Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
NUTRITION
Effects of high selenium and fat supplementation on growth performance and thyroid hormones concentration of broilers Stella E. Chadio a,∗ , Athanasios C. Pappas b , Anastasios Papanastasatos a , Dionysia Pantelia a , Aikaterini Dardamani b,1 , Konstantinos Fegeros b , George Zervas b a b
Department of Anatomy and Physiology of Domestic Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
a r t i c l e
i n f o
Article history: Received 10 December 2013 Accepted 24 September 2014 Keywords: Broiler Glutathione peroxidase Selenium Thyroid Zinc L-selenomethionine
s u m m a r y A total of 400, as hatched, broilers were used to investigate the effect of increase of selenium and energy intake on thyroid hormone metabolism, growth and liver fatty acid profile. There were 5 replicates of 4 dietary treatments namely, TA (0.289 mg Se per kg diet and adequate energy content), TB (0.583 mg Se per kg diet and adequate energy content), TC (0.267 mg Se per kg diet and 9% increase of energy content) and TD (0.576 mg Se per kg diet and 9% increase of energy content). Diets were isonitrogenous. Zinc Lselenomethionine complex was used to increase Se content and corn oil was used to increase the energy content. The experiment lasted 42 days. Broiler growth performance was not significantly affected by dietary treatments. Liver glutathione peroxidase (GPx) activity increased (P < 0.05) in broilers fed high Se and energy diets compared to other ones. Whole blood GPx activity was higher in Se supplemented groups however, it was reduced by age. Thyroid hormone concentrations were unaffected by dietary treatments. A significant increase of linoleic and arachidonic acid concentration (P < 0.001) was observed in the liver of broilers fed diets with moderately increased energy content and supplemented with Se compared to those fed diets with moderately increased energy content alone. In conclusion, zinc Lselenomethionine complex and moderate increase of energy content did not affect growth rate or thyroid hormone metabolism but led to increased liver fatty acid content and hepatic GPx activity. © 2014 Elsevier GmbH. All rights reserved.
Introduction Selenium (Se) is a nutrient of fundamental importance to mammalian and avian biology. As selenocysteine, Se is a component of selenoproteins with important enzymatic functions. Among them glutathione peroxidases play an antioxidant defence role, preventing lipid-free radical chain reactions that cause peroxidative damage [1]. Se is also required for the expression of the selenoenzymes type I iodothyronine deiodinase (ID-I) and type II iodothyronine deiodinase (ID-II), which are crucial in the generation of the active hormone 3,3 5-tri-iodothyronine (T3 ) [2–4].
∗ Corresponding author at: Department of Anatomy and Physiology of Domestic Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos Str, 11855, Athens, Greece. Tel.: +30 2105294428; fax: +30 2105294388. E-mail address: [email protected] (S.E. Chadio). 1 Present address: Ministry of Rural Development and Food, Directorate of Agricultural Extension, 207 Patision and 19 Skalistiri, 112 53, Athens, Greece. http://dx.doi.org/10.1016/j.jtemb.2014.09.010 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
Se status alterations may affect proper thyroid function. Although the effects of Se deficiency on thyroid hormone metabolism have been studied extensively in a number of animals [5,6] including chicken [7], very little is known about the influence of excess Se on thyroid hormone metabolism and selenoenzyme activities. In humans, Se supplementation led to variable results, depending on the Se status of the examined population group, extending from non to substantial changes in thyroid function [8–12]. Under this context, the form of Se (inorganic or organo-Se compounds) may also affect body Se reserves built and bioavailability [13,14]. According to the National Research Council (NRC) published recommendations [15] the Se concentration of poultry diet should be 0.15 mg Se per kg. However, recent European Union (EU) legislation approves concentration up to 0.5 mg Se per kg [16], whereas commercially used concentration is usually higher than NRC and lower than the EU one [17,18]. High energy intake appeared to be another factor influencing thyroid hormones concentration [19]. In rats, obesity induced by high-fat diets increased ID-I activity and caused normal circulating concentrations of T4 and T3 , but increased reverse T3 levels [20]. Furthermore in humans, without a history of thyroid disease,
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
203
Table 1 The diet formulation, calculated analysis and determined levels of selenium of the experimental diets. Energy content Adequate
Ingredients (g/kg) Maize Soybean meal (45%) Wheat bran Corn oil Limestone Dicalcium phosphate Sodium chloride Methionine Lysine Premixa Calculated analysis (g/kg) ME (MJ/kg) Crude protein Sodium Ca Available P Methionine + Cystine Lysine Determined analysis (g/kg) Treatmentb TA TB TC TD
Increased
Starter diet (0–21 d)
Grower diet (22–42 d)
Starter diet (0–21 d)
Grower diet (22–42 d)
622.6 326.4 7.7 – 11.2 19.2 5.6 2.5 0.8 4.0
716.9 229.0 16.2 – 10.4 14.9 4.5 2.4 1.7 4.0
566.1 341.7 50.0 10.9 19.6 5.6 2.1 – 4.0
666.0 243.0 3.1 50.0 10.1 15.4 4.5 2.5 1.4 4.0
12.00 210.00 2.20 10.00 5.00 8.90 12.00
12.47 175.00 1.80 8.50 4.20 7.80 10.00
13.10 210.00 2.20 10.00 5.00 8.54 11.65
13.61 175.00 1.80 8.50 4.20 7.80 10.00
Se addedc – 300 – 300
Se determined 289 ± 14 583 ± 19 267 ± 29 576 ± 21
a Premix supplied per kg of diet: 12000 IU vitamin A (retinyl acetate), 2000 IU vitamin D3 (cholecalciferol), 440 mg vitamin E (DL-␣-tocopheryl acetate), 4 mg vitamin K3, 3 mg thiamin, 6 mg riboflavin, 4 mg vitamin B6, 0.03 mg vitamin B12, 30 mg nicotinic acid, 12 mg pantothenic acid, 1.5 mg folic acid, 0.08 mg biotin, 200 mg vitamin C, 350 mg choline, 2 mg iodine, 40 mg iron, 100 mg manganese, 15 mg copper, 0.25 mg cobalt, 0.2 mg selenium, 80 mg zinc. b In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet). c Availa-Se 1000, Zinc L-selenomethionine complex, (Zinpro Corporation, USA) was added only in Se-supplemented diets (TB and TD) to supply additionally 0.3 mg Se per kg of diet.
body mass index and waist circumference were positively associated with serum thyroid-stimulating hormone (TSH) and free T3 , but not free T4 [21]. Data on the effects of simultaneous increase of Se and energy intake in avian species are limited. Given the fact that development of modern broiler strains has increased their growth potential and the need for high energy intake [22], a study was designed to investigate whether concomitant increase of Se and energy content in broiler diets affects thyroid hormone levels and selenoenzyme activity. Materials and methods Four hundred (400), as hatched, day-old, Cobb broilers were used in total. The broilers were obtained from a commercial hatchery. All animals were cared for according to applicable recommendations of directive 2010/63/EU of the European Parliament and the Council of the European Union. There were five replicate pens of four dietary treatments namely TA, TB, TC and TD, randomly allocated in the house. Pen was the experimental unit. Each replicate was assigned to a clean concrete floor pen (2 m2 ) and birds were raised on a wheat straw shavings litter. There were 20 broilers per pen, 100 per treatment. In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet). Diets were
isonitrogenous. Zinc L-selenomethionine complex (ZnSeMet) was used to increase Se content (Availa-Se 1000, Zinpro Corporation, Eden Prairie, Minnesota, USA) and corn oil was used to increase the energy content. The duration of the experiment was 42 days with housing and care of broilers, conforming to the guidelines of the Faculty of Animal Science and Aquaculture of the Agricultural University of Athens. The broilers were raised in a house where light and ventilation were controlled. The lighting program was 23 h of light and 1 h of darkness. Heat was provided with a heating lamp per pen. The broilers were fed a starter diet to the 21st day of their life and a grower diet to the 42nd day (Table 1). Feed and water were provided ad libitum. At the end of the 2nd, 4th and 6th week of the study, one broiler per replicate pen was sacrificed with electrical stunning so that liver and whole blood samples were collected for determination of enzymatic activity and thyroid hormones concentration. Whole blood samples were collected in EDTA treated tubes (Aptaca, Canelli, Italy). Furthermore, for the detection of T3 and T4 levels blood was collected in heparin treated tubes (Aptaca, Canelli, Italy) and centrifuged at 1700 × g at 4 ◦ C for 10 min (Hereaus Biofuge stratos, Kendro Laboratory Products, Langenselbold Germany) and the obtained plasma samples were kept at −20 ◦ C until analysis. During the experimental period, body weight and feed intake were recorded weekly and at the end of the experimental period body mass gain, feed consumption and feed to gain ratio (FCR) were calculated. Se was determined in feed using inductively coupled plasma mass spectrometry, ICP-MS (Perkin Elmer, Elan 9000, Perkin Elmer Life and Analytical Sciences Inc., Waltham, MA, USA) as described previously [23] (Table 1).
204
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
Table 2 Dietary treatment and broiler age effects on the concentration of cholesterol, triglycerides, thyroid hormones and activity of GPx and ID-I. Factor examined
Age (wk)
Treatmenta
Source of variation
TA Total T4 (nmol/L)
Total T3 (nmol/L)
Whole blood GPx activity (U/mg Hb)
GPx activity in Liver (U/mg prot)
ID-I activity in Liver (pmol/min/mg prot)
Cholesterol (mg/dl)
Triglyerides (mg/dl)
2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th
50.0 41.8 47.0 3.5 3.8 3.0 429.1ax 396.3ax 318.5ay 2.3 ax 2.0 ax 2.6 ax 157.5 115.2 160.3 135.7 ax 99.8 ay 106.0 ay 94.8 ax 70.4 ay 64.8 ay
TB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.2 3.3 5.2 0.2 0.4 0.1 45.3 26.8 12.9 0.3 0.1 0.3 23.8 10.4 26.2 8.5 6.2 7.6 8.6 6.1 7.4
TC
47.2 40.1 51.2 3.9 3.8 3.2 480.1ax 507.9bx 352.9ay 3.3 ax 2.2 ax 2.6ax 171.5 101.5 134.2 133.5 ax 116.0ax 112.7 ay 100.0 ax 84.8 ax 70.8 ay
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2 6.4 2.3 0.4 0.6 0.4 36.7 41.6 13.7 0.5 0.2 0.3 21.7 12.3 17.3 5.0 10.9 9.3 6.3 9.9 7.1
45.7 39.9 48.3 4.7 3.9 3.5 453.9ax 407.2ax 327.7ay 2.5 ax 2.8ax 2.8 ax 132.2 133.8 116.2 138.9 ax 117.8 ax 129.7bx 100.6 ax 70.4 ay 78.0 ay
TD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.7 3.1 1.7 0.6 0.9 0.2 34.0 29.9 23.5 0.2 0.2 0.1 3.3 16.6 13.7 7.4 6.3 10.1 4.4 6.1 8.2
37.5 47.6 42.9 5.0 3.8 4.0 431.6ax 482.4bx 416.5ax 2.6 ax 3.2 bx 3.6by 130.3 180.2 175.8 145.8 ax 116.2 ay 130.2 bx 99.6 ax 91.7 ax 79.3 ax
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 3.3 2.3 0.8 0.3 0.4 26.5 56.5 68.2 0.3 0.2 0.4 12.3 20.7 40.8 8.4 2.4 8.9 12.1 6.5 16.9
Trt P-value
Age
Age × Trt
NS
NS
NS
NS
NS
NS
0.04
0.014
NS
0.009
NS
0.019
NS
NS
NS
0.043
0.001
NS
NS
0.001
NS
Values are means ± standard error of the mean (SEM) of five replicates. NS: non significant. Within each row, means between columns with different superscripts (a, b, c) are different at P < 0.05 unless otherwise stated (treatment effects). Within each column, means between rows with different superscripts (x, y, z) are different at P < 0.05 unless otherwise stated (age effects). a In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet).
Whole blood and liver cytosolic glutathione peroxidase (GPx) enzyme activity was determined according to Paglia and Valentine [24]. Units of enzyme activity were expressed as per mg hemoglobin (Hb) or per mg of liver protein (prot). Briefly, livers were minced in 0.9% NaCl, washed twice with 0.125 M phosphate buffer, pH 7.4, containing 1.0 mM EDTA (PBS-EDTA) and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY, USA) for 1 min in PBS-EDTA (3 ml/g of liver tissue) at 4 ◦ C. Hemoglobin concentration was determined spectrophotometrically using Drabkin’s reagent (Sigma–Aldrich, MI, USA). Liver protein concentration was determined according to Bradford [25] using commercially available kit (BioRad, CA, USA). ID-I activitiy was determined in tissue homogenates as previously described [26] with 3 M 125 I rT3 as substrate. Plasma T3 and T4 concentrations were determined by radioimmunoassay, using commercially available kits (Biocode, Liege, Belgium). The sensitivity for the T3 assay was 0.1 ng/ml, whereas that for T4 1.8 ng/ml. Intra- and inter assay coefficients of variation were 2.9 and 8.4 for T3 and 3.27 and 4.94 for T4 , respectively. Cholesterol (mg/dl) and triglycerides (mg/dl) were determined using an automated ABX Pentra 400 bench top analyzer (Horiba-ABX, Montpellier, France). The fatty acids concentration in liver was determined using gas chromatography (GC). Extraction and methylation procedure was performed as previously described [27]. Specific GC conditions were described previously [28]. Individual fatty acids were expressed as % of total fatty acids. Statistical analysis The statistical analysis was performed using SAS software (SAS Institute Inc., Cary NC, USA). All variates were analyzed by ANOVA. The fixed factors included treatment, age and their potential interactions. All interactions were analyzed and all pair-wise comparisons were tested. Descriptive statistics, including mean and standard error of the mean (SEM), are presented. Percentage data (data of fatty acids) were subjected to angular transformation prior
to analysis and are presented as mean and post model standard error (SE). The statements of significance presented in this study were based on P ≤ 0.05 unless otherwise stated. Results The mean body weight gain, during the 42 day growing period, of broilers fed the TA, TB, TC and TD diet was 2057.5, 2320.5, 2082.2 and 2350.7 g, respectively (SE = 91.3 g). A tendency for higher weight gain (P = 0.063) was observed for broilers fed the Se supplemented diets compared to those fed diets with adequate Se content. However, mean feed consumption of broilers did not differ statistically between the four experimental treatments (data not shown). Finally, no difference was observed for the feed conversion ratio of broilers being for TA, TB, TC and TD, 1.79, 1.71, 1.82 and 1.73, respectively (SE = 0.17). The activity of GPx differed between the four dietary treatments (Table 2). By the end of 4th week of age, whole blood GPx activity was significantly higher in broilers fed diets with supplemental Se (treatments TB and TD) compared to those fed the unsupplemented diets (treatments TA and TC), while a reduction in enzymatic activity was observed with age. Treatment with both Se and corn oil (TD) resulted in significantly higher liver GPx activity compared to other three treatments (Table 2). Cholesterol levels showed a significant increase in those broilers fed diets supplemented with corn oil, while no effect was observed for triglycerides levels. As the age of broilers increased concentration of cholesterol and triglycerides decreased. Plasma thyroid hormone concentration (total T3 and T4 ) were not affected by dietary treatments (Table 2). A numerically higher activity for liver ID-I was observed in broilers supplemented with both Se and corn oil, but without reaching significance. The major fatty acids present in the liver of broilers included but not limited to oleic (C18:1n-9), palimitic (C16:0), stearic (C18:0) and linoleic (C18:2n-6) acids (Table 3). The liver fatty acid profile of broilers fed diets with increased energy content (treatments TC, TD) was altered
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
205
Table 3 Dietary treatment and broiler age effect on the concentration of fatty acids, as a percentage of total fatty acids, in the liver. Factor studied
Treatmenta TA TB TC TD Age (wk) 2nd 4th 6th SE Source of variation Treatment Age Treatment × Age
C14:0 Myristic acid
C16:0 Palmitic acid
C16:1 n-7 Palmitoleic acid
C18:0 Stearic acid
C18:1 n-9 Oleic acid
C18:2 n-6 Linoleic acid
C18:3 n-3 ␣- Linolenic acid
C20:4 n-6 Arachidonic acid
C20:5 n-3 C22:6 n-3 Eicosapentaenoic Docosahexaenoic acid acid
1.04 1.02 0.88 0.95
25.68 24.98 24.21 25.32
3.55a 3.62a 2.67ab 2.39b
11.86a 12.46a 13.56a 15.63b
46.82a 46.18a 43.84a 37.42b
7.29a 7.54a 9.81b 11.03c
0.10 0.12 0.10 0.11
3.18a 3.56a 4.58a 6.55b
0.16 0.23 0.12 0.10
0.23 0.25 0.15 0.41
0.79a 0.78a 1.10b 0.064
24.15 25.26 25.73 0.735
3.14 2.92 3.12 0.243
12.87 14.51 12.75 0.858
45.84 41.53 43.33 2.033
8.57 9.34 8.84 0.557
4.06 4.85 4.49 0.481
0.12 0.16 0.18 0.066
0.27 0.23 0.29 0.069
NS
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
NUTRITION
Effects of high selenium and fat supplementation on growth performance and thyroid hormones concentration of broilers Stella E. Chadio a,∗ , Athanasios C. Pappas b , Anastasios Papanastasatos a , Dionysia Pantelia a , Aikaterini Dardamani b,1 , Konstantinos Fegeros b , George Zervas b a b
Department of Anatomy and Physiology of Domestic Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece
a r t i c l e
i n f o
Article history: Received 10 December 2013 Accepted 24 September 2014 Keywords: Broiler Glutathione peroxidase Selenium Thyroid Zinc L-selenomethionine
s u m m a r y A total of 400, as hatched, broilers were used to investigate the effect of increase of selenium and energy intake on thyroid hormone metabolism, growth and liver fatty acid profile. There were 5 replicates of 4 dietary treatments namely, TA (0.289 mg Se per kg diet and adequate energy content), TB (0.583 mg Se per kg diet and adequate energy content), TC (0.267 mg Se per kg diet and 9% increase of energy content) and TD (0.576 mg Se per kg diet and 9% increase of energy content). Diets were isonitrogenous. Zinc Lselenomethionine complex was used to increase Se content and corn oil was used to increase the energy content. The experiment lasted 42 days. Broiler growth performance was not significantly affected by dietary treatments. Liver glutathione peroxidase (GPx) activity increased (P < 0.05) in broilers fed high Se and energy diets compared to other ones. Whole blood GPx activity was higher in Se supplemented groups however, it was reduced by age. Thyroid hormone concentrations were unaffected by dietary treatments. A significant increase of linoleic and arachidonic acid concentration (P < 0.001) was observed in the liver of broilers fed diets with moderately increased energy content and supplemented with Se compared to those fed diets with moderately increased energy content alone. In conclusion, zinc Lselenomethionine complex and moderate increase of energy content did not affect growth rate or thyroid hormone metabolism but led to increased liver fatty acid content and hepatic GPx activity. © 2014 Elsevier GmbH. All rights reserved.
Introduction Selenium (Se) is a nutrient of fundamental importance to mammalian and avian biology. As selenocysteine, Se is a component of selenoproteins with important enzymatic functions. Among them glutathione peroxidases play an antioxidant defence role, preventing lipid-free radical chain reactions that cause peroxidative damage [1]. Se is also required for the expression of the selenoenzymes type I iodothyronine deiodinase (ID-I) and type II iodothyronine deiodinase (ID-II), which are crucial in the generation of the active hormone 3,3 5-tri-iodothyronine (T3 ) [2–4].
∗ Corresponding author at: Department of Anatomy and Physiology of Domestic Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos Str, 11855, Athens, Greece. Tel.: +30 2105294428; fax: +30 2105294388. E-mail address: [email protected] (S.E. Chadio). 1 Present address: Ministry of Rural Development and Food, Directorate of Agricultural Extension, 207 Patision and 19 Skalistiri, 112 53, Athens, Greece. http://dx.doi.org/10.1016/j.jtemb.2014.09.010 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
Se status alterations may affect proper thyroid function. Although the effects of Se deficiency on thyroid hormone metabolism have been studied extensively in a number of animals [5,6] including chicken [7], very little is known about the influence of excess Se on thyroid hormone metabolism and selenoenzyme activities. In humans, Se supplementation led to variable results, depending on the Se status of the examined population group, extending from non to substantial changes in thyroid function [8–12]. Under this context, the form of Se (inorganic or organo-Se compounds) may also affect body Se reserves built and bioavailability [13,14]. According to the National Research Council (NRC) published recommendations [15] the Se concentration of poultry diet should be 0.15 mg Se per kg. However, recent European Union (EU) legislation approves concentration up to 0.5 mg Se per kg [16], whereas commercially used concentration is usually higher than NRC and lower than the EU one [17,18]. High energy intake appeared to be another factor influencing thyroid hormones concentration [19]. In rats, obesity induced by high-fat diets increased ID-I activity and caused normal circulating concentrations of T4 and T3 , but increased reverse T3 levels [20]. Furthermore in humans, without a history of thyroid disease,
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
203
Table 1 The diet formulation, calculated analysis and determined levels of selenium of the experimental diets. Energy content Adequate
Ingredients (g/kg) Maize Soybean meal (45%) Wheat bran Corn oil Limestone Dicalcium phosphate Sodium chloride Methionine Lysine Premixa Calculated analysis (g/kg) ME (MJ/kg) Crude protein Sodium Ca Available P Methionine + Cystine Lysine Determined analysis (g/kg) Treatmentb TA TB TC TD
Increased
Starter diet (0–21 d)
Grower diet (22–42 d)
Starter diet (0–21 d)
Grower diet (22–42 d)
622.6 326.4 7.7 – 11.2 19.2 5.6 2.5 0.8 4.0
716.9 229.0 16.2 – 10.4 14.9 4.5 2.4 1.7 4.0
566.1 341.7 50.0 10.9 19.6 5.6 2.1 – 4.0
666.0 243.0 3.1 50.0 10.1 15.4 4.5 2.5 1.4 4.0
12.00 210.00 2.20 10.00 5.00 8.90 12.00
12.47 175.00 1.80 8.50 4.20 7.80 10.00
13.10 210.00 2.20 10.00 5.00 8.54 11.65
13.61 175.00 1.80 8.50 4.20 7.80 10.00
Se addedc – 300 – 300
Se determined 289 ± 14 583 ± 19 267 ± 29 576 ± 21
a Premix supplied per kg of diet: 12000 IU vitamin A (retinyl acetate), 2000 IU vitamin D3 (cholecalciferol), 440 mg vitamin E (DL-␣-tocopheryl acetate), 4 mg vitamin K3, 3 mg thiamin, 6 mg riboflavin, 4 mg vitamin B6, 0.03 mg vitamin B12, 30 mg nicotinic acid, 12 mg pantothenic acid, 1.5 mg folic acid, 0.08 mg biotin, 200 mg vitamin C, 350 mg choline, 2 mg iodine, 40 mg iron, 100 mg manganese, 15 mg copper, 0.25 mg cobalt, 0.2 mg selenium, 80 mg zinc. b In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet). c Availa-Se 1000, Zinc L-selenomethionine complex, (Zinpro Corporation, USA) was added only in Se-supplemented diets (TB and TD) to supply additionally 0.3 mg Se per kg of diet.
body mass index and waist circumference were positively associated with serum thyroid-stimulating hormone (TSH) and free T3 , but not free T4 [21]. Data on the effects of simultaneous increase of Se and energy intake in avian species are limited. Given the fact that development of modern broiler strains has increased their growth potential and the need for high energy intake [22], a study was designed to investigate whether concomitant increase of Se and energy content in broiler diets affects thyroid hormone levels and selenoenzyme activity. Materials and methods Four hundred (400), as hatched, day-old, Cobb broilers were used in total. The broilers were obtained from a commercial hatchery. All animals were cared for according to applicable recommendations of directive 2010/63/EU of the European Parliament and the Council of the European Union. There were five replicate pens of four dietary treatments namely TA, TB, TC and TD, randomly allocated in the house. Pen was the experimental unit. Each replicate was assigned to a clean concrete floor pen (2 m2 ) and birds were raised on a wheat straw shavings litter. There were 20 broilers per pen, 100 per treatment. In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet). Diets were
isonitrogenous. Zinc L-selenomethionine complex (ZnSeMet) was used to increase Se content (Availa-Se 1000, Zinpro Corporation, Eden Prairie, Minnesota, USA) and corn oil was used to increase the energy content. The duration of the experiment was 42 days with housing and care of broilers, conforming to the guidelines of the Faculty of Animal Science and Aquaculture of the Agricultural University of Athens. The broilers were raised in a house where light and ventilation were controlled. The lighting program was 23 h of light and 1 h of darkness. Heat was provided with a heating lamp per pen. The broilers were fed a starter diet to the 21st day of their life and a grower diet to the 42nd day (Table 1). Feed and water were provided ad libitum. At the end of the 2nd, 4th and 6th week of the study, one broiler per replicate pen was sacrificed with electrical stunning so that liver and whole blood samples were collected for determination of enzymatic activity and thyroid hormones concentration. Whole blood samples were collected in EDTA treated tubes (Aptaca, Canelli, Italy). Furthermore, for the detection of T3 and T4 levels blood was collected in heparin treated tubes (Aptaca, Canelli, Italy) and centrifuged at 1700 × g at 4 ◦ C for 10 min (Hereaus Biofuge stratos, Kendro Laboratory Products, Langenselbold Germany) and the obtained plasma samples were kept at −20 ◦ C until analysis. During the experimental period, body weight and feed intake were recorded weekly and at the end of the experimental period body mass gain, feed consumption and feed to gain ratio (FCR) were calculated. Se was determined in feed using inductively coupled plasma mass spectrometry, ICP-MS (Perkin Elmer, Elan 9000, Perkin Elmer Life and Analytical Sciences Inc., Waltham, MA, USA) as described previously [23] (Table 1).
204
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
Table 2 Dietary treatment and broiler age effects on the concentration of cholesterol, triglycerides, thyroid hormones and activity of GPx and ID-I. Factor examined
Age (wk)
Treatmenta
Source of variation
TA Total T4 (nmol/L)
Total T3 (nmol/L)
Whole blood GPx activity (U/mg Hb)
GPx activity in Liver (U/mg prot)
ID-I activity in Liver (pmol/min/mg prot)
Cholesterol (mg/dl)
Triglyerides (mg/dl)
2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th 2nd 4th 6th
50.0 41.8 47.0 3.5 3.8 3.0 429.1ax 396.3ax 318.5ay 2.3 ax 2.0 ax 2.6 ax 157.5 115.2 160.3 135.7 ax 99.8 ay 106.0 ay 94.8 ax 70.4 ay 64.8 ay
TB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.2 3.3 5.2 0.2 0.4 0.1 45.3 26.8 12.9 0.3 0.1 0.3 23.8 10.4 26.2 8.5 6.2 7.6 8.6 6.1 7.4
TC
47.2 40.1 51.2 3.9 3.8 3.2 480.1ax 507.9bx 352.9ay 3.3 ax 2.2 ax 2.6ax 171.5 101.5 134.2 133.5 ax 116.0ax 112.7 ay 100.0 ax 84.8 ax 70.8 ay
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.2 6.4 2.3 0.4 0.6 0.4 36.7 41.6 13.7 0.5 0.2 0.3 21.7 12.3 17.3 5.0 10.9 9.3 6.3 9.9 7.1
45.7 39.9 48.3 4.7 3.9 3.5 453.9ax 407.2ax 327.7ay 2.5 ax 2.8ax 2.8 ax 132.2 133.8 116.2 138.9 ax 117.8 ax 129.7bx 100.6 ax 70.4 ay 78.0 ay
TD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.7 3.1 1.7 0.6 0.9 0.2 34.0 29.9 23.5 0.2 0.2 0.1 3.3 16.6 13.7 7.4 6.3 10.1 4.4 6.1 8.2
37.5 47.6 42.9 5.0 3.8 4.0 431.6ax 482.4bx 416.5ax 2.6 ax 3.2 bx 3.6by 130.3 180.2 175.8 145.8 ax 116.2 ay 130.2 bx 99.6 ax 91.7 ax 79.3 ax
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.0 3.3 2.3 0.8 0.3 0.4 26.5 56.5 68.2 0.3 0.2 0.4 12.3 20.7 40.8 8.4 2.4 8.9 12.1 6.5 16.9
Trt P-value
Age
Age × Trt
NS
NS
NS
NS
NS
NS
0.04
0.014
NS
0.009
NS
0.019
NS
NS
NS
0.043
0.001
NS
NS
0.001
NS
Values are means ± standard error of the mean (SEM) of five replicates. NS: non significant. Within each row, means between columns with different superscripts (a, b, c) are different at P < 0.05 unless otherwise stated (treatment effects). Within each column, means between rows with different superscripts (x, y, z) are different at P < 0.05 unless otherwise stated (age effects). a In TA treatment, broilers were fed a commercial diet with adequate Se and energy content (0.289 mg Se per kg diet), in TB, broilers were fed the TA diet with 0.3 mg added Se per kg of diet (0.583 mg Se per kg diet), in TC, broilers were fed the TA diet with 9% increase of energy content (0.267 mg Se per kg diet) and in TD treatment, broilers were fed the TA diet with 0.3 mg added Se per kg of diet and 9% increase of energy content (0.576 mg Se per kg diet).
Whole blood and liver cytosolic glutathione peroxidase (GPx) enzyme activity was determined according to Paglia and Valentine [24]. Units of enzyme activity were expressed as per mg hemoglobin (Hb) or per mg of liver protein (prot). Briefly, livers were minced in 0.9% NaCl, washed twice with 0.125 M phosphate buffer, pH 7.4, containing 1.0 mM EDTA (PBS-EDTA) and homogenized with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY, USA) for 1 min in PBS-EDTA (3 ml/g of liver tissue) at 4 ◦ C. Hemoglobin concentration was determined spectrophotometrically using Drabkin’s reagent (Sigma–Aldrich, MI, USA). Liver protein concentration was determined according to Bradford [25] using commercially available kit (BioRad, CA, USA). ID-I activitiy was determined in tissue homogenates as previously described [26] with 3 M 125 I rT3 as substrate. Plasma T3 and T4 concentrations were determined by radioimmunoassay, using commercially available kits (Biocode, Liege, Belgium). The sensitivity for the T3 assay was 0.1 ng/ml, whereas that for T4 1.8 ng/ml. Intra- and inter assay coefficients of variation were 2.9 and 8.4 for T3 and 3.27 and 4.94 for T4 , respectively. Cholesterol (mg/dl) and triglycerides (mg/dl) were determined using an automated ABX Pentra 400 bench top analyzer (Horiba-ABX, Montpellier, France). The fatty acids concentration in liver was determined using gas chromatography (GC). Extraction and methylation procedure was performed as previously described [27]. Specific GC conditions were described previously [28]. Individual fatty acids were expressed as % of total fatty acids. Statistical analysis The statistical analysis was performed using SAS software (SAS Institute Inc., Cary NC, USA). All variates were analyzed by ANOVA. The fixed factors included treatment, age and their potential interactions. All interactions were analyzed and all pair-wise comparisons were tested. Descriptive statistics, including mean and standard error of the mean (SEM), are presented. Percentage data (data of fatty acids) were subjected to angular transformation prior
to analysis and are presented as mean and post model standard error (SE). The statements of significance presented in this study were based on P ≤ 0.05 unless otherwise stated. Results The mean body weight gain, during the 42 day growing period, of broilers fed the TA, TB, TC and TD diet was 2057.5, 2320.5, 2082.2 and 2350.7 g, respectively (SE = 91.3 g). A tendency for higher weight gain (P = 0.063) was observed for broilers fed the Se supplemented diets compared to those fed diets with adequate Se content. However, mean feed consumption of broilers did not differ statistically between the four experimental treatments (data not shown). Finally, no difference was observed for the feed conversion ratio of broilers being for TA, TB, TC and TD, 1.79, 1.71, 1.82 and 1.73, respectively (SE = 0.17). The activity of GPx differed between the four dietary treatments (Table 2). By the end of 4th week of age, whole blood GPx activity was significantly higher in broilers fed diets with supplemental Se (treatments TB and TD) compared to those fed the unsupplemented diets (treatments TA and TC), while a reduction in enzymatic activity was observed with age. Treatment with both Se and corn oil (TD) resulted in significantly higher liver GPx activity compared to other three treatments (Table 2). Cholesterol levels showed a significant increase in those broilers fed diets supplemented with corn oil, while no effect was observed for triglycerides levels. As the age of broilers increased concentration of cholesterol and triglycerides decreased. Plasma thyroid hormone concentration (total T3 and T4 ) were not affected by dietary treatments (Table 2). A numerically higher activity for liver ID-I was observed in broilers supplemented with both Se and corn oil, but without reaching significance. The major fatty acids present in the liver of broilers included but not limited to oleic (C18:1n-9), palimitic (C16:0), stearic (C18:0) and linoleic (C18:2n-6) acids (Table 3). The liver fatty acid profile of broilers fed diets with increased energy content (treatments TC, TD) was altered
S.E. Chadio et al. / Journal of Trace Elements in Medicine and Biology 29 (2015) 202–207
205
Table 3 Dietary treatment and broiler age effect on the concentration of fatty acids, as a percentage of total fatty acids, in the liver. Factor studied
Treatmenta TA TB TC TD Age (wk) 2nd 4th 6th SE Source of variation Treatment Age Treatment × Age
C14:0 Myristic acid
C16:0 Palmitic acid
C16:1 n-7 Palmitoleic acid
C18:0 Stearic acid
C18:1 n-9 Oleic acid
C18:2 n-6 Linoleic acid
C18:3 n-3 ␣- Linolenic acid
C20:4 n-6 Arachidonic acid
C20:5 n-3 C22:6 n-3 Eicosapentaenoic Docosahexaenoic acid acid
1.04 1.02 0.88 0.95
25.68 24.98 24.21 25.32
3.55a 3.62a 2.67ab 2.39b
11.86a 12.46a 13.56a 15.63b
46.82a 46.18a 43.84a 37.42b
7.29a 7.54a 9.81b 11.03c
0.10 0.12 0.10 0.11
3.18a 3.56a 4.58a 6.55b
0.16 0.23 0.12 0.10
0.23 0.25 0.15 0.41
0.79a 0.78a 1.10b 0.064
24.15 25.26 25.73 0.735
3.14 2.92 3.12 0.243
12.87 14.51 12.75 0.858
45.84 41.53 43.33 2.033
8.57 9.34 8.84 0.557
4.06 4.85 4.49 0.481
0.12 0.16 0.18 0.066
0.27 0.23 0.29 0.069
NS
