The Changes of Serum Clinical Biochemistry Values in Ostrich Chicken from 4 to 12 Weeks of Age

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1 J Anim Diagn Med (2011) 1: Research Article The Changes of Serum Clinical Biochemistry Values in Ostrich Chicken from 4 to 12 Weeks of Age Yieng-How Chen 1, Shu-Yin Wang 2, and Chau-Loong Tsang 3, 4 * 1 Department of Animal Science, College of Agriculture, Tunghai University, Taichung, Taiwan 2 Department of Animal Science, Chinese Culture University, Taipei, Taiwan 3 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan 4 Veterinary Teaching Hospital, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan Received: November 18, 2010 / Revised: January 23, 2011 / Accepted: February 20, 2011 A B S T R A C T The objective of this experiment was to establish the normal serum clinical biochemistry values in ostrich chicken from 4 to 12 weeks of age. The effects of age and sex on affecting the changes of clinical biochemistry values in ostrich were also studied. Thirty 4-wk-old ostriches (male, 14 birds and female, 16 birds) were used in this study. Feed and water were supplied ad libutum throughout the entire experimental period. Blood samples were taken in tube from superficial planter metatarsal vein in 4, 6, 8, 10 and 12 wks of age, and those samples were used for measurement of serum enzymes activities of clinical biochemistry, serum protein-relation of clinical biochemistry, total cholesterol, triglyceride, total billirubin and glucose. The results indicated that the most of mean clinical biochemistry values were within an acceptable range of those in the published literatures about ostriches. The highest of serum total cholesterol, triglyceride, total billirubin and glucose levels, BUN, CREA and UA values appeared in 4 or 6 weeks of age; however, ALB and A/G values in 4-week-old ostrich were the lowest among the week of age in ostrich (P < 0.05). The sex of ostrich had no infuence on various serum biochemistry values except ALP, LDH, total cholesterol. On the contrary, age affected all parameters values; however, TP values of female ostrich were in exception. Keywords: Clinical biochemistry, Ostrich I N T R O D U C T I O N Ostrich is a developing livestock enterprise in Taiwan and birds had high ecnomic value for human because birds are good sources of leather, meat and feather. However, young ostrich had a low survival rate (Chen et al., 2003; Verwoerd et al., 1999), meanwhile the survival rate of the chicks to three months of age were (Deeming et al., 1993). Clinical hematology and biochemistry are useful aid for the diagnosis of disease in birds (Woerpel and Rosskopf, 1984; Ho et al., 2002). Therefore, it is important to establish clinical biochemistry reference values in ostrich in order to have baseline informa- * Corresponding author: Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan address: cltsang@mail.vm.nchu.edu.tw tion (Palomeque et al., 1991). Though, Levy et al. (1989), Okotie-Eboh et al. (1992) and Palomeque et al. (1984) had established blood biochemistry reference values of ostrich, those ostriches were not all from the same group kept under the same condition (Levy et al., 1989; Okotie- Eboh et al., 1992), and the data of clinical bio- chemistry reference values in ostrich from 4 to 12 weeks of age is absent (Levy et al., 1989; Okotie-Eboh et al., 1992; Palomeque et al., 1991). Perelman (1999) suggested that the best way to obtain accutate reference values to compare with a clinical case was to take a significant number of samples from apparently healthy birds from the same group kept under the same condition. Bogin (1999) pointed out clinical biochemistry reference values of birds were affected by nutrition and environment conditions. Therefore, the purpose of this study is to establish the blood biochemistry reference values in ostrich, a native breed of ostrich in Taiwan, and study the

2 40 Yieng-How Chen et al. effect of age and gender on blood biochemistry reference values in ostrich from 4 to 12 weeks of age. M A T E R I A L S A N D M E T H O D S Experimental Animals Thirty 4-week-old ostrich (14 males and 16 females) at the Cha-nan ostrich farm, Chia-Yi Hsein, south of Taiwan, were as experimental animals and were brooded in an electric brooder from 0 to 6 wks of age. Ostrich was fed on high floor pen in close house (area 3 m 2 m) from 0 to 4 week of age, fed on wire floor pen open house (area 10 m 6 m) from 5 to 8 week of age, and fed on floor pen (area 30 m 20 m) from 9 to 12 week of age. The ostrich chickens at 0-8 wks of age fed commercial starter diets (crude protein: 18%; ME:2447 kcal/kg; crude fiber:6%) and at wks of age will feed grower diets (crude protein:15 % ; ME:2690 kcal/kg; crude fiber:8 % ) and mixed alfalfal (crude protein: 17%; crude fiber:28%) as the ratio 1:1. Feed and water were supplied ad libitum for a 8-wk experimental period (4-12 wks of age). The plastic number band were put on leg of 4-week-old orstrich and New castle vacine were muscle injected at 1 and 2 month of age. Analysis of clinical biochemistry values of serum samples The blood samples of all birds at 4, 6, 8, 10 and 12 weeks of age were collected by superficial plantar metatrsal vein puncture with 21-gauge needle syringes after feed was deprived off for 8 hours. To reduce the variation associated with diurnal changes in blood, the blood samples were obtained from the vein at the same time period (9:00 to 12:00). Blood samples was centrifuged at 600 xg, 15 min to separate serum and serum samples were stored in refrigerator under -20 to supply the analysis of clinical biochemistry values. The of identification the gender of ostrich chicken were used by PCR (polymerase chain reaction) method (Horing and Huang, 2003). Serum enzymes activities of clinical biochemistry: alanine aminotransferase (ALT), alkaline phosphatase (ALP),γ-glutamyltransferase (GGT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH); serum protein-relation of clinical biochemistry: total protein (TP), albumin (ALB),blood urea nitrogen (BUN), creatinine (CREA), uric acid (UA) and albumin/globumin (A/G). total cholesterol, triglyceride, total bilirubin and glucose. All blood parameters concentration were determined using commercial kits by clinical analyzer (Express plus, Ciba Corning, England). Table 1. The changes of serum enzymes activities of clinical biochemistry in ostrich from 4 to 12 wks of age. Age (wks) N ALT ALP GGT AST LDH CK u / L ±2.6 a 803±218 bc 0.4±0.5 a 455.3±60.0 a 1107±212 a 3188±1126 a ±3.7 ab 867±221 c 1.2±0.7 b 500.5±63.2 a 1494±296 ba 4359±766 b ±2.3 ab 681±182 ab 1.6±1.0 b 515.2±51.7 a 1236±210 aa 4048±996 b ±6.4 b 725±200 abc 1.3±1.1 b 648.4±306.7 b 1700±572 b 3984±1319 b ±4.4 ab 621±211 a 1.1±0.8 b 495.8±70.1 a 1165±293 a 3663±829 ab Mean 7.0± ±218 A 1.1± ± ±412 A 3872± ±2.2 a 888±223 bc 0.6±0.9 a 486.8±85.5 a 1373±251 a 3193±696 a ±2.4 ab 929±215 c 1.4±0.7 b 522.1±42.8 a 1824±320 bb 3299±1510 ab ±2.6 ab 766±185 a 1.3±0.7 b 548.7±76.1 a 1528±226 ab 4416±716 b ±5.2 b 798±224 ab 0.7±0.8 b 644.8±168.0 b 1898±421 b 3929±612 ab ±1.3 a 696±158 a 1.0±0.8 ab 487.4±55.8 a 1300±350 a 3435±1160 ab Mean 6.1± ±220 B 1.1± ± ±393 B 3806±1064 ALT: alanine aminotransferase; ALP: alkaline phosphatase; GGT: γ- glutamyltransferase; AST: aspartate aminotransferase; LDH: lactate dehydrogenase; CK: creatine kinase. a, b,c Means within the same column without the same superscripts differ significantly among different ages in the same sex of ostrich (P < 0.05). A, B Means between sexes differ significantly at same week (P < 0.05). F: female ostrich ; M: male ostrich.

3 J Anim Diagn Med (2011) 1: Table 2. The changes of serum protein-relation of clinical biochemistry in ostrich from 4 to 12 wks of age. Age (wks) N TP (g/dl) ALB (g/dl) A/G BUN CREA UA ± ±0.07 a 0.92±0.11 a 1.56±0.88 b 0.21±0.05 b 8.72±0.05 b ± ±0.18 b 1.49±0.12 b 1.59±0.91 b 0.21±0.03 b 8.46±6.40 b ± ±0.17 bc 1.77±0.15 d 1.46±0.81 ab 0.16±0.05 a 5.26±2.61 a ± ±0.22 c 1.69±0.13 cd 1.18±0.81 ab 0.15±0.05 a 5.17±2.08 a ± ±0.30 b 1.56±0.22 bc 0.45±0.49 a 0.16±0.05 a 5.14±1.76 a Mean 2.88± ± ± ± ± ± ±0.22 a 1.27±0.14 a 0.85±0.10 a 1.98±0.55 b 0.21±0.03 b 8.47±4.18 b ±0.28 a 1.62±0.23 b 1.51±0.26 b 1.65±0.75 b 0.22±0.06 b 9.01±5.08 b ±0.26 a 1.83±0.16 cd 1.89±0.23 d 1.18±0.81 ab 0.15±0.05 a 5.17±2.08 a ±0.26 b 1.91±0.21 d 1.71±0.25 c 1.06±0.50 a 0.13±0.06 a 4.53±1.40 a ±0.32 a 1.72±0.20 c 1.55±0.20 b 0.20±0.14 a 0.13±0.05 a 4.24±1.45 a Mean 2.84± ± ± ± ± ±3.78 TP: total protein; ALB: albumin; BUN: blood urea nitrogen; CREA: creatinine; UA: uric acid: A/G: albumin/globulin. a, b,c Means within the same column without the same superscripts differ significantly among different ages in the same sex of ostrich (P < 0.05). A, B Means between sexes differ significantly at same week (P < 0.05). F: female ostrich ; M: male ostrich. Statisticaal analysis All datas were subjected to analysis of variance using the General Line Model (GLM) of SAS (Statistical Analysis System) (1988). The Least Squares Means were used to compare and estimate the difference among the weeks of age and between the gender. R E S U L T S A N D D I S C U S S I O N The changes of serum enzymes activities of clinical biochemistry in ostrich from 4 to 12 wks of age were shown Table 1. The serum enzymes activities of clinical biochemistry including ALT, ALP, GGT, AST, LDH and CPK were flunctuated with week of age. There were significant difference in the values of ALT, ALP, GGT, AST, LDH and CPK among the weeks of age in ostrich (P <0.05); however, no significant difference were found in those biochemistry values between sexes of the birds at same week of age, except LDH. The female ostrich chick had higher LDH values than that of male ostrich at 6 and 8 week of age (P <0.05). The changes of serum protein-relation of clinical biochemistry in ostrich from 4 to 12 wks of age were presented in Table 2. Significant differences were observed in the TP, ALB, A/G, BUN, CREA and UA of both gender and in TP of female ostriches among the weeks of age. All of these parameters, expect TP, ALB, and A/G werethe highest in 4 or 6 weeks of agein ostrich, however, ALB and A/G values in 4-week old ostriches, the lowest among the week of age. There was no significant difference between sexes of ostrich in the above mentioned parameters. Those parameters including BUN, CREA, and UA were significantly decrease with age (P<0.05) and Uric acid were significant correlation with BUN, CREA (P<0.001) and the correlation values (r) were 0.32 and 0.31, respectively. The changes of serum total cholesterol, triglyceride, and total bilirubin and glucose concentrations in ostrich from 4 to 12 wks of age are shown in Table 3. The 4-week-old ostriches in both gender had the highest of serum total cholesterol, triglyceride, total bilirubin and glucose values among the week of age, and the highest values of total billirubin in in male and female ostrich appeared at 4 and 6 weeks of age, respectively. There were no significant difference in the values of certain parameters including triglyceride, total bilirubin and glucose between sexes of orstrich; however, the female ostriches had higher total cholesterol values than those of male at 4-week-old and the whole study period (P <0.05).

4 42 Yieng-How Chen et al. Table 3. The changes of serum total cholesterol, triglyceride, total bilirubin and glucose concentrations in ostrich from 4 to 12 wks of age. Age (wks) N Total cholesterol Triglyceride Total bilirubin Glucose m g/dl ±172 ca 388±190 b 0.8±0.4 b 210±17 c ±47 b 332±123 b 0.9±0.8 b 195±35 c ±24 a 193±99 a 0.4±0.2 a 172±31 b ±54 ab 131±68 a 0.4±0.1 a 151±34 a ±46 ab 129±65 a 0.4±0.2 a 164±23 ab Mean 183±101 A 224± ± ± ±170 cb 392±163 b 1.0±0.4 b 228±28 d ±42 b 328±130 b 0.9±0.4 b 198±19 c ±43 a 152±83 a 0.4±0.2 a 174±28 b ±63 ab 106±51 a 0.3±0.1 a 152±38 a ±57 ab 107±74 a 0.3±0.2 a 160±25 ab Mean 243±124 B 211± ± ±38 a, b, c, d Means within the same column without the same superscripts differ significantly among different ages in the same sex of ostrich (P < 0.05). A, B Means between sexes differ significantly at same week (P < 0.05). F: female ostrich; M: male ostrich. Comparison of GGT values in ostrich, broiler and Leghron of egg type chick at 6-8 weeks of age, GGT values of ostrich in this study is very low and less than the 4% value in broiler and Leghron ( vs and 45.0 U). At the similar age, the means of ALT, ALP, CK, AST values in this study, except means of GGT and LDH values, were higher than those of ostriches tested by Levy et al. (1989). The difference might result from environment or other factors, e.g. sodium heparin and EDTA. Some parameters may be affected by the kind of anticoagulant used and the way the sample is handled during collection and processing (Palomeque et al., 1984), and Levy et al. (1989) took blood samples with heparin for clinical biochemistry. Nevertheless, most of parametres values in this study are within an acceptance range of those in the literatures (Levy et al. 1989; Lumeij, 1997; Olowookorun and Makinde, 1998.Okotie-Eboh et al., 1992; Palomeque et al., 1984). The total protein levels of ostrich from 4 to 12 weeks of age was lower than those reported by Palomeque et al. (1991) for ostrich, also lower than those of mule duck (Pai et al., 1996) and broiler (Ross et al., 1984) at similar age; however, total protein values was within the normal range for ostrich (Okotie-Eboh et al., 1992). Altough Perelman (1999) pointed that total protein levels lower than 3.0 g/dl probably suggesting hypoproteinaemia and probaly hypoalbuminaemia which may occur in unhealthy condition; however, basing on appearance of bird, ostriches were in health status in this study. ostrich had the higest of TP concentration at 10 weeks of age, this might be associated with high metabolic rate in relation to rapid tissue and feather growth and as a consequence of high protein demand (Palomeque et al., 1991). Though female chicks grew faster than male chicks before 3 months of age (Deeming et al., 1993), it did not reflect the phenomenon concerning that female chicks had higher TP values than that of male chicks in this study. ALB and A/G level increase with age from 4 to 8 wks of age; then, on the contrary, those parameter values decrease with age from 10 to 12 wks of age. Whether this phenomenon relates to nutrient absorption or another physiology is unkown. This study suggests that further research is required to resolve this problem. According to the report of Okotie-Eboh et al. (1992) and Palomeque et al. (1984), the blood urea nitrogen, creatinine, uric acid level was within the normal range in the ostrich; meanwhile 12-week-old ostrich in both gender had the lowest concentrations in this study. This might be explained by its relation with diet. Bell and Sturkie (1965) pointed that elevated uric acid level occurs in animals on high protein diets. In this experiment, ostrich fed crude protein level in the diet decreased from 18% to 16%

5 J Anim Diagn Med (2011) 1: after 10 weeks of age, and there were positive correlation among blood urea nitrogen, creatinine, uric acid values. In this study, total cholesterol and triglyceride values in ostrich were higher than those of ostrich from 1 to 3 month of age in the report by Levy et al (1989). The Phenomenon of female ostriches had higher total choles- terol values than those of male ostriches at 4-week-old is interesting and the reason could not be explained in this study, it need to be researched in advance. Total bi- lirubin level in this experimental ostrich from 4 to 12 weeks of age was higher than those of ostrich for 5-month-old and 1-yr-old (Palomeque et al., 1991) as well as other avian species, e.g. mallard (Chen et al., 2002) and Tsaiya duck (Ho et al., 2002). Nevertheless, the means of total bilirubin concentration of ostrich on both gender was similar to the average values of 1-3 month of age in ostrich described (Levy et al., 1989). The glucose values obtained were close to the report for juveniles ostriches (263.5 mg/dl) (Palomeque et al., 1991) and for brooding white Tsaiya duckling (214 mg/dl) (Ho et al., 2002). In this study, glucose level decreased with age, and this result was in accordance with the Palomeque et al. (1991) find that juvenile had higher glucose level than that of adult in ostrich. This parameter was wide variation from 4 to 12 wks of age and the reason could be a consequence of the absorptive state of the animal (Palomeque et al., 1991), also a seasonal rhythm of bird (Bairlein, 1983). Plasma glucose levels in ostriches range from mg/dl (Funge, 1996), and glucose levels lower than 120 mg/dl are suggestive of hypoglycaemia (Palomeque et al., 1991). Glucose values of ostriches in this study was mg/dl, therefore, birds were in normal status. A C K N O W L E D G M E N T S The authors wish to thank professor M. C. Huang of Department of Animal Science, National Chung-Hsing University for his help with sexual identification in this research, and also give our thanks to Cha-Nan Ostrich Co. for suppling ostrich chicks as experimental animal. R E F E R E N C E S Bairlein F Season variations of serum glucose levels in a migratory songbird, Silvia borin. Comp Biochem Physiol. 76A: Bell DJ, Sturkie PD Chemical constituents of blood. In: Sturkie PD, ed. Avian physiology, Comstock Publications Associate Division, Cornell University Press, Ithaca, New York. pp Bogin E Clinical biochemistry of waterfowl, the first World Waterfowl Conference Proceedings. Taiwan, ROC. pp Chen YH, Kou MJ, Tsang CL, Shiu CH, Wang SY Studies on the change of clinical biochemistry values of mallard ducks during the moulting period. Tunghai J (College of Agricuture). 43: 7-14,. Chen YH, Feng CW, Kuo MJ, Lin DT, Shiau JR, Tsang CL The changes of hematology in ostrich from 4 to 12 weeks of age. 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