The Influence of Age on Clinical Biochemical Parameters in Pure-bred Arabian Mares

The Influence of Age on Clinical Biochemical Parameters in Pure-bred Arabian Mares

ORIGINAL RESEARCH The Influence of Age on Clinical Biochemical Parameters in Pure-bred Arabian Mares Sema Y. Gurgoze, Professor,a and Hasan Icen, PhDb...

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ORIGINAL RESEARCH The Influence of Age on Clinical Biochemical Parameters in Pure-bred Arabian Mares Sema Y. Gurgoze, Professor,a and Hasan Icen, PhDb

ABSTRACT Specific reference intervals are needed for each animal species for the appropriate interpretation of the results obtained from serum biochemical tests. The aim of this study was to investigate the blood composition of pure-bred Arabian mares to evaluate the need for defining reference values for different age groups. In all, 90 clinically healthy mares from five farms were used in the trial. For the determination of the effect of age, 90 mares were assigned to six groups. Groups A, B, C, D, E, and F comprised 15 foals aged 5 months (mean  SD: 2.45  1.88), 15 foals aged 6 to 18 months (mean  SD: 9.33  2.93), 15 mares aged 2 to 5 years (mean  SD: 3.91  0.7), 15 mares aged 6 to 12 years (mean  SD: 8.66  2.3), 15 mares aged 14 to 20 years (mean  SD: 17.81  2.13), and 15 mares aged >20 years (mean  SD: 22.8  1.93), respectively. Glucose, blood urea nitrogen (BUN), albumin, creatinine, total bilirubin, direct bilirubin, total protein, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), calcium, iron, magnesium, and phosphorus concentrations were measured in the blood serum samples. There were significant age-related alterations for most of the biochemical parameters, except for ALT, LDH activities, and glucose, albumin, magnesium, and iron levels. The results of the present study showed that direct bilirubin (P < .01), ALP, and phosphorus decreased significantly (P < .001), whereas BUN, calcium (P < .01), creatinine, total protein, and total bilirubin increased (P < .001) with age. In foals, ALP activity, direct bilirubin, and phosphorus levels were higher, whereas BUN, creatinine, total protein, total bilirubin, and calcium levels were lower as compared with older animals. Biochemical values determined in the present study serve as reference ranges for pure-bred Arabian mares and can be used for health control and diagnosis of diseases.

From the Department of Biochemistry, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, Turkeya; and Department of Internal Medicine, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, Turkeyb. Reprint requests: Sema Y. Gurgoze, Professor, Department of Biochemistry, Faculty of Veterinary Medicine, Dicle University, Diyarbakır, Turkey. 0737-0806/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jevs.2010.09.006

Journal of Equine Veterinary Science  Vol 30, No 10 (2010)

Keywords: Pure-bred Arabian mare; Age; Serum biochemistry; Reference value

INTRODUCTION Hematological and biochemical parameters are used in horses as an aid in the clinical diagnosis of organic, infectious, and several parasitic diseases. They are also used in monitoring the recovery during treatment and in the assessment of metabolic conditions of a single animal or an entire herd.1 Specific reference intervals are needed for each animal species for the appropriate interpretation of serum biochemical results. Sometimes, a distinct reference value is needed for an analyte from a specific age or breed of animal. Many values vary with the age of the animal, with major changes occurring before puberty.2 Consequently, some analytes require different reference intervals for different age groups.2 Some researchers3,4 demonstrated that parameters such as total serum protein, globulins, and g-glutamyltransferase were influenced by the intake of the first colostrum, thereby increasing their concentrations in the serum of the calves.5 Furthermore, Steihardt and Thielscher6,7 reported that the total serum protein, albumin, and inorganic phosphorus concentrations were influenced not only by age but also by the nutrition of calves. However, a limited number of observations have been reported on the effects of age on the biochemical values in horse breeds.1,8 To our knowledge, no study related to the effects of age on biochemical values in pure-bred Arabian mares has been performed. Taking into consideration all the aforementioned information, the present study was performed to determine the effects of age on the reference values of some biochemical parameters (such as, glucose, blood urea nitrogen [BUN], albumin, creatinine, total bilirubin, direct bilirubin, total protein, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), calcium, iron, magnesium, and phosphorus) in pure-bred Arabian mares. We aimed to verify the hypothesis that age induces some biochemical alterations in pure-bred Arabian mares.

MATERIALS AND METHODS In all, 90 clinically healthy, pure-bred Arabian mares from five farms located in the Sanliurfa region of Turkey were

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Table 1. The distribution of six different age groups of pure-bred Arabian mares for farms (groups A, B, C, D, E, and F) (n ¼ 15) Age Groups

Farm 1

Farm 2

Farm 3

Farm 4

Farm 5

23 days–5-month-old foals (group A) 6–13-month-old foals (group B) 2–5-year-old mares (group C) 6–12-year-old mares (group D) 14–20-year-old mares (group E) >20-year-old mares (group F)

2 4 1 7 – 3

6 1 5 – 4 4

5 2 3 6 5 –

2 2 1 1 4 3

– 6 5 1 2 5

used in the trial. To determine the effect of the age of the animal on the normal ranges of serum biochemical parameters, mares were allocated to six different age groups. The distribution of the pure-bred Arabian mares, assigned to the six different age groups, on the basis of the farms they were obtained from is shown in Table 1. The first group (group A) comprised 15 foals aged 5 months (mean  SD: 2.45  1.88 months), the second (group B) 15 horses aged 6 to 18 months (mean  SD: 9.33  2.93 months), the third (group C) 15 horses aged 2 to 5 years (mean  SD: 3.91  0.7 years), the fourth (group D) 15 horses aged 6 to 12 years (mean  SD: 8.66  2.3 years), the fifth (group E) 15 horses aged 14 to 20 years (mean  SD: 17.81  2.13 years), and the sixth (group F) 15 horses above the age of 20 years (mean  SD: 22.8  1.93 years). The farms selected were representative samples of the farms located in the Sanliurfa region, and were determined to apply an appropriate nutrition regimen and to have a good health status. All horses were healthy and showed no sign of abnormality during the study period. Before the start of the trial, the mares, which were reproductively active, were confirmed not to be pregnant by ultrasonography and were also ascertained not to be lactating (groups C, D, and E); the mares included in groups A and B were reproductively inactive. The mares in group F were those that were not used for breeding purposes. Among the foals 5 months of age, two were 23- and 27-days old, three were 2 months, three were 3 months, three were 4 months, and four were 5 months. In all, 90 blood samples were collected from the animals between April and May 2009, and these were handled with care to minimize stress-induced effects. To reduce circadian variations, all samples were collected between 8:00 AM and 12:00 AM, before the animals were fed. The animals were fed twice a day with hay and oats. They did not receive any mineral supplements in their diet. Drinking water was given ad libitum. None of the animals performed physical exercise before blood samples were collected. All samples were drawn from the jugular vein of each animal into vacuum glass tubes without anticoagulant. After clotting, the serum was separated by centrifugation at 3,000 rpm

for 15 minutes at 48 C and the aliquots were kept frozen at 208 C until the analysis. Concentrations of serum glucose, BUN, albumin, creatinine, total bilirubin, direct bilirubin, total protein, ALP, AST, ALT, LDH, calcium, iron, magnesium, and phosphorus were measured by commercial kits (Biolabo SA, France) using a biochemical autoanalyser (AIRONE 200, Medisis Medical Systems Ltd., Italy), known to be appropriate for veterinary use.9-11 All data were analyzed using the statistical program SPSS, version 11.5, for Windows (SPSS Inc., SPSS 11.5, Chicago, IL, USA). For the evaluation of the effect of age, the data were analyzed using the ANOVA procedure, and Turkey’s multiple range test was used to determine the statistical significance of differences among the experimental groups.12 A significance level of P < 0.05 was used. All values were presented.

RESULTS Tables 2 and 3 present the mean values of some biochemical parameters and mineral levels in pure-bred Arabian mares according to age. Significant variations were observed in the values of BUN, direct bilirubin, calcium (P < .01), ALP, creatinine, total protein, total bilirubin, and phosphorus (P < .001) between different age groups. In foals, ALP activity, direct bilirubin, and phosphorus levels were higher, whereas BUN, creatinine, total protein, total bilirubin, and calcium levels were lower as compared with older animals. Mean serum concentrations of glucose, albumin, iron, and magnesium, and mean serum activities of AST, ALT, and LDH did not differ significantly among the different age groups (P > .05).

DISCUSSION Reference values for biochemical variables in the blood may vary per breed and are influenced by age and, to a certain extent, by rearing conditions.1,8 Several studies15-17 have reported a decrease in serum albumin level with advancing age in sheep and calves,

Parameters ALT (U/L) AST (U/L) ALP (U/L) LDH (U/L) BUN (mmol/L) Creatinine (mmol/L) Glucose (mmol/L) Albumin (g/L) Total protein (g/L) Direct bilirubin (mmol/L) Total bilirubin (mmol/L)

£5 Months Old (Group A)

6–13 Months Old (Group B)

2–5 Years Old (Group C)

6–12 Years Old (Group D)

14–20 Years Old (Group E)

12.90  2.82 31.66  10.44 4.50  0.64 7.66  2.96 4.22  0.82 295.40  21.98 287.16  8.41 266.41  30.66 266.33  36.91 243.00  19.23 590.30  45.83a 589.41  34.19a 283.16  29.35b 326.50  41.82b 323.44  44.12b 412.06  69.6 456.85  46.17 243.68  12.36 236.66  14.81 416.77  122.58 9.74  0.78a,b 11.82  0.83a,b 12.47  1.62a,b 14.14  0.55a,b 9.09  1.02a 73.37  5.30a,b 86.63  5.30b 66.30  9.72a,b 41.54  10.60a 38.01  5.30a 4.72  0.47 4.12  0.32 3.24  0.23 3.99  0.45 3.72  0.42 35.00  0.95 36.08  0.82 35.75  0.81 38.00  1.41 34.77  0.59 64.00  0.73a,b 65.50  0.74b 67.16  1.04b 64.77  1.55a,b 58.00  2.15a a a,b a,b b 2.39  0.17 1.71  0.17 1.53  0.00 1.88  0.34a,b 3.07  0.17 2.39  0.51a,b

2.22  0.51a,b

4.10  0.17b,c

4.95  0.17c

3.07  0.34a,b,c

>20 Years Old (Group F)

Probability Physiological of Difference Levels*

5.50  1.50 226.00  30.00 285.00  99.00b 317.63  115.68 14.63  1.07b 97.24  17.68b 3.87  0.08 35.50  1.50 64.00  1.00a,b 1.71  0.68a,b

P < .01 NS P < .001 NS P < .01 P < .001 NS NS P < .001 P < .01

4–12 152–294 143–395 162–412 4.28–9.28 88.4–167.96 4.12–5.75 27–42 52–79 0–6.84

1.53  0.85a

P < .001

0–34.2

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Table 2. Serum values of the biochemical parameters measured in pure-bred Arabian mares of six different age groups (groups A, B, C, D, E, and F) (n ¼ 15/group)

NS, not significant. a,b,c Significant differences within a row are indicated by different superscript alphabets. * Physiological levels were kindly obtained from some researchers.2,13,14

Table 3. Serum mineral levels measured in pure-bred Arabian mares of six different age groups (groups A, B, C, D, E, and F) (n ¼ 15/group) Parameters

£5 Months Old (Group A)

6–13 Months Old (Group B)

2–5 Years Old (Group C)

6–12 Years 14–20 Years >20 Years Old (Group D) Old (Group E) Old (Group F)

Calcium (mmol/L) 2.96  0.05a,b 3.10  0.07a,b 2.96  0.04a,b 3.32  0.18a 2.80  0.07b 3.03  0.03a,b 1.58  0.03a 1.09  0.08b 1.01  0.06b 0.84  0.05b 0.82  0.11b Phosphorus (mmol/L) 1.73  0.07a Magnesium (mmol/L) 0.84  0.05 0.79  0.06 0.85  0.08 1.29  0.09 1.11  0.17 1.00  0.14 Iron (mmol/L) 19.45  1.24 22.68  1.53 18.55  2.02 16.97  4.93 22.31  3.98 21.48  5.72

Probability of Difference

Physiological Levels*

P < .01 P < .001 P < .05 NS

2.8–3.4 1.0–1.8 0.53–0.82 13.06–25.06

NS, not significant. a,b Significant differences within a row are indicated by different superscript alphabets. * Physiological levels were kindly obtained from some researchers.2,13,14

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whereas Nakai et al18 and Jordana et al19 determined that albumin levels increased significantly with age in cats and donkeys, respectively. Furthermore, some researchers20,21 found that albumin levels in sheep increase with age. In contrast, other studies4,22-25 reported that there were no significant differences observed in albumin levels with age. In the present study, serum albumin levels did not change in all age groups of mares, in agreement with reports in published data,4,22-25 and albumin was within the reference range without any age-dependent changes. Mean serum concentrations of total protein were significantly affected by age, as they were lower in foals (group A) but were within the reference range for adult mares. Similarly, Zinkl et al23 and Nakai et al18 found that total protein tended to increase with advancing age in donkeys and cats, respectively. In contrast, Alonso et al26 reported that total proteins decreased with age. Baumgartner and Pernthaner15 determined that the serum total protein level was not significantly affected by age in sheep aged <1 year. Similarly, Roil et al22 observed no effect of age on the level of total proteins in the serum. The variety of results obtained for the effect of age on this parameter cannot be easily explained. It is possibly related to the breed of the animals tested as well as to rearing conditions. The mean values of BUN were significantly affected by age, as they were higher in adult mares (group F). In mares, BUN values exceeded the reference range after 2 to 5 years of age. In contrast, Hilleman et al,27 Baumgartner and Pernthaner,15 and Roubies et al21 did not detect any significant difference in BUN values between young and old sheep. Also, Mohri et al25 reported that urea levels in calves were within the reference range for adult cattle. Although Levy et al28 reported that BUN increased, Nakai et al18 reported that no alteration occurred with age in kittens. During the growth period, the rate of protein synthesis is greater than that of protein breakdown, whereas during ageing, the rate of protein breakdown exceeds that of protein synthesis. Increase in the intake of dietary protein during the senescent period does not reverse this process.2 The increase observed in the blood urea level of pure-bred Arabian mares with age is thought to be related to the intake of dietary protein and the decrease in renal filtration. Some authors4,17,25 have reported that creatinine levels in calves were higher than the adult reference range at birth, after which it declined to the reference value and remained stable up to 84 days of age. Jordana et al19 suggested that serum creatinine levels in young donkeys were significantly lower than those of adults, whereas Roubies et al21 found that this parameter was not affected significantly by age in sheep. Levy et al28 reported that creatinine increased with age in kittens. The mean serum creatinine concentration was significantly affected by the age of mares and was higher in groups D and F as compared with groups A and B. At an age of 6 to 12 years and >20

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years, it was observed that the creatinine level was within the reference range, but at other ages it was below the reference value. Our data were in agreement with those reported for donkeys,21 perhaps, reflecting differences in feeding conditions because creatinine level depends on the total body content of creatine, which in turn depends on dietary intake and muscle mass.16 The mean serum concentrations of total bilirubin and direct bilirubin were significantly affected by age, as they were higher in groups A and D, respectively. In the present study, the direct bilirubin levels of the mares were always within the reference range for horses. However, in groups C and D, the total bilirubin level was higher than the reference value. Mohri et al25 reported that bilirubin levels in calves were always within the reference range for adult cattle. In contrast, Egli and Blum4 concluded that during the first days of life, bilirubin levels of calves were higher than the adult reference range. Alteration of bilirubin levels with age has also been reported in kittens.18,28 The main sources of energy in cell cultures are glucose and fatty acids. Cells can modify the use of these sources throughout the cell cycle. The ageing process can have major effect in these modifications, changing the metabolism of glucose and fatty acids.29 Roubies et al21 showed that serum glucose levels were higher in lambs compared with ewes. Similarly, some authors4,17,25,30 reported that glucose levels in calves were higher than the reference range for adult cattle. Similarly, Zinkl et al23 reported that glucose levels of older donkeys were significantly lower than those of younger animals. A similar age-dependent change has also been observed in cats.18 The results of the present study showed that serum glucose levels in foals were not significantly different as compared with its levels in adult mares, despite there being a tendency for it to decrease. In all age groups, glucose levels were within the reference range for horses. The variations in the reports of different researchers are considered to may have arisen from species, geographical and nutritional factors, timing of blood sampling, and the methodology and equipment used by laboratories. AST and LDH activities remained within the reference range throughout the study. Although ALP activity was higher than the reference value in groups A and B, it declined to the adult range during the course of the trial. ALT activity exceeded the reference range at 6 to 13 months of age and remained within the reference range at other ages. Liver enzyme activities were not significantly affected by the age of the animals, which is in accordance with the results of Baumgartner and Pernthaner.15 Some studies4,17,25 found that ALP activity in Holstein calves was always above the adult reference range. Similarly, Zinkl et al23 and Nakai et al18 determined a decrease in ALP activity with ageing in donkeys and cats, respectively. Some authors4,18,25 reported that AST activity did not change with age. In contrast, Levy et al28 detected an increase in

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AST activity with age in cats. Nakai et al18 reported that ALT activity in adult cats was significantly higher compared with kittens. A significant decrease in ALP levels with age was observed in pure-bred Arabian mares. We did not detect any significant difference between the six age groups of mares in the levels of AST, ALT, and LDH. Throughout the study, calcium and iron concentrations remained within the previously reported reference range. Phosphorus concentrations were within the reference range for horses in groups A, B, C, and D and were below the reference range in other age groups. Magnesium levels were within the reference range, except groups D, E, and F, which displayed levels higher than the reference range. In the present study, it was demonstrated that the age of the mares influenced markedly serum macroelements, excluding iron and magnesium. Calcium concentrations determined in group D were significantly higher (P < .01) compared with group E. It was demonstrated that phosphorus concentration decreased significantly with age in pure-bred Arabian mares (P < .001). Similar results were also reported by some authors for donkeys,19,23 suggesting that this age-related decrease probably reflects decreased bone metabolism as animals grow older. Baumgartner and Pernthaner,15 Alonso et al,26 and Roubies et al21 reported similar results for serum phosphorus levels. Furthermore, it has been proven that younger animals absorb dietary calcium more efficiently and achieve much higher maximum rates of absorption for both calcium and phosphorus in comparison with older animals.31 In contrast, Nakai et al18 reported that there is no apparent effect of ageing on calcium level in cats.

CONCLUSIONS It seems that age significantly influences biochemical parameters and mineral levels in pure-bred Arabian mares. The data obtained in the present study can enhance our understanding of biochemical parameters in this species, which in turn will allow veterinarians to establish an appropriate interpretation of laboratory data and give these animals appropriate care. ACKNOWLEDGMENTS The authors would like to thank Dr. Abuzer Kaffar Zonturlu (Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Harran University, Sanlıurfa, Turkey) and the staff of the farms in Sanliurfa region, from which the pure-bred Arabian mares were obtained, for their valuable contribution.

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2. Meyer DJ, Harvey JW. Veterinary laboratory medicine: interpretation and diagnosis, 3rd ed. St. Louis, MO: Saunders; 2004. 3. Boediker R. Die bestimmung der gamma-glutamyl-transferase (GGT) im serum als indikator fu¨r die kolostralmilch-versorgung des kalbes. Tieraerztl Umsch 1991;46:190–194. 4. Egli CP, Blum JW. Clinical, haematological, metabolic and endocrine traits during the first three months of life of suckling Simmentaler calves held in a cow-calf operation. J Vet Med A 1998;45: 99–118. 5. Jezek J, Klopcic M, Klinkon M. Influence of age on biochemical parameters in calves. Bull Vet Inst Pulawy 2006;50:211–214. 6. Steihardt M, Thielscher HH. Physiologische variablen und wachstumsleistung bei saugkalbern der mutterkuhhaltung in den ersten beiden lebensmonaten. Tieraerztl Umsch 2000;7:380–389. 7. Steihardt M, Thielscher HH. Reaktionen von milchrindkalbern auf flu¨ssignahrungaufanhme an verschiedenenen alterspunkten von und wahrend der aufzuncht. Tieraerztl Umsch 2000;55:663–673. 8. Satue K, Blanco O, Munoz A. Age-related differences in the hematological profile of Andalusian broodmares of Carthusian strain. Vet Med Czech 2009;54:175–182. 9. Gurgoze SY, Zonturlu AK, Ozyurtlu N, Ic¸en H. Investigation of some biochemical parameters and mineral substance during pregnancy and postpartum period in awassi ewes. Kafkas Univ Vet Fak Derg 2009;15:957–963. 10. Lankin VZ, Ivanova MV, Konovalova GG, Tikhaze AK, Kaminnyi AI, Kukharchuk VV. Effect of b-hydroxy-b-methylglutaryl coenzyme A reductase inhibitors and antioxidant vitamins on free radical lipid oxidation in rat liver. Bull Exp Biol Med 2007;143:414–417. 11. Almeida MAO, Jesus EEV, Sousa-Atta MLB, Alves LC, Berne MEA, Atta AM. Clinical and serological aspects of visceral leishmaniasis in Northeast Brazilian dogs naturally infected with Leishmania chagasi. Vet Parasitol 2005;127:227–232. 12. SPSS. Statistical package for social sciences for Windows. Chicago, IL: SPSS Inc; 1999. 13. Turgut K. Veteriner klinik laboratuar tes‚his. 2. baskı. Konya, Bahc¸ıvanlar Basım Sanayi A.S‚; 2000. 14. Gurgoze SY, Cetin H. The investigation of some biochemical parameters in purebred Arabian mares with healthy and endometritis. F U Sag Bil Derg 2004;18:127–130. 15. Baumgartner W, Pernthaner A. Influence of age, season and pregnancy upon blood parameters in Austrian Karakul sheep. Small Ruminant Res 1994;13:147–151. 16. Kaneko JJ, Harvey JW, Bruss ML. Clinical biochemistry of domestic animals, 5th ed. New York/London: Academic Press; 1997. 17. Knowles TG, Edwards N, Butterworth A, Warriss PD. Changes in the blood biochemical and haematological profile of neonatal calves with age. Vet Rec 2000;147:593–598. 18. Nakai N, Nawa K, Maekawa M, Nagasawa H. Age-related changes in hematological and serum biochemical values in cats. Jikken Dobutso 1992;41:287–294.

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