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RETRACTED: Comparative biochemical studies on steroidogenic compounds in chickens
Research in Veterinary Science 89 (2010) 168–173
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Comparative biochemical studies on steroidogenic compounds in chickens Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Egypt Ministry of Agriculture, Veterinary Clinic, Sharkia Province, Egypt
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Article history: Accepted 8 March 2010
Keywords: Estrogen Nordette Lutofolone Serum enzymes Chickens
Comparative studies of the effects of Nordette and Lutofolone on 15 days old chicken were carried out to determine their effects on growth performance, biochemical parameters and an analysis of hormonal residues in the liver and muscle. Sixty chickens were equally divided into three groups. Group 1 was served as a control. Groups 2 and 3 were treated daily with Nordette (1 mg/kg B.W.) mixed in the ration and Lutofolone (0.5 mg/kg B.W.) orally via a bent stainless steel feeding tube, respectively, for 30 days (from the 15th till the 45th day old). Then these treated groups were left for another 15 days without any treatment. Blood samples were collected at 45 and 60 days old and used for biochemical studies, while liver and muscles were excised from each chicken and used to prepare tissue homogenate for estimation of hormonal residues (estrogen and progesterone). Both drugs caused a gain in body weight. They also increased several serum variables, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, creatine kinase (CK), creatinine and uric acid, and reduced total proteins, albumin and globulin levels at 30 days post-administration. Moreover, this study exhibited a significant increase in the levels of estrogen residues in the liver and muscle. Estrogen level was much higher in the liver than muscles. However, some of these findings were insignificant changed at 15 days post-stopping of the hormones. Data on the biochemical parameters and residue levels obtained from these results clearly indicate that anabolic agents may entail a special risk to the chickens and probably to the consumer. Ó 2010 Published by Elsevier Ltd.
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1. Introduction
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Mohamed O.T. Badr a, Mohamed A. Hashem a,*, Nissreen N. Gado b
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Chicken meat is considered one of the more desirable sources of animal protein for human consumption (Gary, 2002). This requirement is one of the reasons behinds the development of poultry industry in both private and governmental farms to encounter the increasing necessity for protein production. Yet some poultry producers deliberately ignore some health and safety rules in their process of producing and making the highest profits at the shortest time. There are now several hormones and hormone-like agents with marked ability to improve the rate of growth and efficiency of feed intake for farm animals. The Joint (FAO/WHO) Codex Alimentarius Commission, in particular its Codex Committee for Veterinary Drug Residues in Food, has approved the use of a number of natural and synthetic growth promoters for use in food-producing animals, including the endogenous growth promoters estradiol-17 beta, progesterone and testosterone, and the synthetic growth promoters, trenbolone acetate and zeranol (Sadek et al., 1998). It has been clearly indicated that the administration of hormonal
* Corresponding author. Tel.: +20 552295984, +20 172594311 (mobile); fax: +20 552283683. E-mail address: [email protected] (M.A. Hashem). URL: http://www.staff.zu.edu.eg/elnady_vet (M.A. Hashem). 0034-5288/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2010.03.010
replacement therapy is concomitant functional disturbances of liver and kidney together with alterations in the serum concentration of enzymes (Ganong, 1995; AL-Rawi, 2002). A variety of changes have been noted concerning the effect of estradiol and progesterone on plasma protein, triglycerides, cholesterol and uric acid levels in the serum (Hagan et al., 1984; El-Allawy et al., 1984; Rath et al., 1996). Estrogen administration to chickens tends to decrease metabolism and fattening effects may be attributed in part to these factors (Khan and Zafar, 2005). The purpose of this study was to show the side effects of Nordette and Lutofolone, used as anabolic agents in chickens, on the growth performance (body weight and gain), and the liver and kidney function tests, with studying the levels of these anabolic agents (hormonal residue) in the liver and muscles.
2. Material and methods 2.1. Chickens Sixty-one day old broiler chickens (Hubbard breed) purchased from Al-Noubaria poultry company in Alexandria province, Egypt. Chickens were housed under proper hygienic conditions and maintained on a commercial well balanced ration and water ad libitum.
M.O.T. Badr et al. / Research in Veterinary Science 89 (2010) 168–173
2.2. Drugs Two hormonal compounds, Nordette (0.15 mg levonorgestrol and 0.03 mg ethinyl estradiol) and Lutofolone (2 mg oestradiol benzoate and 20 mg progesterone) were used in this experiment. Nordette was produced by Nile Co. for Pharmaceuticals Cairo A.R.E. under License of American Home Product, New York. Lutofolone was produced by Misr Co. for Pharmaceutical IND. S.A.E. 2.3. Experimental design
2.6. Statistical analysis
2.4. Evaluation of growth performance
Data were analyzed using the one way ANOVA (SPSS 11.0 for Windows) according to Tamhane and Dunlop (2000). Significant differences found between the means were compared using the Duncan’s Multiple Range Test (Duncan, 1955) and accepted at 5% probability level (P < 0.05). 3. Results
3.1. Body performance
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On 15 days of age, the chicks were divided into three groups randomly (20 chickens per group). The 1st group kept as control, 2nd treated daily with Nordette, mixed in the ration, (1 mg/kg B.W.), and 3rd treated daily by the oral route via a bent stainless steel feeding tube with Lutofolone (0.5 mg/kg B.W.) for 30 days (from the 15th till the 45th day old). Then these treated groups were left for another 15 days without any treatment. The dose and route of treatments were in the same manner as used by poultry growers in Egypt.
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a. Body weight: The birds were weighed individually at the 2 weeks of age, to obtain the average initial body weight and then the body weight was recorded every week for calculating the average body weight developments in each group. b. Body weight gain: It was calculated every week (Brody, 1968). 2.5. Samplings
buffer and stored at 80 °C in small aliquots for further analysis. Protein concentrations were determined in each aliquot used within the same day of the experiment according to the BioRad Bradford assay for microtiter plates (650 nm wavelength on a Molecular Devices Emax plate reader; bovine serum albumin standard at 0.635–0.079 mg/ml). The final supernatant was used for the estimation of hormonal residues (Hoffmann, 1983). The tissue residues for estradiol II (E2) and progesterone II (P2) were determined according to Lichtenberg et al. (1992) and Guillaume et al. (1987), respectively. The estimation of hormones was done exactly as indicated in the instructions by using test-kits of Roche/USA and the electrochemi-luminescence immuno-assay ‘‘ECLIA” 2010 analyzer. The sensitivities of estradiol and progesterone kits were 8.0 and 0.15 pg/g with intra-assays coefficients of variations of 5.30% and 3.9%, respectively.
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All chickens were vaccinated against Newcastle disease by hitchner B1 via water at 7 and 21 days of age and against Gumboro disease via drinking water at 15 days of age (Giambrone and Clay, 1986). The animal experiments have been approved by the Committee of Animal Experimental Ethics of the Faculty of Veterinary Medicine, Zagazig University, Egypt.
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2.5.1. Blood sampling On 45 and 60 days of age, 10 birds were randomly selected from each group, weighed and sacrificed by severing the jugular vein and blood allowed to flow freely into labeled tubes without anticoagulant for separation of serum. The serum was kept deep frozen prior to biochemical analysis. Serum total proteins (T.P.) and albumin were determined according to Doumas et al. (1981) and Drupt (1974), respectively, while serum globulins were calculated as the difference between T.P. and albumin. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined colorimetrically according to Reitman and Frankel (1957). Serum cholesterol (Tietz, 1995) and uric acid (Sanders et al. 1980) were estimated using colorimetric method. Serum creatine kinase (CK) (Gerhardt and Waldenstrom, 1979) and creatinine (Slot, 1965) were determined using kinetic method. 2.5.2. Tissue sample The livers and muscles were removed at the end of experimental periods (at 45 and 60 days of age) and perfused with normal saline (0.9% W/V) to reduce red blood cell contamination. Samples were homogenized in dark place in 100 mM potassium phosphate buffer (pH 7.5) containing 0.15 M KCl to obtain 10% homogenate, using a motor fitted homogenizer. The homogenates were centrifuged at 5500 rpm for 10 min. The pellets were discarded and supernatants were filtered through cheesecloth prewetted with
The average body weight was increased significantly in groups (2 and 3) at the 1st, 2nd, 3rd, 4th, 5th and 6th week, but there were no changes in body gain at the 6th week, comparatively with the control (Tables 1 and 2). The body gain in chickens of groups (2 and 3) showed a significant increase at the 1st, 2nd, 3rd, 4th and 5th week, but there were no changes at the 6th week, comparatively with the control. 3.2. Biochemical analysis The serum total proteins, albumin and globulins levels revealing high significance (P < 0.01) decrease but the serum AST, ALT and CK activities, as well as cholesterol, creatinine and uric acid levels were highly significantly (P < 0.01) increased in the Nordette and Lutofolone groups at 30 days post-administration (Table 3). On day 15 post-stopping administration of hormones (at 60 days old), the serum proteinogram, creatinine and uric acid levels did not differ significantly in all groups, while serum enzymes and cholesterol were still increased significantly (Table 4). 3.3. Hormonal residues in the liver and muscles Administration of Nordette and Lutofolone for 30 days resulted in highly significant increase in estrogen residues in the liver and muscle tissue of chickens, while the progesterone level had non significant residues (Table 5). The same results were shown at 15 days post-stopping the drugs, but with lower concentration (Table 6). 4. Discussion The estrogenic and/or progestagenic compounds are used in poultry farm as anabolic agents to improve the body performance and in turn the economic income (Harrison and Harrison, 1986; Ghouniem, 1987). The present study has been decided to illustrate the biochemical investigation of hormones used for long period in poultry farms and their residues in the body tissues, especially the liver and muscles.
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Table 1 Average body weight (gm)/chicken before and after administration of Nordette at a dose of 1 mg/kg B.W. (gp. 2) and Lutofolone at a dose of 0.5 mg/kg B.W. (gp. 3). Groups
1 2 3 F-test L.S.D.
Weeks Before beginning the experiment
After beginning the experiment 1st week
2nd week
3rd week
4th week
5th week
6th week
238 ± 2.5 249 ± 1.87 238 ± 6.04 N.S. –
353c ± 12.21 538a ± 9.57 439b ± 9.92
480c ± 20.06 978a ± 22.06 714b ± 18.67
624c ± 8.71 1360a ± 10.12 893b ± 17.14
793c ± 14.54 1745a ± 10.12 1100b ± 17.15
955c ± 16.66 2007a ± 26.01 1314b ± 31.59
1102c ± 21.66 2172a ± 23.0 1461b ± 29.55
**
**
**
**
**
**
32.75
62.58
38.67
57.21
78.61
76.96
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Means within the same columns having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 2 Average body gain (gm)/chicken after administration of Nordette (gp. 2) and Lutofolone (gp. 3) as compared with the control (gp. 1). Groups
Weeks 1st week
2nd week
3rd week
1 2 3 F-test L.S.D.
115c ± 10.72 289a ± 8.86 202b ± 8.46
127b ± 9.56 289a ± 8.86 275a ± 18.23
143c ± 13.0 382a ± 14.46 179b ± 20.21
**
**
**
28.96
39.88
26.89
4th week
5th week
6th week
169c ± 6.59 389a ± 18.05 207b ± 19.40
162c ± 3.74 258a ± 15.21 214b ± 28.74
147 ± 12.21 165 ± 3.54 147 ± 4.89
**
**
**
28.59
28.23
–
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Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 3 Serum biochemical parameters at 30 days post-administration of Nordette (gp. 2) and Lutofolone (gp. 3) in chicken (45 days old).
1 2 3 F-test L.S.D.
Parameters T.P. (g/dl)
Albumin (g/dl)
Globulin (g/dl)
AST (U/l)
ALT (U/l)
Cholesterol (mg/dl)
CK (U/l)
Creatinine (mg/dl)
Uric acid (mg/dl)
5.48a ± 0.06 2.50b ± 0.05 2.67b ± 0.09
3.13a ± 0.03 1.24c ± 0.03 1.50b ± 0.06
2.33a ± 0.13 1.16b ± 0.03 1.18b ± 0.15
25.33b ± 6.01 95.67a ± 3.38 91.67a ± 3.18
10.00c ± 0.58 51.00a ± 1.86 40.00b ± 1.15
122.60b ± 1.67 143.33a ± 2.40 143.67a ± 0.67
477.00c ± 4.51 822.67a ± 53.15 608.00b ± 26.86
0.45c ± 0.01 0.73a ± 0.01 0.64b ± 0.01
4.53b ± 0.23 8.40a ± 0.21 7.74a ± 0.29
**
**
*
**
**
*
**
**
**
0.21
0.15
0.40
45.17
4.52
5.99
119.30
0.04
0.85
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Groups
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Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference. T.P., Total protein; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CK, Creatine kinase. * Significant at P < 0.05. ** Highly significant at P < 0.01.
Table 4 Serum biochemical parameters at 15 days post-stopping of Nordette (gp. 2) and Lutofolone (gp. 3) in chicken (60 days old). Parameters
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Groups
1 2 3 F-test L.S.D.
T.P. (g/dl)
Albumin (g/dl)
Globulin (g/dl)
AST (U/l)
ALT (U/l)
Cholesterol (mg/dl)
CK (U/l)
Creatinine (mg/dl)
Uric acid (mg/dl)
5.87 ± 0.17 5.70 ± 0.21 5.77 ± 0.17 N.S. –
3.70 ± 0.09 3.67 ± 0.09 3.53 ± 0.15 N.S. –
2.17 ± 0.15 2.13 ± 0.13 2.14 ± 0.33 N.S. –
18.53b ± 0.29 22.33a ± 0.67 24.00a ± 0.29
12.00b ± 1.50 15.00a ± 2.30 15.40a ± 1.80
117.00b ± 5.00 138.00a ± 6.80 141.10a ± 10.33
421.00b ± 53.00 485.00a ± 64.19 488.00a ± 85.53
*
*
*
*
2.6
1.7
15
61
0.45 ± 0.02 0.50 ± 0.30 0.50 ± 0.25 N.S. –
4.53 ± 0.50 4.40 ± 0.30 4.77 ± 0.90 N.S. –
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. T.P., Total protein; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CK, Creatine kinase. * Significant at P < 0.05.
Administration of Nordette and Lutofolone resulted in a significant increase in the body performance (weight and gain) at the 1st till 5th weeks in comparison with the control. Such improvement in the body weight observed along all periods was in a direct relationship with the time. The increase in body weight and gain of
treated chickens may be a reflection of the increased feed intake, increased utilization or due to the summation of anabolic properties and oedema in between muscle fibers, as reported by Ghouniem (1987). Another suggestion for the increase in the body weight is the increase of deposition of body fat as a result of steroi-
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Groups
1 2 3 F-test L.S.D.
Parameters Estradiol (pg/g tissue)
Progesterone (pg/ g tissue)
Liver
Muscle
Liver
Muscle
612.43c ± 6.56 772.73b ± 5.19 1047.20a ± 26.19
77.60c ± 3.40 102.93b ± 2.02 155.63a ± 3.45
**
**
54.95
10.49
1.23 ± 0.14 1.11 ± 0.05 1.20 ± 0.13 N.S. –
0.20 ± 0.001 0.20 ± 0.004 0.21 ± 0.004 N.S. –
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 6 Hormonal residues in the liver and muscle tissues at 15 days post-stopping of hormones in chicken (60 days old).
1 2 3 F-test L.S.D.
Parameters Estradiol (pg/g tissue)
Progesterone (pg/ g tissue)
Liver
Muscle
Liver
Muscle
444.53b ± 4.76 564.67a ± 8.95 555.45a ± 11.59
20.93c ± 0.59 25.38b ± 0.74 29.40a ± 0.46
2.09 ± 0.07 2.04 ± 0.21 2.06 ± 0.06 N.S. –
0.15 ± 0.01 0.14 ± 00.01 0.135 ± 0.03 N.S. –
**
**
30.46
2.09
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Groups
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
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dogenic compounds administration. Our opinion was supported by Ranney and Chaikoff (1951), Lone (1997) and Wagner et al. (2008). They reported that estrogen increases the deposition of abdominal and liver fat, with lesser increase in other organs. Previous reports (Stake et al., 1980; Hermier et al., 1996) indicated that administering exogenous estrogen dramatically increased the body weight, fatty liver and abdominal fat. The authors attributed their results to the hypervolemia due to increased water retention by the kidney, in combination with the friability of the liver due to increased liver lipid. Moreover, Thayer et al. (1945) mentioned that a slight over weight of the estrogenated chickens is the result of increased abdominal fat and not actual growth and it also improves the grade of carcass in broilers, roasters and cocks. Hill et al. (1958) showed that increased gains in live weight in diethylstilbestrol-treated chickens was found to be due primarily to increased fat production, energy consumption and to a lesser extent to preferential synthesis of fat at the expense of protein tissue. Under the influence of estrogen, total tissue gain was increased, and was composed of greater fat gain and lower protein gain. Also, Nesheim (1976) stated that estrogens markedly stimulate food intake and at times, weight gain as well as, there is a marked increase in fattening in chickens fed estrogenic compounds. Concerning the biochemical study in the present work, severe changes in the hepatic and renal function tests were seen. The proteinogram of treated chickens revealed hypoproteinemia, hypoalbuminemia and hypoglobulinemia at 30 days postadministration. Our results may be attributed to disturbance of metabolism by the liver, in addition to the cummulative effect of hormones on the kidneys which leads to proteinuria (albuminuria). The hypoproteinemia and hypoalbuminemia may be due to longstanding albuminuria due to glomerulonephropathy or hepatic damage due to hormones administration (Harrison and Harrison, 1986; Harcourt, 2002). Moreover, Nesheim (1976) stated that there
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seems to be a particular stimulation of lipogenesis of estrogenic compounds to the extent that protein synthesis is depressed. Morgan (1975) recorded a significant decrease in the plasma albumin in two non laying hens injected subcutaneously with 17b-oestradiol dissolved in propylene glycol (20 mg/kg B.W.) at two doses with 3 day intervals. Nearly similar results were obtained by Williams et al. (1978) in roosters chicken given estrogen (0.005 mg/ kg B.W.) daily for 12 days, and Mahmoud et al. (1998) in chickens administered estrogen/progesterone combination for 35 days. They suggested that the decrease in hepatic albumin synthesis after hormone treatment is due to an estrogen-mediated decrease in the content of albumin mRNA. The present study showed a significant increase in the serum AST and ALT activities at 30 days post-administration of Nordette or Lutofolone. The increased activities have been associated with hepatocellular damage (Chandra et al., 1984; Coles, 1986). The elevation of enzymes is in accordance with El-Allawy et al. (1984) who attributed this increase to hepatocellular damage induced by estrogen and progesterone combination. This explanation was ensured by Ganong (1995) who reported an increase in the serum activities of transaminases after hormonal therapy (estrogen and progesterone). The increase in ALT activity is as a result of intrahepatic cholestasis by steroidogenic compounds administered for long periods. This leads to alteration in the cell membrane permeability and allows the escape of the enzyme into serum in abnormal high levels (Benjamin 1961). Evaluation of enzymes provides a clue of liver cell disorders previously reported by Abdel-Kader et al. (1979). On the other hand, the results of several studies have described the appearance of hepatocellular carcinoma in women using oral contraceptive steroids for prolonged periods of time (Henderson et al., 1983). Although stopping the administration of hormones, the enzyme activities were still significantly increased and this indicating the liver damage. The serum total cholesterol level was increased with administration of Nordette or Lutofolone. Such increase may be attributed to the hepatic cholestasis induced by steroidogenic compounds which leads to regurgitation of all cholesterol contents to the blood, resulting in hypercholesterolemia. This result revealed that, the steroidogenic compounds were hepatotoxic as evidenced by Stake et al. (1980) and Ghouniem (1987) in chickens. Also, hyperlipemia (Gilbert, 1962) with increase the serum triglycerides and cholesterol (Hagan et al., 1984; Rath et al., 1996) were produced by estrogen. Hermier et al. (1996) reported a dramatic increase in VLDL concentration and hepatic lipid content in estrogenized chickens. On contrary, Kovanen et al. (1981) reported that the fall in total plasma cholesterol was attributable to a marked increase in the number of LDL receptors in the liver, with consequent rapid clearance of lipoproteins from plasma. Ma et al. (1986) reported that, daily administration of 17b-ethinyl estradiol in a dose of 5 mg/kg subcutaneously to rabbits for 10 days resulted in a significant decrease in the plasma cholesterol level comparatively with the control. The difference may be attributed to the dose, species, treatment-duration and route of sex steroids administration. Although the preparation used in this study may accept as estrogen-dominant, there may be a synergic interaction between the estrogen and progesterone components of combination. For this reason, estrogen/progesterone combination used for long-time caused an increase in total cholesterol levels. This observation coincides with the previous reports of Varley et al. (1980). The increased serum CK in treated chickens may be attributed to muscles damage and other tissue damage which lead to alteration in cell membrane permeability and allows the escape of the enzyme in the serum in abnormal high amount (Benjamin, 1961). The serum CK activity does not be elevated unless the material is highly irritating (Coles, 1986) and this was in line with dangerous effect of estrogens on the muscles. These results were in
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Table 5 Hormonal residues in the liver and muscle tissues at 30 days post-administration of hormones in chicken (45 days old).
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accordance with Harrison and Harrison (1986), Coles (1997) and Mahmoud et al. (1998). Evaluation of the renal function in treated chickens with Nordette or Lutofolone revealed significant changes only in 45 days old but insignificant changes at 60 days old. In the present study the serum creatinine and uric acid levels were significantly increased comparatively with the control, but the increase was higher with Nordette than Lutofolone – treated chickens. Varley et al. (1980) reported that increased serum values of creatinine and uric acid were usually associated with increased estrogen level in the blood. Coles (1997) mentioned that the prominent rise of creatinine and uric acid in serum without its efficient excretion supplies a further confirmation regarding to harmful effects of repeated injections of both estrogens and progesterone hormones on the kidney. All changes in the renal parameters associated with renal damage were most probably due to salt and water retention accompanying steroidogenic compounds administration. This was supported by Ghouniem (1987) and Mahmoud et al. (1998). Hyperuricemia may be either due to decrease the rate of tubular excretion accompanying the renal damage or increase the uric acid production due to catabolism of body tissues (Coles, 1986; Tully et al., 2000). The combined usage of two hormones enhanced the hepatic and renal disturbances as previously reported by El-Allawy et al. (1984) and Zilva et al. (1988). At 15 days post-stopping the administration of drugs, slight improvement in the kidney function was occurred. Regarding the hormonal residues evaluation in chicken’s tissues, the present study revealed a significant increase in the estrogen concentration in the liver and muscles at 30 days postadministration of either Nordette or Lutofolone. The estrogen residues in the liver samples were higher than in muscles. Similar findings were obtained by Sadek et al. (1995). Several workers have suggested that oral contraceptive steroids can act as promoters (Sadek and Abdel-Meguid, 1986). However, insignificant change in progesterone residues in the liver and muscle tissues was shown. This is meaning that the estrogen was the dominant, and having strong activity in the tissues. Similar findings and comments were reported by Sturkie and Textor (1965), Houston et al. (1995) and Ketterson and Nolan (1999). Concerning the determination of estrogen and progesterone in the liver and muscles poststopping of Nordette or Lutofolone for 15 days, the same results were obtained but with lower levels than at 30 days post-administration. This remaining level of estrogen in the tissue is considered as a risk factor that entails a special risk to the animals (Sadek et al., 1995), and probably to the consumer. Since some countries strictly prohibit the application of sex hormones-like activities as anabolic agents and in particular, the use of estrogens (Hoffmann, 1983; Hoffmann and Evers, 1986). Finally it could be concluded that both Nordette and Lutofolone, each containing estrogen and progesterone, improved the body performance, leaving hormonal residues in the tissues, and altered the liver and kidney functions. Therefore, the use of these hormones in chickens as anabolic agents by some poultry growers entails a special risk to chickens, and probably to the consumer. It is recommended that the laws and regulations concerning the use of these anabolic agents, particularly estrogen, in chickens should be updated to prohibit their use.
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Conflict of interest None. Acknowledgements We thank Prof. Dr. Amany A. Abdalla, Clinical Pathology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt, for
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Sadek, I.A., Ismail, H.M., Sallam, H.N., Salem, M., 1998. Survey of hormonal levels in meat and poultry sold in Alexandria, Egypt. Eastern Mediterranean Health Journal 4, 239–243. Sanders, G.T., Pasman, A.J., Hoek, F.J., 1980. Determination of uric acid with uricase and peroxidase. Clinical Chemistry Acta 101, 299–303. Slot, C., 1965. Determination of serum creatinine. Journal Clinical Investigation 17, 381–390. Stake, P., Fredrickson, T., Bourdean, C., 1980. Induction of fatty liver haemorrhagic syndrome in laying hens by exogenous b-estradiol. Avian Disease 25, 410– 422. Sturkie, P.D., Textor, K., 1965. Effect of estrogen administration on growth fat deposition and storage in tissue. Avian Physiology 2, 853–860. Tamhane, A.C., Dunlop, D.D., 2000. Statistic and Data Analysis from Elementary to Intermediate. Upper Saddle River, USA. pp. 85. Thayer, R.H., Jacop, R.G., Penquit, R., 1945. Fattening chicken by feeding estrogens. Poultry Science 24, 483–489. Tietz, N.W., 1995. Clinical Guide to Laboratory Tests, third ed., WB Saunders Co., Philadelphia. pp. 610–611. Tully, T.N., Lawton, M.P.C., Dorrestein, G.M., 2000. Avian Medicine, first ed. Butterworth Heinemann, Oxford. Varley, H., Gowenlock, A.H., Bell, M., 1980. Practical Clinical Biochemistry. William Heinman Medical Books Ltd., London. 15th ed. Wagner, E.C., Prevolsek, J.S., Wynne-Edward, K.E., Williams, T.D., 2008. Hematological changes associated with egg production, estrogen dependence and repeatability. Journal of Experimental Biology 211, 400–408. Williams, D., Wang, S.Y., Klett, H., 1978. Decrease in functional albumin mRNA during estrogen-induced vitellogenin biosynthesis in avian liver. Processing National Academic Science, USA 75, 5974–5978. Zilva, J.F., Pannall, P.R., Mayne, P.D., 1988. Clinical Chemistry in Diagnosis and Treatment, fifth ed. Edward Amold PG, Asian Ed., Singapore.
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Lone, K.P., 1997. Natural sex steroids and their xenobiotic analogs in animal production: growth, carcass quality, pharmacokinetics, metabolism, mode of action, residues, methods, and epidemiology. Crit Revision Food Science Nutrition 37, 93–209. Ma, Y.T., Goldstein, J.L., Brown, M., 1986. Increase mRNA for low density lipoprotein receptor in livers of rabbits treated with 17b-ethinyl estradiol. Medical Science 83, 792–796. Mahmoud, A.Z., Salem, D.A., Ibrahiem, A., Abd EL-Naser, M., Eman, E.G., 1998. Evaluation of prolonged administration of some growth promotors and sex hormones on the effect on tissue residue levels. Assiut Journal of Veterinary Science 2, 95–111. Morgan, E.H., 1975. Plasma iron transport during egg laying and after estrogen administration in the domestic fowl (Gallus domesticus). Quarterly Journal of Experimental Physiology Cognitive Medical Science 60, 233–247. Nesheim, M.C., 1976. Some observations on the effectiveness of anabolic agents in increasing the growth rate of poultry. Environmental Quality Saf Supplement 5, 110–114. Ranney, R.E., Chaikoff, I.L., 1951. Effect of functional hepatectomy upon estrogeninduced lipemia in the fowl. American Journal of Physiology 165, 600–603. Rath, N.C., Huff, W.E., Balog, J.M., Bayyari, G.R., 1996. Effect of gonadal steroids on bone and other physiological parameters of male broiler chickens. Poultry Science 75, 556–562. Reitman, S., Frankel, S., 1957. A colorimetric method for determination of serum glutamic oxalacetic and pyruvic transaminases. American Journal of Clinical Pathology 28, 56–63. Sadek, I.A., Abdel-Meguid, N., 1986. Enhancement of liver tumour in the Egyptian toad by oral contraceptive steroids. Journal of Nutrition Growth and Cancer 3, 239–244. Sadek, I.A., Ismail, H., Sallam, H., Salem, M., 1995. Some aspects of the use of oral contraceptives as growth-promoting agents in poultry. Eastern Mediterranean Health Journal 1, 241–247.
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Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Comparative biochemical studies on steroidogenic compounds in chickens Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Egypt Ministry of Agriculture, Veterinary Clinic, Sharkia Province, Egypt
a r t i c l e
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Article history: Accepted 8 March 2010
Keywords: Estrogen Nordette Lutofolone Serum enzymes Chickens
Comparative studies of the effects of Nordette and Lutofolone on 15 days old chicken were carried out to determine their effects on growth performance, biochemical parameters and an analysis of hormonal residues in the liver and muscle. Sixty chickens were equally divided into three groups. Group 1 was served as a control. Groups 2 and 3 were treated daily with Nordette (1 mg/kg B.W.) mixed in the ration and Lutofolone (0.5 mg/kg B.W.) orally via a bent stainless steel feeding tube, respectively, for 30 days (from the 15th till the 45th day old). Then these treated groups were left for another 15 days without any treatment. Blood samples were collected at 45 and 60 days old and used for biochemical studies, while liver and muscles were excised from each chicken and used to prepare tissue homogenate for estimation of hormonal residues (estrogen and progesterone). Both drugs caused a gain in body weight. They also increased several serum variables, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol, creatine kinase (CK), creatinine and uric acid, and reduced total proteins, albumin and globulin levels at 30 days post-administration. Moreover, this study exhibited a significant increase in the levels of estrogen residues in the liver and muscle. Estrogen level was much higher in the liver than muscles. However, some of these findings were insignificant changed at 15 days post-stopping of the hormones. Data on the biochemical parameters and residue levels obtained from these results clearly indicate that anabolic agents may entail a special risk to the chickens and probably to the consumer. Ó 2010 Published by Elsevier Ltd.
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1. Introduction
a b s t r a c t
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Mohamed O.T. Badr a, Mohamed A. Hashem a,*, Nissreen N. Gado b
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Chicken meat is considered one of the more desirable sources of animal protein for human consumption (Gary, 2002). This requirement is one of the reasons behinds the development of poultry industry in both private and governmental farms to encounter the increasing necessity for protein production. Yet some poultry producers deliberately ignore some health and safety rules in their process of producing and making the highest profits at the shortest time. There are now several hormones and hormone-like agents with marked ability to improve the rate of growth and efficiency of feed intake for farm animals. The Joint (FAO/WHO) Codex Alimentarius Commission, in particular its Codex Committee for Veterinary Drug Residues in Food, has approved the use of a number of natural and synthetic growth promoters for use in food-producing animals, including the endogenous growth promoters estradiol-17 beta, progesterone and testosterone, and the synthetic growth promoters, trenbolone acetate and zeranol (Sadek et al., 1998). It has been clearly indicated that the administration of hormonal
* Corresponding author. Tel.: +20 552295984, +20 172594311 (mobile); fax: +20 552283683. E-mail address: [email protected] (M.A. Hashem). URL: http://www.staff.zu.edu.eg/elnady_vet (M.A. Hashem). 0034-5288/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2010.03.010
replacement therapy is concomitant functional disturbances of liver and kidney together with alterations in the serum concentration of enzymes (Ganong, 1995; AL-Rawi, 2002). A variety of changes have been noted concerning the effect of estradiol and progesterone on plasma protein, triglycerides, cholesterol and uric acid levels in the serum (Hagan et al., 1984; El-Allawy et al., 1984; Rath et al., 1996). Estrogen administration to chickens tends to decrease metabolism and fattening effects may be attributed in part to these factors (Khan and Zafar, 2005). The purpose of this study was to show the side effects of Nordette and Lutofolone, used as anabolic agents in chickens, on the growth performance (body weight and gain), and the liver and kidney function tests, with studying the levels of these anabolic agents (hormonal residue) in the liver and muscles.
2. Material and methods 2.1. Chickens Sixty-one day old broiler chickens (Hubbard breed) purchased from Al-Noubaria poultry company in Alexandria province, Egypt. Chickens were housed under proper hygienic conditions and maintained on a commercial well balanced ration and water ad libitum.
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2.2. Drugs Two hormonal compounds, Nordette (0.15 mg levonorgestrol and 0.03 mg ethinyl estradiol) and Lutofolone (2 mg oestradiol benzoate and 20 mg progesterone) were used in this experiment. Nordette was produced by Nile Co. for Pharmaceuticals Cairo A.R.E. under License of American Home Product, New York. Lutofolone was produced by Misr Co. for Pharmaceutical IND. S.A.E. 2.3. Experimental design
2.6. Statistical analysis
2.4. Evaluation of growth performance
Data were analyzed using the one way ANOVA (SPSS 11.0 for Windows) according to Tamhane and Dunlop (2000). Significant differences found between the means were compared using the Duncan’s Multiple Range Test (Duncan, 1955) and accepted at 5% probability level (P < 0.05). 3. Results
3.1. Body performance
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On 15 days of age, the chicks were divided into three groups randomly (20 chickens per group). The 1st group kept as control, 2nd treated daily with Nordette, mixed in the ration, (1 mg/kg B.W.), and 3rd treated daily by the oral route via a bent stainless steel feeding tube with Lutofolone (0.5 mg/kg B.W.) for 30 days (from the 15th till the 45th day old). Then these treated groups were left for another 15 days without any treatment. The dose and route of treatments were in the same manner as used by poultry growers in Egypt.
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a. Body weight: The birds were weighed individually at the 2 weeks of age, to obtain the average initial body weight and then the body weight was recorded every week for calculating the average body weight developments in each group. b. Body weight gain: It was calculated every week (Brody, 1968). 2.5. Samplings
buffer and stored at 80 °C in small aliquots for further analysis. Protein concentrations were determined in each aliquot used within the same day of the experiment according to the BioRad Bradford assay for microtiter plates (650 nm wavelength on a Molecular Devices Emax plate reader; bovine serum albumin standard at 0.635–0.079 mg/ml). The final supernatant was used for the estimation of hormonal residues (Hoffmann, 1983). The tissue residues for estradiol II (E2) and progesterone II (P2) were determined according to Lichtenberg et al. (1992) and Guillaume et al. (1987), respectively. The estimation of hormones was done exactly as indicated in the instructions by using test-kits of Roche/USA and the electrochemi-luminescence immuno-assay ‘‘ECLIA” 2010 analyzer. The sensitivities of estradiol and progesterone kits were 8.0 and 0.15 pg/g with intra-assays coefficients of variations of 5.30% and 3.9%, respectively.
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All chickens were vaccinated against Newcastle disease by hitchner B1 via water at 7 and 21 days of age and against Gumboro disease via drinking water at 15 days of age (Giambrone and Clay, 1986). The animal experiments have been approved by the Committee of Animal Experimental Ethics of the Faculty of Veterinary Medicine, Zagazig University, Egypt.
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2.5.1. Blood sampling On 45 and 60 days of age, 10 birds were randomly selected from each group, weighed and sacrificed by severing the jugular vein and blood allowed to flow freely into labeled tubes without anticoagulant for separation of serum. The serum was kept deep frozen prior to biochemical analysis. Serum total proteins (T.P.) and albumin were determined according to Doumas et al. (1981) and Drupt (1974), respectively, while serum globulins were calculated as the difference between T.P. and albumin. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were determined colorimetrically according to Reitman and Frankel (1957). Serum cholesterol (Tietz, 1995) and uric acid (Sanders et al. 1980) were estimated using colorimetric method. Serum creatine kinase (CK) (Gerhardt and Waldenstrom, 1979) and creatinine (Slot, 1965) were determined using kinetic method. 2.5.2. Tissue sample The livers and muscles were removed at the end of experimental periods (at 45 and 60 days of age) and perfused with normal saline (0.9% W/V) to reduce red blood cell contamination. Samples were homogenized in dark place in 100 mM potassium phosphate buffer (pH 7.5) containing 0.15 M KCl to obtain 10% homogenate, using a motor fitted homogenizer. The homogenates were centrifuged at 5500 rpm for 10 min. The pellets were discarded and supernatants were filtered through cheesecloth prewetted with
The average body weight was increased significantly in groups (2 and 3) at the 1st, 2nd, 3rd, 4th, 5th and 6th week, but there were no changes in body gain at the 6th week, comparatively with the control (Tables 1 and 2). The body gain in chickens of groups (2 and 3) showed a significant increase at the 1st, 2nd, 3rd, 4th and 5th week, but there were no changes at the 6th week, comparatively with the control. 3.2. Biochemical analysis The serum total proteins, albumin and globulins levels revealing high significance (P < 0.01) decrease but the serum AST, ALT and CK activities, as well as cholesterol, creatinine and uric acid levels were highly significantly (P < 0.01) increased in the Nordette and Lutofolone groups at 30 days post-administration (Table 3). On day 15 post-stopping administration of hormones (at 60 days old), the serum proteinogram, creatinine and uric acid levels did not differ significantly in all groups, while serum enzymes and cholesterol were still increased significantly (Table 4). 3.3. Hormonal residues in the liver and muscles Administration of Nordette and Lutofolone for 30 days resulted in highly significant increase in estrogen residues in the liver and muscle tissue of chickens, while the progesterone level had non significant residues (Table 5). The same results were shown at 15 days post-stopping the drugs, but with lower concentration (Table 6). 4. Discussion The estrogenic and/or progestagenic compounds are used in poultry farm as anabolic agents to improve the body performance and in turn the economic income (Harrison and Harrison, 1986; Ghouniem, 1987). The present study has been decided to illustrate the biochemical investigation of hormones used for long period in poultry farms and their residues in the body tissues, especially the liver and muscles.
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Table 1 Average body weight (gm)/chicken before and after administration of Nordette at a dose of 1 mg/kg B.W. (gp. 2) and Lutofolone at a dose of 0.5 mg/kg B.W. (gp. 3). Groups
1 2 3 F-test L.S.D.
Weeks Before beginning the experiment
After beginning the experiment 1st week
2nd week
3rd week
4th week
5th week
6th week
238 ± 2.5 249 ± 1.87 238 ± 6.04 N.S. –
353c ± 12.21 538a ± 9.57 439b ± 9.92
480c ± 20.06 978a ± 22.06 714b ± 18.67
624c ± 8.71 1360a ± 10.12 893b ± 17.14
793c ± 14.54 1745a ± 10.12 1100b ± 17.15
955c ± 16.66 2007a ± 26.01 1314b ± 31.59
1102c ± 21.66 2172a ± 23.0 1461b ± 29.55
**
**
**
**
**
**
32.75
62.58
38.67
57.21
78.61
76.96
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Means within the same columns having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 2 Average body gain (gm)/chicken after administration of Nordette (gp. 2) and Lutofolone (gp. 3) as compared with the control (gp. 1). Groups
Weeks 1st week
2nd week
3rd week
1 2 3 F-test L.S.D.
115c ± 10.72 289a ± 8.86 202b ± 8.46
127b ± 9.56 289a ± 8.86 275a ± 18.23
143c ± 13.0 382a ± 14.46 179b ± 20.21
**
**
**
28.96
39.88
26.89
4th week
5th week
6th week
169c ± 6.59 389a ± 18.05 207b ± 19.40
162c ± 3.74 258a ± 15.21 214b ± 28.74
147 ± 12.21 165 ± 3.54 147 ± 4.89
**
**
**
28.59
28.23
–
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Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 3 Serum biochemical parameters at 30 days post-administration of Nordette (gp. 2) and Lutofolone (gp. 3) in chicken (45 days old).
1 2 3 F-test L.S.D.
Parameters T.P. (g/dl)
Albumin (g/dl)
Globulin (g/dl)
AST (U/l)
ALT (U/l)
Cholesterol (mg/dl)
CK (U/l)
Creatinine (mg/dl)
Uric acid (mg/dl)
5.48a ± 0.06 2.50b ± 0.05 2.67b ± 0.09
3.13a ± 0.03 1.24c ± 0.03 1.50b ± 0.06
2.33a ± 0.13 1.16b ± 0.03 1.18b ± 0.15
25.33b ± 6.01 95.67a ± 3.38 91.67a ± 3.18
10.00c ± 0.58 51.00a ± 1.86 40.00b ± 1.15
122.60b ± 1.67 143.33a ± 2.40 143.67a ± 0.67
477.00c ± 4.51 822.67a ± 53.15 608.00b ± 26.86
0.45c ± 0.01 0.73a ± 0.01 0.64b ± 0.01
4.53b ± 0.23 8.40a ± 0.21 7.74a ± 0.29
**
**
*
**
**
*
**
**
**
0.21
0.15
0.40
45.17
4.52
5.99
119.30
0.04
0.85
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Groups
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Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference. T.P., Total protein; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CK, Creatine kinase. * Significant at P < 0.05. ** Highly significant at P < 0.01.
Table 4 Serum biochemical parameters at 15 days post-stopping of Nordette (gp. 2) and Lutofolone (gp. 3) in chicken (60 days old). Parameters
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Groups
1 2 3 F-test L.S.D.
T.P. (g/dl)
Albumin (g/dl)
Globulin (g/dl)
AST (U/l)
ALT (U/l)
Cholesterol (mg/dl)
CK (U/l)
Creatinine (mg/dl)
Uric acid (mg/dl)
5.87 ± 0.17 5.70 ± 0.21 5.77 ± 0.17 N.S. –
3.70 ± 0.09 3.67 ± 0.09 3.53 ± 0.15 N.S. –
2.17 ± 0.15 2.13 ± 0.13 2.14 ± 0.33 N.S. –
18.53b ± 0.29 22.33a ± 0.67 24.00a ± 0.29
12.00b ± 1.50 15.00a ± 2.30 15.40a ± 1.80
117.00b ± 5.00 138.00a ± 6.80 141.10a ± 10.33
421.00b ± 53.00 485.00a ± 64.19 488.00a ± 85.53
*
*
*
*
2.6
1.7
15
61
0.45 ± 0.02 0.50 ± 0.30 0.50 ± 0.25 N.S. –
4.53 ± 0.50 4.40 ± 0.30 4.77 ± 0.90 N.S. –
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. T.P., Total protein; AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CK, Creatine kinase. * Significant at P < 0.05.
Administration of Nordette and Lutofolone resulted in a significant increase in the body performance (weight and gain) at the 1st till 5th weeks in comparison with the control. Such improvement in the body weight observed along all periods was in a direct relationship with the time. The increase in body weight and gain of
treated chickens may be a reflection of the increased feed intake, increased utilization or due to the summation of anabolic properties and oedema in between muscle fibers, as reported by Ghouniem (1987). Another suggestion for the increase in the body weight is the increase of deposition of body fat as a result of steroi-
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Groups
1 2 3 F-test L.S.D.
Parameters Estradiol (pg/g tissue)
Progesterone (pg/ g tissue)
Liver
Muscle
Liver
Muscle
612.43c ± 6.56 772.73b ± 5.19 1047.20a ± 26.19
77.60c ± 3.40 102.93b ± 2.02 155.63a ± 3.45
**
**
54.95
10.49
1.23 ± 0.14 1.11 ± 0.05 1.20 ± 0.13 N.S. –
0.20 ± 0.001 0.20 ± 0.004 0.21 ± 0.004 N.S. –
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
Table 6 Hormonal residues in the liver and muscle tissues at 15 days post-stopping of hormones in chicken (60 days old).
1 2 3 F-test L.S.D.
Parameters Estradiol (pg/g tissue)
Progesterone (pg/ g tissue)
Liver
Muscle
Liver
Muscle
444.53b ± 4.76 564.67a ± 8.95 555.45a ± 11.59
20.93c ± 0.59 25.38b ± 0.74 29.40a ± 0.46
2.09 ± 0.07 2.04 ± 0.21 2.06 ± 0.06 N.S. –
0.15 ± 0.01 0.14 ± 00.01 0.135 ± 0.03 N.S. –
**
**
30.46
2.09
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Groups
Means within the same column having different superscript letters are significantly different. L.S.D., Least significant difference; N.S., Non significant difference. ** Highly significant at P < 0.01.
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dogenic compounds administration. Our opinion was supported by Ranney and Chaikoff (1951), Lone (1997) and Wagner et al. (2008). They reported that estrogen increases the deposition of abdominal and liver fat, with lesser increase in other organs. Previous reports (Stake et al., 1980; Hermier et al., 1996) indicated that administering exogenous estrogen dramatically increased the body weight, fatty liver and abdominal fat. The authors attributed their results to the hypervolemia due to increased water retention by the kidney, in combination with the friability of the liver due to increased liver lipid. Moreover, Thayer et al. (1945) mentioned that a slight over weight of the estrogenated chickens is the result of increased abdominal fat and not actual growth and it also improves the grade of carcass in broilers, roasters and cocks. Hill et al. (1958) showed that increased gains in live weight in diethylstilbestrol-treated chickens was found to be due primarily to increased fat production, energy consumption and to a lesser extent to preferential synthesis of fat at the expense of protein tissue. Under the influence of estrogen, total tissue gain was increased, and was composed of greater fat gain and lower protein gain. Also, Nesheim (1976) stated that estrogens markedly stimulate food intake and at times, weight gain as well as, there is a marked increase in fattening in chickens fed estrogenic compounds. Concerning the biochemical study in the present work, severe changes in the hepatic and renal function tests were seen. The proteinogram of treated chickens revealed hypoproteinemia, hypoalbuminemia and hypoglobulinemia at 30 days postadministration. Our results may be attributed to disturbance of metabolism by the liver, in addition to the cummulative effect of hormones on the kidneys which leads to proteinuria (albuminuria). The hypoproteinemia and hypoalbuminemia may be due to longstanding albuminuria due to glomerulonephropathy or hepatic damage due to hormones administration (Harrison and Harrison, 1986; Harcourt, 2002). Moreover, Nesheim (1976) stated that there
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seems to be a particular stimulation of lipogenesis of estrogenic compounds to the extent that protein synthesis is depressed. Morgan (1975) recorded a significant decrease in the plasma albumin in two non laying hens injected subcutaneously with 17b-oestradiol dissolved in propylene glycol (20 mg/kg B.W.) at two doses with 3 day intervals. Nearly similar results were obtained by Williams et al. (1978) in roosters chicken given estrogen (0.005 mg/ kg B.W.) daily for 12 days, and Mahmoud et al. (1998) in chickens administered estrogen/progesterone combination for 35 days. They suggested that the decrease in hepatic albumin synthesis after hormone treatment is due to an estrogen-mediated decrease in the content of albumin mRNA. The present study showed a significant increase in the serum AST and ALT activities at 30 days post-administration of Nordette or Lutofolone. The increased activities have been associated with hepatocellular damage (Chandra et al., 1984; Coles, 1986). The elevation of enzymes is in accordance with El-Allawy et al. (1984) who attributed this increase to hepatocellular damage induced by estrogen and progesterone combination. This explanation was ensured by Ganong (1995) who reported an increase in the serum activities of transaminases after hormonal therapy (estrogen and progesterone). The increase in ALT activity is as a result of intrahepatic cholestasis by steroidogenic compounds administered for long periods. This leads to alteration in the cell membrane permeability and allows the escape of the enzyme into serum in abnormal high levels (Benjamin 1961). Evaluation of enzymes provides a clue of liver cell disorders previously reported by Abdel-Kader et al. (1979). On the other hand, the results of several studies have described the appearance of hepatocellular carcinoma in women using oral contraceptive steroids for prolonged periods of time (Henderson et al., 1983). Although stopping the administration of hormones, the enzyme activities were still significantly increased and this indicating the liver damage. The serum total cholesterol level was increased with administration of Nordette or Lutofolone. Such increase may be attributed to the hepatic cholestasis induced by steroidogenic compounds which leads to regurgitation of all cholesterol contents to the blood, resulting in hypercholesterolemia. This result revealed that, the steroidogenic compounds were hepatotoxic as evidenced by Stake et al. (1980) and Ghouniem (1987) in chickens. Also, hyperlipemia (Gilbert, 1962) with increase the serum triglycerides and cholesterol (Hagan et al., 1984; Rath et al., 1996) were produced by estrogen. Hermier et al. (1996) reported a dramatic increase in VLDL concentration and hepatic lipid content in estrogenized chickens. On contrary, Kovanen et al. (1981) reported that the fall in total plasma cholesterol was attributable to a marked increase in the number of LDL receptors in the liver, with consequent rapid clearance of lipoproteins from plasma. Ma et al. (1986) reported that, daily administration of 17b-ethinyl estradiol in a dose of 5 mg/kg subcutaneously to rabbits for 10 days resulted in a significant decrease in the plasma cholesterol level comparatively with the control. The difference may be attributed to the dose, species, treatment-duration and route of sex steroids administration. Although the preparation used in this study may accept as estrogen-dominant, there may be a synergic interaction between the estrogen and progesterone components of combination. For this reason, estrogen/progesterone combination used for long-time caused an increase in total cholesterol levels. This observation coincides with the previous reports of Varley et al. (1980). The increased serum CK in treated chickens may be attributed to muscles damage and other tissue damage which lead to alteration in cell membrane permeability and allows the escape of the enzyme in the serum in abnormal high amount (Benjamin, 1961). The serum CK activity does not be elevated unless the material is highly irritating (Coles, 1986) and this was in line with dangerous effect of estrogens on the muscles. These results were in
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Table 5 Hormonal residues in the liver and muscle tissues at 30 days post-administration of hormones in chicken (45 days old).
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her valuable help and facilities which made the work calmly achieved. References Abdel-Kader, M.M., Zaky, A.H., Sawsan, A., Abdel-Halim, M.A., Soliman, F.A., 1979. The effect of some long acting steroid contraceptives on some enzymes in goats and rabbits. Egyptian Journal of Veterinary Science 16, 151–158. AL-Rawi, M.M., 2002. Biochemical and histological studies on the effect of hormonal replacement therapy in the old female rats (Rattus norvegicus). Journal of King Saud University 15, 59–69. Benjamin, M.M., 1961. Outline of Veterinary Clinical Pathology, second ed. Iowa University Press, Ames., Iowa, USA. Brody, S., 1968. Bioenergestics and Growth. Hafner Publ. Com., New York. Chandra, M., Singh, B., Soni, G.L., Ahuja, S.P., 1984. Renal and biochemical changes produced in broilers by high protein, high calcium, urea containing and vitamin A deficient diets. Avian Disease 28, 1–11. Coles, E.H., 1986. Veterinary Clinical Pathology, second ed. W.B. Saunders Company, Philadelphia and London. Coles, B.H., 1997. Avian Medicine and Surgery, second ed. Blackwell Science, USA. Doumas, B.T., Bayso, D.D., Carter, R.J., Peters, T., Shaffer, R., 1981. Determination of serum total protein. Clinical Chemistry 27, 1642. Drupt, F., 1974. Colorimetric method for determination of serum albumin. Pharmcological Biology 9, 777. Duncan, D.B., 1955. Multiple range and multiple F-test. Biometrics 11, 1–42. El-Allawy, R.M., Said, M.M., Ibrahim, M.H., Zohni, M.S., Attalla, A.L., 1984. Certain metabolic changes as influenced by five ovulatory steroids. Journal of Drug Research 15, 74–86. Ganong, W.F., 1995. Review of Medical Physiology, 17th ed. Appleton and Hang Nor Walk, Royal Society of Canada. pp. 459–490. Gary, G.P., 2002. The future of animal protein in poultry diets. International Journal of Poultry Science 5, 662–665. Gerhardt, W., Waldenstrom, J., 1979. Creatine kinase B-subunit activity in serum after immunoinhibition of M-subunit activity. Clinical Chemistry 25, 1274– 1280. Ghouniem, M.H., 1987. Histopathological, biochemical and toxicological investigation of contraceptive pills (Anovlar, Nordette) and estrogenic compound folone and their residues in chicken. Ph.D. Thesis, Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University. Giambrone, J.J., Clay, R.P., 1986. Vaccination of day old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines. Avian Disease 30, 557–562. Gilbert, A.B., 1962. Sedimentation rate of erythrocytes in the domesticated cock. Poultry Science 41, 784–788. Guillaume, J., Benjamin, F., Sicuranza, B., Wang, C.F., Garcia, A., Friberg, J., 1987. Maternal serum levels of estradiol, progesterone and human chorionic gonadotropin in ectopic pregnancy and their correlation with endometrial histological findings. Surgical Gynecological Obstetric Research 165, 9–12. Hagan, R.C., Leszczynski, D.E., Kummerow, F.A., 1984. Comparative plasma lipid response of pullets and laying hens to estradiol and progesterone. Toxicological Applied Pharmacology 76, 483–489. Harcourt, B.F., 2002. Text Book of Rabbit Medicine, first ed. Butter worth Heinemann, Oxford, Boston. pp. 89. Harrison, G.J., Harrison, L.R., 1986. Clinical Avian Medicine and Surgery. W.B. Saunders Company, Philadelphia, London, Toronto. pp. 203. Henderson, B.E., Preston, M.S., Edmondson, H.A., Peters, R.L., Pike, M.C., 1983. Hepatocellular carcinoma and oral contraceptives. British Journal of Cancer 48, 437–440. Hermier, D., Catheline, D., Legrand, P., 1996. Relationship between hepatic fatty acid desaturation and lipid secretion in the estrogenized chicken. Comparative Biochemical Physiological A Physiology 115, 259–264. Hill, F.W., Carew, L.B., Van Tienhoven, A., 1958. Effect of diethylstilbestrol on utilization of energy by the growing chick. American Journal of Physiology 195, 654–658. Hoffmann, B., 1983. Use of radio immunoassay procedures for the determination of sex hormones in animal tissues. Journal of Steroid Biochemistry 19, 947–951. Hoffmann, B., Evers, P., 1986. Anabolic agents with sex-hormone like activities: problems of residues. In: Rico, G. (Ed.), Drug Residues in Animal. Veterinary Science and Comparative Medicine. Academic Press, New York, pp. 111–146. Houston, D.C., Donnan, D., Jones, P., Hamilton, I., Osberne, D., 1995. Changes in the muscle condition of female zebra finches Poephila-guttata during egg-laying. Journal of Experimental Biology 211, 400–408. Ketterson, E.D., Nolan, V., 1999. Hormones and life histories – an integrative approach. American Nature 140, 533–562. Khan, T.A., Zafar, F., 2005. Haematological study in response to varying doses of estrogen in broiler chicken. International Journal of Poultry Science 4, 748–751. Kovanen, P.T., Brown, M.S., Basu, S.K., Bilheimer, D.W., Goldstein, J.L., 1981. Evaluation of prolonged administration of some growth promotors and sex hormones on the effect on tissue residue concentration. Processing National Academic Science, USA 78, 1396–1400. Lichtenberg, V., Schulte-Baukloh, A., Lindne, C., Braendle, W., 1992. Discrepancies between results of serum 17b-oestradiol determinations carried out using different immunoassay kits in women receiving estrogen replacement therapy. Laboratory Medicine 16, 412–416.
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accordance with Harrison and Harrison (1986), Coles (1997) and Mahmoud et al. (1998). Evaluation of the renal function in treated chickens with Nordette or Lutofolone revealed significant changes only in 45 days old but insignificant changes at 60 days old. In the present study the serum creatinine and uric acid levels were significantly increased comparatively with the control, but the increase was higher with Nordette than Lutofolone – treated chickens. Varley et al. (1980) reported that increased serum values of creatinine and uric acid were usually associated with increased estrogen level in the blood. Coles (1997) mentioned that the prominent rise of creatinine and uric acid in serum without its efficient excretion supplies a further confirmation regarding to harmful effects of repeated injections of both estrogens and progesterone hormones on the kidney. All changes in the renal parameters associated with renal damage were most probably due to salt and water retention accompanying steroidogenic compounds administration. This was supported by Ghouniem (1987) and Mahmoud et al. (1998). Hyperuricemia may be either due to decrease the rate of tubular excretion accompanying the renal damage or increase the uric acid production due to catabolism of body tissues (Coles, 1986; Tully et al., 2000). The combined usage of two hormones enhanced the hepatic and renal disturbances as previously reported by El-Allawy et al. (1984) and Zilva et al. (1988). At 15 days post-stopping the administration of drugs, slight improvement in the kidney function was occurred. Regarding the hormonal residues evaluation in chicken’s tissues, the present study revealed a significant increase in the estrogen concentration in the liver and muscles at 30 days postadministration of either Nordette or Lutofolone. The estrogen residues in the liver samples were higher than in muscles. Similar findings were obtained by Sadek et al. (1995). Several workers have suggested that oral contraceptive steroids can act as promoters (Sadek and Abdel-Meguid, 1986). However, insignificant change in progesterone residues in the liver and muscle tissues was shown. This is meaning that the estrogen was the dominant, and having strong activity in the tissues. Similar findings and comments were reported by Sturkie and Textor (1965), Houston et al. (1995) and Ketterson and Nolan (1999). Concerning the determination of estrogen and progesterone in the liver and muscles poststopping of Nordette or Lutofolone for 15 days, the same results were obtained but with lower levels than at 30 days post-administration. This remaining level of estrogen in the tissue is considered as a risk factor that entails a special risk to the animals (Sadek et al., 1995), and probably to the consumer. Since some countries strictly prohibit the application of sex hormones-like activities as anabolic agents and in particular, the use of estrogens (Hoffmann, 1983; Hoffmann and Evers, 1986). Finally it could be concluded that both Nordette and Lutofolone, each containing estrogen and progesterone, improved the body performance, leaving hormonal residues in the tissues, and altered the liver and kidney functions. Therefore, the use of these hormones in chickens as anabolic agents by some poultry growers entails a special risk to chickens, and probably to the consumer. It is recommended that the laws and regulations concerning the use of these anabolic agents, particularly estrogen, in chickens should be updated to prohibit their use.
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Conflict of interest None. Acknowledgements We thank Prof. Dr. Amany A. Abdalla, Clinical Pathology Department, Faculty of Veterinary Medicine, Zagazig University, Egypt, for
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