEMATOLOGICAL AND SERUM BIOCHEMICAL REFERENCE VALUES FOR THE POSAVINA AND CROATIAN COLDBLOOD HORSE BREEDS PAĐEN Lana¹, GOMERČIĆ Tomislav2, ĐURAS Martina3, ARBANASIĆ Haidi4, GALOV Ana4* Department of Physiology and Radiobiology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia; 2Department of Biology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia; 3Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia; 4Department of Animal Physiology, Faculty of Science, Division of Biology, University of Zagreb, Zagreb, Croatia 1
(Received 31 January; Accepted 04 April 2014) In this study the hematological and biochemical parameters in the blood of indigenous Croatian working horse breeds were investigated. The Posavina and Croatian Coldblood horses are adapted to harsh environmental conditions and their blood parameters might differ from other horse breeds. The study was carried out on 100 mares and 12 stallions of ages from 2 to 19 years. Fifteen hematological and 19 biochemical parameters were analysed. Values of 22 parameters showed considerable overlapping with values obtained for other horse breeds, and substantial resemblance is evident with values reported for Pakistani working horses. Several reference values showed statistically significant effect of sex (Hb, ALB, CREA, TBIL and Ca where significantly higher in stallions; EOS, CK, ALP, UREA and Mg were significantly higher in mares). None of the parameters studied showed any differences associated with age. The adaptation of Posavina and Croatian Coldblood horses to the harsh environment of flooded pastures and the way of breeding might be reflected in their specific reference values. The obtained results enabled the establishment of hematological and biochemical reference values in the blood of Posavina and Croatian Coldblood horses that will in turn support clinical diagnosis and further research into horse physiology. Key words: biochemistry, coldblood horses, hematology, Posavina horse, Croatian Coldblood horse, reference values
INTRODUCTION The Posavina and Croatian Coldblood are indigenous Croatian working horse breeds. Posavina horse originated from uncontrolled crossing of mares of Slavonian - Posavina horse and other mares of unknown origin with warm-blooded (Arab, Lipizzan, * Corresponding author: e-mail:
[email protected]
200
Pađen et al.
Nonius, Thoroughbred), and later, cold-blooded (Brabant, Ardennes, Percheron and Norico) stallions [1]. The history of Posavina horse is tightly interweaved with the history of the Croatian Coldblood. Ljubesic et al. [2] wrote about the Posavina horse as a type of Croatian Coldblood. The notion that Posavina horse is a variety or type of Croatian Coldblood and not a distinct breed is further emphasized by Mason [3]. In recent history, in the 1990s, due to economical and political reasons Posavina horses influenced heavily breeding of Croatian Coldbloods, as exclusively owners of Posavina horses received annual premiums [1]. In 1991 the Croatian Livestock Centre started creating Studbooks for Posavina and Croatian Coldblood horses. The Studbook for the Posavina horse breed was closed in 2004 [4], while creation of the Studbook for Croatian Coldbloods still proceeded until its formal closure in 2008 [5]. Although considered as different breeds, Ivankovic et al. [6] found, based on mitochondrial D-loop sequence variation, that the Posavina and the Croatian Coldblood horses were the most closely related populations. Furthermore, using microsatellite markers, Galov et al. [7] did not observe genetic differentiation between these two breeds. According to the Annual Report of horse breeding in the Republic of Croatia [8], in the 2010 the number of Posavina horses was 4909, with the largest number of animals bred in the area of Sisak-Moslavina County (69.81%). Apart from this County, a growing area of this breed is also Zagreb County. In the same year, the number of Croatian Colblood horses was 6304, with 902 animals in the area of Sisak-Moslavina County [8]. In Europe, the coldblood autochthonous horse breeds have become endangered, as they lost their traditional use value as working animals [6]. However, floodplain areas along the Sava River and its tributaries are a rare eco-system with preserved traditional way of horse breeding and herding in general [9]. Posavina and Croatian Coldblood horses are kept together in coexistence with cows and pigs. All animals are fed on feeds that they find themselves. The mares are released in early spring on an open area of flooded pastures and before winter returned to the barn, so most of the time they are not under the owners’ supervision. Breeding and health status are poorly controlled by owners and are therefore more exposed to natural selection. Because of the way of breeding, keeping and feeding, these horses are highly adapted to specific environment and climatic conditions in the region, and have characteristics that might be reflected in their physical status and consequently their hematological and serum biochemical parameters. Hematological parameters, such as RBC (red blood cells), MCV (mean corpuscular volume), Hb (hemoglobin concentration), PCV (packed cell volume) and total and differential WBC (white blood cell) count, as well as biochemical parameters such as different enzyme activities, mineral concentrations and other serum substrates help clinicians to evaluate the health status of the specimen and diagnose infectious and some parasitic diseases, as well as to observe a recovery period. They also provide important information about the response to therapy, strength and systemic effect of disease and the metabolic state of the individual animal or herd [10]. Some of the hematological and biochemical values are of great interest in horses subjected to
201
Acta Veterinaria-Beograd 2014, 64 (2), 200-212
standardized field exercise tests [11-13]. The hematological and serum biochemical reference values of various horse breeds may differ due to genetic factors and/or various environmental factors (e.g. nutritional quality, availability of water, parasites and climate). For instance, coldblood horse breeds have a lower number of erythrocytes, lower hemoglobin, hematocrit and blood volume than hotblood horses. Hematological data are available for several horse breeds, such as American miniature horse [14], Andalusian horse [15], Arab horse [16], Thoroughbred horse [17], Lipizzan horse [18], Quarter horse [19], Yugoslav Trotter [20], as well as for horses in general [21]. For Croatian working horse breeds, some hematological parameters are available for Murinsulaner horses [22]. However, no data on hematological and biochemical parameters have been published for the Posavina and Croatian Coldblood horse breeds. The aim of this study was to determine hematological and biochemical reference values for Posavina and Croatian Coldblood horse breeds combined, as blood samples were collected prior to formal closure of their respective studbooks and all the individuals were kept together under the same conditions. Moreover, no genetic differentiation between them was observed [7]. Our aim was also to compare those reference values with already known reference values for other breeds of coldblood horses, hotblood horses and horses in general. We further aimed to determine any differences associated with sex and age of the studied horses.
MATERIALS AND METHODS This study was carried out on 112 horses (100 mares and 12 stallions) of two Croatian working horse breeds - Posavina and Croatian Coldblood. Age of studied animals ranged between 2 and 19 years, with an average age of 8 years. Effects of age were analysed by dividing the animals into three (3-4, 5-6 and 11-12 years; stallions) and four (2-5, 6-10, 11-13 and 14-19 years; mares) age groups. Blood samples were collected during June and July 1999 and 2001 on six different locations in central Croatia (Figure 1). Stallions were housed in individual stalls under conditions of natural photoperiod and natural indoor temperature and under constant supervision of the owners, while mares were kept in an open system biocenosis of woods and marshy meadows. The animals were clinically healthy. Blood samples were taken by jugular vein puncture (vena jugularis externa) directly into Vacutainer® tubes with gel for biochemical analysis and Vacutainer ® tubes for whole blood (EDTA). In the whole blood samples the number of red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (PCV), average volume of red blood cells in the blood (MCV), and total number of leukocytes (WBC) were determined using an automatic cell counter (model ZF, Coulter Electronics. Ltd., UK). Differential blood count (neutrophils – NEU, eosinophils – EOS, basophils – BAS, monocytes – MON, lymphocytes – LYM) was determined on a blood smear using a light microscope at x 400 magnification. Blood serum was separated by centrifugation at 1600 g for 20 min. The serum 202
Pađen et al.
samples were stored at -20°C until further analysis. Biochemical analysis included the activity of enzymes gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and amylase (AMYL), concentration of glucose (GLUC), albumin (ALB), total protein (TP), cholesterol (CHOL), triglycerides (TRI), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), urea (UREA), creatinine (CREA) and total bilirubin (TBIL). Samples for biochemical analysis were processed using standard procedures with the biochemical analyzer Olympus AU 560 (Olympus Hamburg, Germany), using tests for Olympus. Statistical analyses were made using the computer program Statistica, version 9th (StatSoft, Inc., Tulsa, USA). After testing the normality (Kolmogorov-Smirnov and Shapiro-Wilk test) t - test were made for dependent samples. The significance level of 0.05 was adjusted by means of Bonferroni correction procedure.
Figure 1. Geographical map of Croatia with black circles indicating areas where samples of Posavina and Croatian Coldblood horses were collected. The dark grey area indicates approximate distribution of the two Croatian working horse breeds
RESULTS Mean value, standard deviation and range for the blood erythrocyte parameters (RBC, MCV, Hb, PCV), total and differential WBC count, enzymes activity (GGT, AST, ALT, CK, ALP, LDH, AMYL), mineral concentrations (Ca, P, Mg, Fe) and other biochemical parameters (GLUC, ALB, TP, CHOL, TRI, UREA, CREA, TBIL) are presented in Tables 1 and 2.
203
204
Stallions1 (N=12)
0.0286
0.7177
0.1987
0.0004*
0.4109
0.3069
0.2076
0.2643
0.0024*
0.0262
0.4508
0.1306
0.0020*
0.5538
0.0183
0.0061
p – value (difference between sexes) 1 Mares and General Murinsulaner values stallions for 1 2 horses combined horses3,4 (N=112) 8.23±3.69 (2-19) 5.95±0.82 6.98±1.08 (6.0-12)3 (4.18-8.25) A,B 56.90±6.21 3 (44.9-72.0) A,B 54.51±8.15 (34-58) 104.84±13.14 125±18.26 (100-180) 3 (56-135) A 33.34±4.49 37.55±5.50 (32-48) 3 (22.80-49.50)A 11.64±2.93 6.47±1.91 (6.0-12) 3 (4.47-23.65) 57.21±12.94 62.10±11.92 (30-75) 3 (20-90) 7.78±5.54 1.85±2.01 (0-0.8) 3 (0-30) 0.67±0.99 0 (0-0.3) 3 (0-5)C 1.49±2.12 0 (0-0.6) 3 (0-10) C 32.84±13.37 36.15±11.00 (25-60) 3 (2-71) 6.46±2.35 (3.0-6.0) 3 (0.00-12.56) 0.85±0.61 (1.0-10) 3 (0.00-3.20) A 0.08±0.13 (0.0-3.0) 3 (0.00-0.65) 0.15±0.24 (1.0-8.0) 3 (0.00-1.41) A 3.74±1.82 (1.5-5) 3 (0.00-9.37)A -
(2 – 10) (15 – 50)
-
-
-
-
(1.5–5.5)
(0.0–0.2)
-
(0.1–0.6)
(2.7–6.7)
-
-
(2 – 12) (0 – 3)
-
(35 – 75)
-
(25.0–40.3)
(89–139)
(43.9–55.6)
(4.97–8.18)
-
39.5±3.8
129.8±11.7
-
9±0.84
-
(1.44–6.78)
(0.00–0.62)
-
(0.00–1.13)
(2.22–7.25)
-
-
-
-
-
3.89±1.28
0.28±0.23
0
0.14±0.14
5.41±1.32
-
-
-
-
-
(6.0–12.0) (5.72–13.71) 9.73±1.71
(35–46)
(6.0 – 12)
(110–180)
(41–49)
(8.5–12.5)
-
(80 – 140)
-
(5.5 – 9.5)
-
General Pakistani values for UK horses6 cold-blood Thoroughbred7 cold-blood horses6 horses5
1
This study; 2[22]; 3[36]; 4[24]; 5[21]; 6[23]; 7[25]; Aoverlapping of values from this study with Pakistani coldblood horses [23]; Boverlapping of values from this study with Murinsulaner horses [22]; Coverlapping of values from this study with coldblood horses [21]; * - significant after Bonferroni correction procedure
age (year)
8.56±3.64 5.5±2.97 (2-19) (3-12) 5.89±0.76 6.48±1.1 RBCx10¹²/L (4.18-8.04) (4.51-8.25) 57.12±6.34 55.99±4.68 MCV fl (44.9-72) (47.7-61) 103.48±12.61 115.75±13.33 Hb g/L (56-135) (80-133) 33.15±4.47 35.23±4.44 PCV % (22.8-49.5) (25.4-39.8) 11.7±2.73 11.02±4.39 WBCx109/L (4.47-19.3) (7.51-23.65) 56.31±12.73 65.45±13.06 NEU % (20-90) (45-90) 8.26±5.56 3±1.95 EOS % (0-30) (0-6) 0.72±1.01 0.36±0.67 BAS % (0-4.55) (0-2) 1.42±1.98 2.27±3.17 MON % (0-10) (0-9) 33.29±13.32 28.91±14.4 LYM % (2-71) (5-50) 6.51±2.34 5.92±2.57 NEUx109/L (0-12.56) (0-10.55) 0.92±0.6 0.27±0.21 EOSx109/L (0-3.2) (0-0.54) 0.09±0.13 0.04±0.07 BASx109/L (0-0.65) (0-0.23) 0.15±0.24 0.18±0.26 MONx109/L (0-1.41) (0-0.7) 3.87±1.82 2.65±1.69 LYMx109/L (0-9.37) (0-5.06)
Parameter
Mares1 (N=100)
Table 1. Reference values of hematological parameters of Croatian Coldblood and Posavina horse breeds (mean ± SD, range is given in parenthesis) and available values for other horse breeds used for comparison
Acta Veterinaria-Beograd 2014, 64 (2), 200-212
Stallions1 (N=12) 5.5±2.97 (3-12) 18.25±6.38 (11-32) 300.92±76.81 (213-471) 9.17±3.16 (4-16) 143.5±33.36 (101-207) 490.67±182.58 (252-996) 905.83±212.15 (644-1233) 3.5±2.32 (1-10) 5.15±0.62 (4-6) 33.81±5.23 (22.5-40) 80.1±4.49 (73.58-86.12)
Mares1 (N=100)
8.56±3.64 (2-19)
21.42±51.24 (6-511)
311.91±81.8 (186-629)
7.77±2.91 (1-21)
285.05±127.64 (105-1007)
698.86±206.3 (378-1398)
926.77±228.18 (547-1662)
3.47±1.9 (0-10)
4.58±0.66 (3.18-7.06)
28.53±2.18 (24-34.4)
85.04±7.46 (59.15-99.39)
Parameter
age (year)
GGT U/L
AST U/L
ALT U/L
CK U/L
ALP U/L
LDH U/L
AMYL U/L
GLUC mmol/L
ALB g/L
TP g/L
0.0273
0.0000 *
0.0055
0.9564
0.7632
0.0012 *
0.0002*
0.1212
0.6594
0.8316
0.0061
p – value (difference between sexes) 1
84.55±7.33 (59.15-99.39)
29.02±2.95 (22.5-40.0)D,E
4.64±0.67 (3.18-7.06)A,D,E
3.47±1.95 (0-10)
924.41±224.62 (547-1662)
676.19±212.31 (252-1398)
269.85±128.19 (101-1007)
7.90±2.94 (1-21)D
310.06±80.92 (186-629)A
20.99±48.18 (6-511)
8.23±3.69 (2-19)
Mares and stallions combined1 (N=112)
(52-79)4
(26-37)4
(4.2-6.4)4
(75-150)4
(162-412)4
(143-395)4
-
(3.0-23)4
(226-366)4
(4.3-13.4)4
-
General values for horses3,4
(62–70)
(30–36)
(3.5–6)
-
-
