- Email: [email protected]
Changes of biochemical parameters and enzyme activities in broiler chickens with cold-induced ascites
Changes of biochemical parameters and enzyme activities in broiler chickens with cold-induced ascites M. Daneshyar, H. Kermanshahi,1 and A. Golian Excellence Center for Animal Sciences Research and Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, PO Box 91775-1163, Mashhad, Iran
Key words: ascites, biochemical parameter, enzyme activity, broiler 2009 Poultry Science 88:106–110 doi:10.3382/ps.2008-00170 Before a bird exhibits gross ascites syndrome lesions, commonly anatomical and also hematological changes can be detected (Maxwell et al., 1986, 1987). A right ventricle-to-total ventricle ratio (RV/TV) of more than 0.27 is considered an accurate measure of the onset of ascites (Huchzermeyer and De Ruyck, 1986; Paecock et al., 1988). The changes of hematocrit and hemoglobin (Cueva et al., 1974; Maxwell et al., 1986, 1987; Yersin et al., 1992; Yahav et al., 1997; Wideman et al., 1998), red blood cell counts (Cueva et al., 1974; Maxwell et al., 1986, 1987; Yersin et al., 1992), and blood gas volume (Olkowski et al., 1999; Daneshyar et al., 2007) between healthy and ascitic birds were investigated. In addition, some other variables may be affected by hypoxemia and ascites. Tankson et al. (2002) observed different serum protein and cholesterol between healthy and Enterococcus faecalis-challenged chickens to inducing pulmonary hypertension syndrome. Diaz-Cruz et al. (1996) detected a greater concentration of glucose in the liver of broilers with ascites. Yersin et al. (1992) observed a decrease of serum albumin concentration with ascites. Biswas et al. (1995) reported decreased
INTRODUCTION The incidence of ascites in broilers has become of increasing concern to the poultry industry in many areas of the world (Silva et al., 1988). Despite investigations of the syndrome for many years, it is still a condition that inflicts financial loss on poultry producers around the world (Maxwell and Robertson, 1997). It is a severe cause of loss to the broiler industry in many countries, not only due to high rate of mortality, but also due to decreased weight gain and increased condemnations at slaughter (Julian, 1993). This metabolic disorder accounts for over 25% of overall mortality (Guo et al., 2007); considering that an estimated 40 billion broilers are produced annually around the world, it is evident that losses due to ascites are significant (Solis de los Santos et al., 2005). ©2009 Poultry Science Association Inc. Received April 26, 2008. Accepted September 8, 2008. 1 Corresponding author: [email protected]
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ol, triglyceride, activity of lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were determined. Throughout the study, the right ventricle-to-total ventricle ratio and total mortality percentage due to ascites of CT-treated birds at the end of experiment was greater (P ≤ 0.05) than those of NTtreated ones. Fasting blood sugar of CT-treated birds in wk 4 and 6 was greater (P ≤ 0.05) than NT-treated birds. Total blood protein of CT treatment was lower than NT-treated birds in every week and whole period, but this difference was only significant (P ≤ 0.05) in wk 6. There was not a significant difference between 2 treatments for triglyceride and cholesterol, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase. It was concluded that cold-induced ascites could affect serum protein and fasting blood sugar of broiler chickens.
ABSTRACT An experiment with 250 one-day-old male broilers (Ross 308) was conducted to investigate the differences of some blood parameters of cold-induced ascitic and healthy broiler chicks in a 6-wk period. The chickens were divided into 2 groups of 5 replicates each. One group of these chickens was raised in normal temperature (NT) treatment and the other in cold temperature (CT) treatment to induce ascites. Mortality was necropsied daily to determine cause of death. At the end of the experiment (wk 6), 5 chickens from each replicate were randomly selected and slaughtered. The heart was removed; the right ventricle was dissected away from the left ventricle and septum. Weights of right and left ventricles were determined separately. Average BW gain and average feed intake were measured weekly, and weekly average feed conversion ratio was calculated. Serum glucose, total protein, cholester-
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Table 1. Composition of experimental diets Item
Grower (21 to 42 d)
52.12 29.94 8.87 5.00 1.20 1.74 0.50 0.47 0.10 0.06 100.00
59.65 27.84 4.55 4.50 1.32 1.22 0.50 0.34 0.03 0.05 100.00
3,200.00 23.00 1.00 0.45 0.20 1.30 0.90 1.10 0.29
3,200.00 20.00 0.90 0.35 0.15 1.19 0.72 1.00 0.26
1 Supplied per kilogram of diet: vitamin A, 10,000 IU; vitamin D3, 9,790 IU; vitamin E, 121 IU; B12, 20 µg; riboflavin, 4.4 mg; calcium pantothenate, 40 mg; niacin, 22 mg; choline, 840 mg; biotin, 30 µg; thiamine, 4 mg; zinc sulfate, 60 mg; manganese oxide, 60 mg.
serum protein in fast-growing chickens experiencing ascites. Balog et al. (2003) did not detect any difference in select hematological parameters between resistant, susceptible, and relaxed lines to ascites except different serum phosphorus and alkaline phosphatase between resistant and susceptible lines. Even though a lot of information about hematocrit, hemoglobin, and blood gases is available, there is little or no information about changes and relationships of other physiological parameters such as fasting blood sugar (FBS), serum protein, cholesterol, and triglyceride in cold-induced ascitic chicks. Therefore, evaluating these parameters was first aim of this study in coldinduced ascitic broiler chickens. Furthermore, effect of cold-induced ascites on activity of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) enzymes were investigated as a second aim. It has been recognized that LDH isoenzymes are released to blood in heart, liver, and pulmonary system damage (Jaffe et al., 1996; Zantop, 1997); AST in heart damage (Wirz et al., 1990) and liver damage (Pratt and Kaplan, 2000); and ALT in liver damage (Zantop, 1997). It also has been found that the liver, heart, and pulmonary system are affected in ascites; therefore, it would be expected that these plasma enzyme activities changes with ascites.
MATERIALS AND METHODS Two hundred fifty 1-d-old male broiler chickens (Ross 308) were used in this experiment. Half of the birds were housed in 5 pens (1 × 2 m2) of 25 each and were reared under normal temperature (NT group) up to 4
wk of age; then, they were exposed to constant 22 ± 1°C until 6 wk of age (Luger et al., 2001). The other half of the birds were housed in a room with 5 pens (1 × 2 m2) of 25 each in a cold temperature condition (CT group). For inducing ascites, the birds of the CT group were raised under 32°C and 30°C during wk 1 and 2, respectively. The house temperature was decreased to 15°C during wk 3 and maintained between 10 and 15 for the rest of the study (Igbal et al., 2001). Feed and water were provided ad libitum. All chickens were supplied with a mash broiler starter diet until 21 d of age and were fed a mash broiler grower diet thereafter (Table 1). Average BW gain (ABWG) and average feed intake (AFI) were measured weekly, and average feed conversion ratio (AFCR) was calculated and reported weekly. Mortality was recorded daily, and all of the dead birds were examined for lesions of heart failure and ascites throughout the study. On d 14, one chicken from each replicate pen was selected randomly, marked, and wing vein blood samples were obtained weekly at 2200 h every Sunday after a 3-h starvation period. Blood samples were immediately aliquoted into nonanticoagulant tubes. These tubes were allowed to clot for 2 h at 37°C, and then serum was decanted and stored at −20°C for later analyses (Tankson et al., 2002). Serum samples were thawed and serum glucose, total protein, cholesterol, triglyceride, and the LDH, AST, and ALT enzyme activities were determined using an autoanalyzer (Autolab, PM 4000, Autoanalyzaer Medical System, Rome, Italy). At the end of the experiment (wk 6), 5 chickens from each replicate (pens) were randomly slaughtered. The heart was removed, and the right ventricle was dissected from the left ventricle and septum. The right and left ventricles were weighed separately.
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Ingredients (%) Corn Soybean meal (44% protein) Corn gluten meal Soybean oil Limestone Dicalcium phosphate Vitamin and mineral premix1 Salt dl-Methionine l-Lysine Total Calculated analysis ME (kcal/kg) CP (%) Calcium (%) Available phosphorus (%) Sodium (%) Arginine (%) Methionine + cystine (%) Lysine (%) Tryptophan (%)
Starter (0 to 21 d)
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Daneshyar et al. Table 2. Growth performance of chickens raised under normal (NT) and cold (CT) environmental temperature Week Treatments
1
Average feed intake (g) NT 107.0 CT 108.0 ±SEM 1.83 P-value 0.818 Average BW gain (g) NT 83.0 CT 80.0 ±SEM 1.31 P-value 0.157 Average feed conversion ratio NT 1.30 CT 1.35 ±SEM 0.033 P-value 0.280
2
3
4
5
6
1 to 6
253.0 261.0 2.81 0.071
535.0a 496.0b 8.98 0.016
826.0a 746.0b 10.19 0.0005
986.0a 886.0b 25.34 0.0235
1,305.0 1,185.0 52.41 0.144
3,914.0a 3,585.0b 14.46 0.027
159.0 166.0 3.00 0.127
296.0 308.0 6.00 0.201
374.0a 329.0b 12.79 0.037
452.0 390.0 20.73 0.068
544.0 484.0 34.98 0.259
1,908.0a 1,758.0b 44.24 0.043
1.60 1.58 0.037 0.766
1.81a 1.61b 0.048 0.020
2.22 2.28 0.067 0.532
2.22 2.31 0.16 0.662
2.43 2.50 0.18 0.816
2.05 2.04 0.07 0.94
Means in each column with no common superscript differ significantly (P ≤ 0.05).
The data were analyzed based on a completely randomized design using the GLM procedure of SAS (SAS Institute, 2002). Duncan’s multiple range test was used to separate the means when treatment means were significant (P ≤ 0.05). The experimental protocols were reviewed and approved by the Animal Care Committee of the Ferdowsi University of Mashhad.
RESULTS Performance Average feed intake, ABWG, and AFCR of NT- and CT-treated birds are shown in Table 2. There were no significant differences for AFI between 2 treatments at the first 2 wk of age, but at wk 3, 4, and 5, AFI of NT-treated birds was greater (P ≤ 0.05) than that of the CT-treated ones. At wk 6, although AFI in the NT group was greater than that of CT, this difference was not significant. For the whole 6 wk, AFI of NTtreated birds was also greater (P ≤ 0.05) than that of the CT group. Although ABWG for NT-treated birds was greater than that of CT-treated ones, this variable was significant (P ≤ 0.05) only at wk 4. Meanwhile, for the whole 6 wk, ABWG of NT-treated birds was significantly (P ≤ 0.05) greater than that of CT-treated birds. Average feed conversion ratio was greater (P ≤ 0.05) just at wk 3 in NT-treated birds, but in other weeks and the whole period of study, this variable was not significantly different.
Biochemical Parameters The FBS and total protein were the biochemical parameters that were significantly (P ≤ 0.05) influenced by cold-induced ascites, at certain times (Table 3). The FBS of CT treatment at all weeks and whole period of the study except wk 2 was greater than NT ones, but these greater values were only significant (P ≤ 0.05) at wk 4 and 6. Although there was no significant difference for FBS between 2 treatments in the whole period (wk 2 to 6), a strong trend (P = 0.058) was detected. Conversely, total protein of CT-treated birds was numerically lower than NT-treated ones at every week and whole period of the study, and this difference was just significant (P ≤ 0.05) at wk 6. Although lower values of triglyceride and cholesterol for CT-treated birds were nearly observed at all weeks, these differences were not significant (P ≥ 0.05).
RV/TV Ratio and Mortality Percentage In our study, cold significantly increased the RV/TV ratio and mortality percentage of birds (Figure 1). The RV/TV ratio of CT-treated birds was greater (P ≤ 0.05) than those of NT-treated ones. Total mortality percentage due to ascites during the whole period of study was significantly (P ≤ 0.05) greater in CT-treated than of NT-treated birds (8.8 vs. 2.4%).
Figure 1. Right ventricle-to-total ventricle ratio (RV/TV) of chickens at wk 6 of age and cumulative mortality percentage due to ascites through 6 wk as a percentage of birds raised under cold (CT) and normal (NT) environmental temperature. Five birds in each pen were slaughtered for heart part analysis.a,bMeans with no common letter are significantly different (P ≤ 0.05).
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a,b
BIOCHEMICAL PARAMETERS AND ENZYME ACTIVITIES IN BROILERS 1
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2
Table 3. Hematological parameters in chickens raised under normal (NT) and cold (CT) environmental temperature Week Treatments
2
4
5
6
2 to 6
195.80 199.40 0.299 2.291
188.00b 207.00a 0.028 5.020
196.60 204.20 0.091 2.808
189.80b 205.20a 0.046 4.614
195.72 204.12 0.058 2.685
2.96 2.88 0.627 0.112
2.95 2.86 0.454 0.081
3.12 3.06 0.856 0.226
85.80 81.80 0.311 2.615
79.40 77.20 0.520 2.313
77.40 74.80 0.480 2.480
72.00 68.20 0.315 2.504
81.88 80.00 0.768 4.362
122.40 118.20 0.373 3.146
132.00 126.60 0.330 3.685
144.60 139.10 0.056 1.742
114.80 110.80 0.429 3.397
124.20 120.54 0.665 5.762
3.46a 3.30b 0.046 0.0480
3.05 2.93 0.506 0.120
a,b
Means in each column with no common superscript differ significantly (P ≤ 0.05). Serum was used for hematological parameters. 2 Blood samples were obtained from 1 bird per pen. 1
Enzyme Activities In general, enzyme activities were similar for CTand NT-treated birds. There were no significant differences in the activity of LDH, AST, and ALT between CT- and NT-treated birds. Mean LDH, mean ALT, and mean AST according to unit of enzyme activity per liter of serum (U/L) were 1,044.88 vs. 1,067.36, 3.092 vs. 3.092, and 228.16 vs. 232.24, respectively, for NT- and CT-treated birds.
DISCUSSION Hypoxia is thought to be the primary cause in the development of ascites; therefore, conditions that impose greater metabolic demand or decreased oxygen consumption increase incidence of ascites (Buys et al., 1999). Cold temperature, despite increasing demand for oxygen consumption, leads to decreased ventilation and decreased oxygen availability in broiler houses (Buys et al., 1999). Hypoxemia initiates a cascade of events that results in ascites and death (Julian, 1993; Wideman and Bottje, 1993). Before death, significant deterioration in the performance of the broilers occurs (Wideman et al., 1998; Luger et al., 2001). Observed decreased AFI and ABWG reduction for CT-treated birds in recent experiments show these deterioration effects. Increase in mortality and RV/TV ratio (Huchzermeyer and De Ruyck, 1986; Paecock et al., 1988) is often accompanied by ascites; therefore, greater RV/TV (0.29 vs. 0.20) and mortality percentage (8.8 vs. 2.4%) of CT-treated birds than NT-treated birds in our study indicated the ascites development.
Moreover, this hypoxemia leads to some hematological changes such as hematocrit, hemoglobin, red blood cell, and blood gas volume changes, and some other parameters could be affected as well. Also, absence of liver glycogen granules (Maxwell et al., 1986), greater concentration of glucose in liver (Diaz-Cruz et al., 1996), decrease of serum albumin concentration (Yersin et al., 1992), and serum protein decrease (Biswas et al., 1995) in birds with ascites were reported. However, the information about changes of serum FBS, triglyceride, and ALT activity and their relations with ascites have not been reported previously. There is little information regarding serum cholesterol, LDH activity, and AST activity with ascites. There were no significant differences between 2 treatments for serum cholesterol and triglyceride. The cold temperature significantly increased serum FBS of birds in wk 4 and 6 and decreased serum protein in wk 6 of the experiment. It might be concluded that the main portion of increased serum FBS is generated from serum proteins by gluconeogenesis, and possibly a slight portion of this glucose might come from triglyceride sources. Diaz-Cruz et al. (1996) detected a greater concentration of glucose in the liver of broilers with altitude-induced ascites. They attributed it to gluconeogenesis, because this pathway uses substrates other than carbohydrates such as lactate and amino acids to produce glucose. They also observed a greater ammonium and as a result greater protein catabolism of liver in the prepared hepatocytes of broilers with ascites in comparison with healthy birds. They suggested that deaminated amino acids might contribute to the high rate of gluconeogenesis found in hepatocytes of broilers
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Fasting blood sugar (mg/dL) NT 208.40 CT 204.80 P-value 0.5946 ±SEM 4.594 Total protein (g/dL) NT 2.76 CT 2.56 P-value 0.304 ±SEM 0.129 Triglyceride (mg/dL) NT 94.80 CT 97.20 P-value 0.659 ±SEM 3.70 Cholesterol (mg/dL) NT 109.60 CT 108.00 P-value 0.683 2.670 ±SEM
3
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Daneshyar et al.
ACKNOWLEDGMENTS We sincerely thank Mohammad Hassanzadeh (College of Veterinary Medicine, University of Tehran, Iran) for his useful advice. We also express our appreciation to Ferdowsi University of Mashhad, Iran, for the financial support.
REFERENCES Balog, J. M., B. D. Kidd, W. E. Huff, J. R. Huff, N. C. Rath, and N. B. Anthony. 2003. Effect of cold stress on broilers selected for resistance or susceptibility to ascites syndrome. Poult. Sci. 82:1383–1387. Biswas, N. K., M. R. Dalapati, and M. K. Bhowmik. 1995. Ascites syndrome in broiler chickens: Observations on certain biochemical and pathological changes. Indian J. Anim. Sci. 65:1068–1072. Buys, N., C. W. Scheele, C. Kwakernaak, J. D. Van Der Klis, and E. Decuypere. 1999. Performance and physiological variables in broiler chicken lines differing in susceptibility to the ascites syndrome: 1. Changes in blood gases as a function of ambient temperature. Br. Poult. Sci. 40:135–139. Cueva, S., H. Sillau, A. Valenzuela, and H. Ploog. 1974. High altitude induced pulmonary hypertension and right heart failure in broiler chickens. Res. Vet. Sci. 16:370–374. Daneshyar, M., H. Kermanshahi, and A. G. Golian. 2007. Changes of blood gases, internal organ weights and performance of broiler chickens with cold induced ascites. Res. J. Biol. Sci. 2:729–735. Diaz-Cruz, A., C. Nava, R. Villanueva, M. Serret, R. Guinzberg, and E. Pina. 1996. Hepatic and cardiac oxidative stress and other metabolic changes in broilers with the ascites syndrome. Poult. Sci. 75:900–903. Guo, J. L., Q. H. Zheng, Q. Q. Yin, W. Cheng, and Y. B. Jiang. 2007. Study on mechanism of ascites syndrome of broilers. Am. J. Anim. Vet. Sci. 2:62–65. Huchzermeyer, F. W., and A. M. C. DeRuyck. 1986. Pulmonary hypertension syndrome associated with ascites in broilers. Vet. Rec. 119:94.
Igbal, M., D. Cawthon, R. F. Wideman Jr., and W. G. Bottje. 2001. Lung mitochondria dysfunction in pulmonary hypertension syndrome II. Oxidative stress and inability to improve function with repeated additions of adenosine diphosphate. Poult. Sci. 80:656–665. Jaffe, A. S., Y. Landt, C. A. Parvin, D. R. Abendschein, E. M. Geltman, and J. H. Ladenson. 1996. Comparative sensitivity of cardiac troponin I and lactate dehydrogenase isoenzymes for diagnosing ascute myocardial infarction. Clin. Chem. 42:1770–1776. Julian, J. R. 1993. Ascites in poultry. Avian Pathol. 22: 419–454. Khajali, F., and D. Qujeq. 2005. Relationship between growth and serum lactate dehydrogenase activity and the development of ascites in broilers subjected to skip-a-day feed restriction. Int. J. Poult. Sci. 4:317–319. Luger, D., D. Shinder, V. Rzepakovsky, M. Rusal, and S. Yahav. 2001. Association between weight gain, blood parameters, and thyroid hormones and development of ascites syndrome in broiler chickens. Poult. Sci. 80:65–71. Maxwell, M. H., and G. W. Robertson. 1997. World broiler ascites survey. Poult. Int. 36:16–30. Maxwell, M. H., G. W. Robertson, and S. Spence. 1986. Studies on an ascitic syndrome in young broilers. 1. Haematology and pathology. Avian Pathol. 15:511–524. Maxwell, M. H., S. G. Tullet, and F. C. Burton. 1987. Haematology and morphological changes in young broiler chicks with experimentally induced hypoxia. Res. Vet. Sci. 43:331–338. Olkowski, A. A., D. Korver, B. Rathgeber, and H. L. Classen. 1999. Cardiac index, oxygen delivery, and tissue oxygen extraction in slow and fast growing chickens, and in chickens with heart failure and ascites: A comparative study. Avian Pathol. 28:137–146. Paecock, A. J., C. K. Picket, K. M. Morris, and J. T. Reeves. 1988. Spontaneous pulmonary hypertension in rapidly growing chickens reared at sea level. Am. Rev. Respir. Dis. 137:106–110. Pratt, D. S., and M. M. Kaplan. 2000. Evaluation of abnormal liver-enzyme results in asymptomatic patients. N. Engl. J. Med. 4:1266–1271. SAS Institute. 2002. SAS Users Guide: Statistics. SAS Institute Inc., Cary, NC. Silva, J. M. L., N. Dale, and J. B. Luchesi. 1988. Effect of pelleted feed on the incidence of ascites in broilers reared at low altitudes. Avian Dis. 32:376–378. Solis de los Santos, F., G. Tellez, M. B. Farnell, J. M. Balog, and N. B. Anthony. 2005. Hypobaric hypoxia in ascites resistant and susceptible broiler genetic lines influences gut morphology. Poult. Sci. 84:145–148. Tankson, J. D., J. P. Thaxton, and Y. Vizzier-Thaxton. 2002. Biochemical and immunological changes in chickens experiencing pulmonary hypertension syndrome caused by Enterococcus faecalis. Poult. Sci. 81:1826–1831. Wideman, R. F., Jr., and W. G. Bottje. 1993. Current understanding of the ascites syndrome and further research directions. Pages1–20 in Nutrition and Technical Symposium Proceedings. Novus International Inc., St. Louis, Mo. Wideman, R. F. Jr., T. Wing, Y. K. Kirby, M. F. Forman, N. Marson, C. D. Tacket, and C. A. Ruiz-feria. 1998. Evaluation of minimally invasive indices for predicting ascites susceptibility in three successive hatches of broilers exposed to cool temperature. Poult. Sci. 77:1565–1573. Wirz, T., U. Brandle, T. Soldati, J. P. Hossle, and J. C. Periarrd. 1990. A unique chicken B-creatine kinase gives rise to two Bcreatine kinase isoproteins with distinct N-termini by alternative splicing. J. Biol. Chem. 265:11656–11666. Yahav, S., A. Straschnow, I. Plavnik, and S. Hurwitz. 1997. Blood system response of chickens to changes in environmental temperature. Poult. Sci. 76:627–633. Yersin, A. G., W. E. Huff, L. F. Kubena, M. A. Elissalde, R. B. Harvey, D. A. Witzel, and L. E. Giroir. 1992. Changes in hematological, blood gas and serum biochemical variables in broilers during exposure to stimulated high altitude. Avian Dis. 36:189–197. Zantop, D. W. 1997. Biochemistries. Pages115–129 in Avian Medicine: Principles and Applications. B. W. Ritchie, G. J. Harrison, and L. R. Harrison, ed. Wingers Publishing Inc., Lake Worth, FL.
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with ascites. Despite chemical reactions like gluconeogenesis, these changes may be related to exudation of serum protein from extra cellular fluids. Tankson et al. (2002) reported that reduction in both serum protein and cholesterol may well be adaptive physiological responses to the impending loss of extra cellular fluids via ascites. Despite slight differences between 2 treatments for enzyme activities, these differences were not significant. The LDH activity result of our study is consistent with those of Khajali and Qujeq (2005), who did not observe any significant difference for LDH activity between healthy and ascitic broilers. Also, results of AST in our experiment are in agreement with those of Tankson et al. (2002), who did not observe any significant difference in activity of AST enzyme in broilers experiencing pulmonary hypertension syndrome caused by E. faecalis challenge for 48 h or in healthy chickens. The greater RV/TV ratio and mortality percentage of CT-treated birds indicated the ascites development. The greater FBS in the CT-treated birds at wk 4 and 6 of age verifies an increase in gluconeogenesis process in ascitic birds. It can be concluded that the main portion of increased serum FBS was generated from serum proteins through gluconeogenesis and possibly a slight portion was generated from triglyceride sources.
Key words: ascites, biochemical parameter, enzyme activity, broiler 2009 Poultry Science 88:106–110 doi:10.3382/ps.2008-00170 Before a bird exhibits gross ascites syndrome lesions, commonly anatomical and also hematological changes can be detected (Maxwell et al., 1986, 1987). A right ventricle-to-total ventricle ratio (RV/TV) of more than 0.27 is considered an accurate measure of the onset of ascites (Huchzermeyer and De Ruyck, 1986; Paecock et al., 1988). The changes of hematocrit and hemoglobin (Cueva et al., 1974; Maxwell et al., 1986, 1987; Yersin et al., 1992; Yahav et al., 1997; Wideman et al., 1998), red blood cell counts (Cueva et al., 1974; Maxwell et al., 1986, 1987; Yersin et al., 1992), and blood gas volume (Olkowski et al., 1999; Daneshyar et al., 2007) between healthy and ascitic birds were investigated. In addition, some other variables may be affected by hypoxemia and ascites. Tankson et al. (2002) observed different serum protein and cholesterol between healthy and Enterococcus faecalis-challenged chickens to inducing pulmonary hypertension syndrome. Diaz-Cruz et al. (1996) detected a greater concentration of glucose in the liver of broilers with ascites. Yersin et al. (1992) observed a decrease of serum albumin concentration with ascites. Biswas et al. (1995) reported decreased
INTRODUCTION The incidence of ascites in broilers has become of increasing concern to the poultry industry in many areas of the world (Silva et al., 1988). Despite investigations of the syndrome for many years, it is still a condition that inflicts financial loss on poultry producers around the world (Maxwell and Robertson, 1997). It is a severe cause of loss to the broiler industry in many countries, not only due to high rate of mortality, but also due to decreased weight gain and increased condemnations at slaughter (Julian, 1993). This metabolic disorder accounts for over 25% of overall mortality (Guo et al., 2007); considering that an estimated 40 billion broilers are produced annually around the world, it is evident that losses due to ascites are significant (Solis de los Santos et al., 2005). ©2009 Poultry Science Association Inc. Received April 26, 2008. Accepted September 8, 2008. 1 Corresponding author: [email protected]
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ol, triglyceride, activity of lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were determined. Throughout the study, the right ventricle-to-total ventricle ratio and total mortality percentage due to ascites of CT-treated birds at the end of experiment was greater (P ≤ 0.05) than those of NTtreated ones. Fasting blood sugar of CT-treated birds in wk 4 and 6 was greater (P ≤ 0.05) than NT-treated birds. Total blood protein of CT treatment was lower than NT-treated birds in every week and whole period, but this difference was only significant (P ≤ 0.05) in wk 6. There was not a significant difference between 2 treatments for triglyceride and cholesterol, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase. It was concluded that cold-induced ascites could affect serum protein and fasting blood sugar of broiler chickens.
ABSTRACT An experiment with 250 one-day-old male broilers (Ross 308) was conducted to investigate the differences of some blood parameters of cold-induced ascitic and healthy broiler chicks in a 6-wk period. The chickens were divided into 2 groups of 5 replicates each. One group of these chickens was raised in normal temperature (NT) treatment and the other in cold temperature (CT) treatment to induce ascites. Mortality was necropsied daily to determine cause of death. At the end of the experiment (wk 6), 5 chickens from each replicate were randomly selected and slaughtered. The heart was removed; the right ventricle was dissected away from the left ventricle and septum. Weights of right and left ventricles were determined separately. Average BW gain and average feed intake were measured weekly, and weekly average feed conversion ratio was calculated. Serum glucose, total protein, cholester-
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Table 1. Composition of experimental diets Item
Grower (21 to 42 d)
52.12 29.94 8.87 5.00 1.20 1.74 0.50 0.47 0.10 0.06 100.00
59.65 27.84 4.55 4.50 1.32 1.22 0.50 0.34 0.03 0.05 100.00
3,200.00 23.00 1.00 0.45 0.20 1.30 0.90 1.10 0.29
3,200.00 20.00 0.90 0.35 0.15 1.19 0.72 1.00 0.26
1 Supplied per kilogram of diet: vitamin A, 10,000 IU; vitamin D3, 9,790 IU; vitamin E, 121 IU; B12, 20 µg; riboflavin, 4.4 mg; calcium pantothenate, 40 mg; niacin, 22 mg; choline, 840 mg; biotin, 30 µg; thiamine, 4 mg; zinc sulfate, 60 mg; manganese oxide, 60 mg.
serum protein in fast-growing chickens experiencing ascites. Balog et al. (2003) did not detect any difference in select hematological parameters between resistant, susceptible, and relaxed lines to ascites except different serum phosphorus and alkaline phosphatase between resistant and susceptible lines. Even though a lot of information about hematocrit, hemoglobin, and blood gases is available, there is little or no information about changes and relationships of other physiological parameters such as fasting blood sugar (FBS), serum protein, cholesterol, and triglyceride in cold-induced ascitic chicks. Therefore, evaluating these parameters was first aim of this study in coldinduced ascitic broiler chickens. Furthermore, effect of cold-induced ascites on activity of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) enzymes were investigated as a second aim. It has been recognized that LDH isoenzymes are released to blood in heart, liver, and pulmonary system damage (Jaffe et al., 1996; Zantop, 1997); AST in heart damage (Wirz et al., 1990) and liver damage (Pratt and Kaplan, 2000); and ALT in liver damage (Zantop, 1997). It also has been found that the liver, heart, and pulmonary system are affected in ascites; therefore, it would be expected that these plasma enzyme activities changes with ascites.
MATERIALS AND METHODS Two hundred fifty 1-d-old male broiler chickens (Ross 308) were used in this experiment. Half of the birds were housed in 5 pens (1 × 2 m2) of 25 each and were reared under normal temperature (NT group) up to 4
wk of age; then, they were exposed to constant 22 ± 1°C until 6 wk of age (Luger et al., 2001). The other half of the birds were housed in a room with 5 pens (1 × 2 m2) of 25 each in a cold temperature condition (CT group). For inducing ascites, the birds of the CT group were raised under 32°C and 30°C during wk 1 and 2, respectively. The house temperature was decreased to 15°C during wk 3 and maintained between 10 and 15 for the rest of the study (Igbal et al., 2001). Feed and water were provided ad libitum. All chickens were supplied with a mash broiler starter diet until 21 d of age and were fed a mash broiler grower diet thereafter (Table 1). Average BW gain (ABWG) and average feed intake (AFI) were measured weekly, and average feed conversion ratio (AFCR) was calculated and reported weekly. Mortality was recorded daily, and all of the dead birds were examined for lesions of heart failure and ascites throughout the study. On d 14, one chicken from each replicate pen was selected randomly, marked, and wing vein blood samples were obtained weekly at 2200 h every Sunday after a 3-h starvation period. Blood samples were immediately aliquoted into nonanticoagulant tubes. These tubes were allowed to clot for 2 h at 37°C, and then serum was decanted and stored at −20°C for later analyses (Tankson et al., 2002). Serum samples were thawed and serum glucose, total protein, cholesterol, triglyceride, and the LDH, AST, and ALT enzyme activities were determined using an autoanalyzer (Autolab, PM 4000, Autoanalyzaer Medical System, Rome, Italy). At the end of the experiment (wk 6), 5 chickens from each replicate (pens) were randomly slaughtered. The heart was removed, and the right ventricle was dissected from the left ventricle and septum. The right and left ventricles were weighed separately.
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Ingredients (%) Corn Soybean meal (44% protein) Corn gluten meal Soybean oil Limestone Dicalcium phosphate Vitamin and mineral premix1 Salt dl-Methionine l-Lysine Total Calculated analysis ME (kcal/kg) CP (%) Calcium (%) Available phosphorus (%) Sodium (%) Arginine (%) Methionine + cystine (%) Lysine (%) Tryptophan (%)
Starter (0 to 21 d)
108
Daneshyar et al. Table 2. Growth performance of chickens raised under normal (NT) and cold (CT) environmental temperature Week Treatments
1
Average feed intake (g) NT 107.0 CT 108.0 ±SEM 1.83 P-value 0.818 Average BW gain (g) NT 83.0 CT 80.0 ±SEM 1.31 P-value 0.157 Average feed conversion ratio NT 1.30 CT 1.35 ±SEM 0.033 P-value 0.280
2
3
4
5
6
1 to 6
253.0 261.0 2.81 0.071
535.0a 496.0b 8.98 0.016
826.0a 746.0b 10.19 0.0005
986.0a 886.0b 25.34 0.0235
1,305.0 1,185.0 52.41 0.144
3,914.0a 3,585.0b 14.46 0.027
159.0 166.0 3.00 0.127
296.0 308.0 6.00 0.201
374.0a 329.0b 12.79 0.037
452.0 390.0 20.73 0.068
544.0 484.0 34.98 0.259
1,908.0a 1,758.0b 44.24 0.043
1.60 1.58 0.037 0.766
1.81a 1.61b 0.048 0.020
2.22 2.28 0.067 0.532
2.22 2.31 0.16 0.662
2.43 2.50 0.18 0.816
2.05 2.04 0.07 0.94
Means in each column with no common superscript differ significantly (P ≤ 0.05).
The data were analyzed based on a completely randomized design using the GLM procedure of SAS (SAS Institute, 2002). Duncan’s multiple range test was used to separate the means when treatment means were significant (P ≤ 0.05). The experimental protocols were reviewed and approved by the Animal Care Committee of the Ferdowsi University of Mashhad.
RESULTS Performance Average feed intake, ABWG, and AFCR of NT- and CT-treated birds are shown in Table 2. There were no significant differences for AFI between 2 treatments at the first 2 wk of age, but at wk 3, 4, and 5, AFI of NT-treated birds was greater (P ≤ 0.05) than that of the CT-treated ones. At wk 6, although AFI in the NT group was greater than that of CT, this difference was not significant. For the whole 6 wk, AFI of NTtreated birds was also greater (P ≤ 0.05) than that of the CT group. Although ABWG for NT-treated birds was greater than that of CT-treated ones, this variable was significant (P ≤ 0.05) only at wk 4. Meanwhile, for the whole 6 wk, ABWG of NT-treated birds was significantly (P ≤ 0.05) greater than that of CT-treated birds. Average feed conversion ratio was greater (P ≤ 0.05) just at wk 3 in NT-treated birds, but in other weeks and the whole period of study, this variable was not significantly different.
Biochemical Parameters The FBS and total protein were the biochemical parameters that were significantly (P ≤ 0.05) influenced by cold-induced ascites, at certain times (Table 3). The FBS of CT treatment at all weeks and whole period of the study except wk 2 was greater than NT ones, but these greater values were only significant (P ≤ 0.05) at wk 4 and 6. Although there was no significant difference for FBS between 2 treatments in the whole period (wk 2 to 6), a strong trend (P = 0.058) was detected. Conversely, total protein of CT-treated birds was numerically lower than NT-treated ones at every week and whole period of the study, and this difference was just significant (P ≤ 0.05) at wk 6. Although lower values of triglyceride and cholesterol for CT-treated birds were nearly observed at all weeks, these differences were not significant (P ≥ 0.05).
RV/TV Ratio and Mortality Percentage In our study, cold significantly increased the RV/TV ratio and mortality percentage of birds (Figure 1). The RV/TV ratio of CT-treated birds was greater (P ≤ 0.05) than those of NT-treated ones. Total mortality percentage due to ascites during the whole period of study was significantly (P ≤ 0.05) greater in CT-treated than of NT-treated birds (8.8 vs. 2.4%).
Figure 1. Right ventricle-to-total ventricle ratio (RV/TV) of chickens at wk 6 of age and cumulative mortality percentage due to ascites through 6 wk as a percentage of birds raised under cold (CT) and normal (NT) environmental temperature. Five birds in each pen were slaughtered for heart part analysis.a,bMeans with no common letter are significantly different (P ≤ 0.05).
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a,b
BIOCHEMICAL PARAMETERS AND ENZYME ACTIVITIES IN BROILERS 1
109
2
Table 3. Hematological parameters in chickens raised under normal (NT) and cold (CT) environmental temperature Week Treatments
2
4
5
6
2 to 6
195.80 199.40 0.299 2.291
188.00b 207.00a 0.028 5.020
196.60 204.20 0.091 2.808
189.80b 205.20a 0.046 4.614
195.72 204.12 0.058 2.685
2.96 2.88 0.627 0.112
2.95 2.86 0.454 0.081
3.12 3.06 0.856 0.226
85.80 81.80 0.311 2.615
79.40 77.20 0.520 2.313
77.40 74.80 0.480 2.480
72.00 68.20 0.315 2.504
81.88 80.00 0.768 4.362
122.40 118.20 0.373 3.146
132.00 126.60 0.330 3.685
144.60 139.10 0.056 1.742
114.80 110.80 0.429 3.397
124.20 120.54 0.665 5.762
3.46a 3.30b 0.046 0.0480
3.05 2.93 0.506 0.120
a,b
Means in each column with no common superscript differ significantly (P ≤ 0.05). Serum was used for hematological parameters. 2 Blood samples were obtained from 1 bird per pen. 1
Enzyme Activities In general, enzyme activities were similar for CTand NT-treated birds. There were no significant differences in the activity of LDH, AST, and ALT between CT- and NT-treated birds. Mean LDH, mean ALT, and mean AST according to unit of enzyme activity per liter of serum (U/L) were 1,044.88 vs. 1,067.36, 3.092 vs. 3.092, and 228.16 vs. 232.24, respectively, for NT- and CT-treated birds.
DISCUSSION Hypoxia is thought to be the primary cause in the development of ascites; therefore, conditions that impose greater metabolic demand or decreased oxygen consumption increase incidence of ascites (Buys et al., 1999). Cold temperature, despite increasing demand for oxygen consumption, leads to decreased ventilation and decreased oxygen availability in broiler houses (Buys et al., 1999). Hypoxemia initiates a cascade of events that results in ascites and death (Julian, 1993; Wideman and Bottje, 1993). Before death, significant deterioration in the performance of the broilers occurs (Wideman et al., 1998; Luger et al., 2001). Observed decreased AFI and ABWG reduction for CT-treated birds in recent experiments show these deterioration effects. Increase in mortality and RV/TV ratio (Huchzermeyer and De Ruyck, 1986; Paecock et al., 1988) is often accompanied by ascites; therefore, greater RV/TV (0.29 vs. 0.20) and mortality percentage (8.8 vs. 2.4%) of CT-treated birds than NT-treated birds in our study indicated the ascites development.
Moreover, this hypoxemia leads to some hematological changes such as hematocrit, hemoglobin, red blood cell, and blood gas volume changes, and some other parameters could be affected as well. Also, absence of liver glycogen granules (Maxwell et al., 1986), greater concentration of glucose in liver (Diaz-Cruz et al., 1996), decrease of serum albumin concentration (Yersin et al., 1992), and serum protein decrease (Biswas et al., 1995) in birds with ascites were reported. However, the information about changes of serum FBS, triglyceride, and ALT activity and their relations with ascites have not been reported previously. There is little information regarding serum cholesterol, LDH activity, and AST activity with ascites. There were no significant differences between 2 treatments for serum cholesterol and triglyceride. The cold temperature significantly increased serum FBS of birds in wk 4 and 6 and decreased serum protein in wk 6 of the experiment. It might be concluded that the main portion of increased serum FBS is generated from serum proteins by gluconeogenesis, and possibly a slight portion of this glucose might come from triglyceride sources. Diaz-Cruz et al. (1996) detected a greater concentration of glucose in the liver of broilers with altitude-induced ascites. They attributed it to gluconeogenesis, because this pathway uses substrates other than carbohydrates such as lactate and amino acids to produce glucose. They also observed a greater ammonium and as a result greater protein catabolism of liver in the prepared hepatocytes of broilers with ascites in comparison with healthy birds. They suggested that deaminated amino acids might contribute to the high rate of gluconeogenesis found in hepatocytes of broilers
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Fasting blood sugar (mg/dL) NT 208.40 CT 204.80 P-value 0.5946 ±SEM 4.594 Total protein (g/dL) NT 2.76 CT 2.56 P-value 0.304 ±SEM 0.129 Triglyceride (mg/dL) NT 94.80 CT 97.20 P-value 0.659 ±SEM 3.70 Cholesterol (mg/dL) NT 109.60 CT 108.00 P-value 0.683 2.670 ±SEM
3
110
Daneshyar et al.
ACKNOWLEDGMENTS We sincerely thank Mohammad Hassanzadeh (College of Veterinary Medicine, University of Tehran, Iran) for his useful advice. We also express our appreciation to Ferdowsi University of Mashhad, Iran, for the financial support.
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with ascites. Despite chemical reactions like gluconeogenesis, these changes may be related to exudation of serum protein from extra cellular fluids. Tankson et al. (2002) reported that reduction in both serum protein and cholesterol may well be adaptive physiological responses to the impending loss of extra cellular fluids via ascites. Despite slight differences between 2 treatments for enzyme activities, these differences were not significant. The LDH activity result of our study is consistent with those of Khajali and Qujeq (2005), who did not observe any significant difference for LDH activity between healthy and ascitic broilers. Also, results of AST in our experiment are in agreement with those of Tankson et al. (2002), who did not observe any significant difference in activity of AST enzyme in broilers experiencing pulmonary hypertension syndrome caused by E. faecalis challenge for 48 h or in healthy chickens. The greater RV/TV ratio and mortality percentage of CT-treated birds indicated the ascites development. The greater FBS in the CT-treated birds at wk 4 and 6 of age verifies an increase in gluconeogenesis process in ascitic birds. It can be concluded that the main portion of increased serum FBS was generated from serum proteins through gluconeogenesis and possibly a slight portion was generated from triglyceride sources.