Effects of the supplemental chromium form on

Effects of the supplemental chromium form on performance and oxidative stress in broilers exposed to heat stress N. Sahin,∗ A. Hayirli,‡ C. Orhan,∗ M. Tuzcu,# F. Akdemir,† J. R. Komorowski,§ and K. Sahin∗,1 ∗

Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, University, 23119 Elazig, Turkey; † Department of Nutrition, Faculty of Fisheries, Inonu University, 44180 Malatya, Turkey; ‡ Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Atat¨ urk University, 25240 Erzurum, Turkey; § Scientific and Regulatory Affairs, Nutrition 21 Inc, 1 Manhattanville Road, Purchase, NY 10577, USA; and # Division of Biology, Faculty of Science, Firat University, 23119 Elazig, Turkey ABSTRACT This experiment was conducted to investigate effects of the organic complex form of supplemental chromium (Cr) on performance, oxidative stress markers, and serum profile in broilers exposed to heat stress (HS). A total of 1,200 10-day-old boilers (Ross308) was divided into one of the 6 treatments (2 environmental temperatures x 3 diets with different Cr forms). The birds were kept in temperature-controlled rooms at either 22 ± 2◦ C 24 h/d (thermoneutral, TN group) or 34 ± 2◦ C for 8 h/d, 08:00 to 17:00 h, followed by 22◦ C for 16 h (HS group) and fed either a basal diet (C) or the basal diet supplemented with Cr (200 μg/kg) through 1.600 mg of CrPic (12.43% Cr) and 0.788 mg of CrHis (25.22% Cr). Feed intake and body weight were recorded weekly. After cervical dislocation, liver samples were harvested to analyze Cr concentration and glucose transporter-2,4 (GLUT-2,4) expression. The breast meat also was sampled for the concentration of Cr and expressions of nuclear factor

erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB). Data were analyzed by 2-way ANOVA. Heat stress caused depressions in feed intake (12.1%) and weight gain (21.1%) as well as elevations in feed conversion (11.2%) and abdominal fat (32.8%). It was also associated with depletion of Cr reserves and increases in serum concentrations of glucose, cholesterol, creatine, and enzymes. Exposure to HS was accompanied by suppression of the expressions of Nrf2 and GLUT-2 in muscle and GLUT-4 in the liver and amplification of the expression of NF-κB in muscle. Both Cr sources partially alleviated detrimental effects of HS on performance and metabolic profile. The efficacy of Cr as CrHis was more notable than Cr as CrPic, which could be attributed to higher bioavailability. In conclusion, CrHis can be added into the diet of broilers during hot seasons to overcome deteriorations in performance and wellbeing related to oxidative stress.

Key words: broiler, chromium-histidinate, chromium-picolinate, heat stress 2017 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pex249 (NF-κB), nuclear factor erythroid 2-related factor 2 (Nrf2), tumor necrosis factor-alpha (TNF-α), and heat shock proteins] as well as serum concentrations of minerals (e.g., Se, Zn, Cu, Mn, and Cr) and vitamins (e.g., vitamins A and E), which are integral components of the antioxidant defense system (Sahin and Kucuk, 2003; Sahin et al., 2009). In response to exposure to HS, increased excretion and reduced bioavailability of nutrients also may contribute to suppressed antioxidant status in broilers (Smith, 1994; Belay and Teeter, 1996). NF-κB is one of the proteins responsible for controlling the DNA transcription and is present in the cytoplasm inactively under normal condition. When cellular stress occurs, it is immediately expressed and enters the nucleus to regulate expressions of some specific genes (Latchman, 1997; Nelson et al., 2004). For example, oxidative stress resulting from exposure to HS negatively affects the membrane structure and function, cell

INTRODUCTION The negative effects of high ambient temperature on survival, performance, and product quality are well described in poultry and continue to be economically detrimental on many farms. Heat stress (HS) causes depression in feed intake, weight gain, carcass weight, and nutrient digestibility and deterioration in feed efficiency and carcass quality (Howlider and Rose, 1987; Sahin and Kucuk, 2003). Moreover, HS was shown to adversely affect immune potency (Ghazi et al., 2012) and expression of transcription factors [e.g., nuclear factor kappa-light-chain-enhancer of activated B cells C 2017 Poultry Science Association Inc. Received May 10, 2017. Accepted August 10, 2017. 1 Corresponding author: [email protected]

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physiology, and DNA transcription (Iwagami, 1996). Nrf2 is another transcription factor that regulates the cellular antioxidant response. It is also located in the cytoplasm. In response to oxidative stress occurring, Nrf2 is relocated into the cell nuclei (Itoh et al., 2004). Several dietary manipulations are available to overcome the detrimental effects of HS in poultry. Chromium (Cr) is considered a preferential mineral in poultry diets due to its strong antioxidant activities to prevent lipid peroxidation caused by HS (Samanta et al., 2008; Sahin et al., 2009; Rao et al., 2012). It has an important role in potentiating insulin action and enhancing the metabolism of nutrients (e.g., carbohydrates, lipids, proteins, and nucleic acid) via activation of enzymes involved in related pathways (Anderson, 1987; Hayirli, 2005). The Cr form is important for efficacy and final biological response. It is well known that Cr chelates with organic compounds have lower toxicity and higher bioavailability than inorganic Cr forms (Kim et al., 1996; Piva et al., 2003). A number of studies dealing with the effects of different Cr chelates [yeast, picolinate (CrPic), nicotinate, propionate, and methionine] on broilers are available. Chromium-histidinate (CrHis) is a newly developed chelate. This experiment was conducted to compare the effects of different Cr-chelates (CrHis vs. CrPic) on performance, oxidative stress markers, serum profile, muscular transcription factors, and glucose transporters in heat-stressed broilers.

MATERIALS AND METHODS Animals, Treatments, and Management The experiment involving 10-day-old mixed-sex chicks (Ross-308, n = 1200) was conducted at Veterinary Control and Research Institute (Elazig, Turkey). The birds were kept in temperature-controlled rooms at either 22 ± 2◦ C 24 h/d (thermoneutral, TN) or 34 ± 2◦ C for 8 h/d, 08:00 to 17:00 h, followed by 22◦ C for 16 h (HS) during the experimental period. The birds were fed on a starter diet from d 10 to d 21 and a grower diet from d 22 to d 42 (NRC, 1994; Table 1). Pens (n = 60) were constructed to contain 20 chicks. Diets for broilers in both the TN and HS groups (n = 30 pen for each) were either not supplemented with Cr (C) or supplemented with Cr (200 μg/kg) through 1.600 mg of CrPic (12.43% elemental Cr, Nutrition 21, Purchase, NY) or 0.788 mg of CrHis (25.22% elemental Cr, Nutrition 21, Purchase, NY). In order to avoid confounding effects of organic matter of the Cr chelates, per kilogram of the diets C, CrPic, and CrHis were added with 1.401 mg picolinic acid plus 0.589 mg histidine, 0.589 mg histidine, and 1.401 mg picolinic acid, respectively. Each treatment was replicated 10 times. Feed and fresh water were offered ad libitum throughout the experimental period. Birds were exposed to an illumi-

Table 1. Ingredients and chemical composition of the basal diet fed to broilers. Ingredient, % Corn Soybean meal 44% CP Corn oil Limestone Dicalcium phosphate Sodium chloride DL-methionine Vitamin-mineral premix1 Nutrient analyses Metabolizable energy, kcal/kg2 Crude protein, % Crude fiber, % Ether extract, % Lysine, % Methionine+cystine, % Ca, % P, %

Starter phase (d 1 to 21)

Grower phase (d 22 to 42)

51.00 36.60 6.00 1.70 1.60 0.40 0.20 2.50

56.90 32.20 5.00 1.30 1.60 0.40 0.10 2.50

3100 22.70 3.85 6.73 1.18 0.90 1.00 0.71

3100 20.60 3.87 6.15 1.00 0.63 0.91 0.66

1 Vitamin-mineral premix provides the following per kilogram: Vitamin A (all-trans-retinyl acetate), 4800 IU; vitamin D3, 1000 IU; Vitamin E (all-rac-α -tocopherol acetate), 16 mg; menadione (menadione sodium bisulphate, 2 mg; riboflavin, 1.4 mg; thiamine (thiamine mononitrate), 1 mg; pyridoxine, 2 mg; niacin, 10 mg; Ca-pantothenate, 6 mg; vitamin B12, 0.01 mg; folic acid, 0.6 mg; d-biotin, 0.02 mg; Mn (MnO), 32 mg; Fe (FeSO4 ), 24 mg; Zn (ZnO), 24 mg; Cu (CuSO4 ), 2 mg; I (KI), 0.4 mg; Co (CoSO4 ), 0.08 mg; Se (NaSe), 0.06 mg. Moreover, the premix was reconstituted at the expense of CaCO3 to contain histidinate (0.236 g) plus picolinate (0.560 g), CrPic (0.640 g) plus histidinate (0.236 g), and CrHis (0.315 g) plus picolinate (0.560 g) in the diets C, CrPic, and CrHis, respectively. 2 Metabolizable energy, lysine, methionine, and cysteine contents were calculated based on their tabular values listed for the feed ingredients (Jurgens, 1996).

nation program providing a light:dark cycle of 23 h:1 h per day.

Sample and Data Collection Feed samples were collected in each growth phase for nutrient analyses. Feed intake and body weights were recorded weekly for all pens. Weight gain and feed conversion ratio (FCR, feed consumed, g:weight gained, g) were then calculated by the pen. At the end of the study (d 42), blood samples were collected from 2 birds randomly chosen from each pen (n = 20 per treatment group). After birds were killed by cervical dislocation, blood samples were put into additive-free vacutainers. Abdominal fat also was removed and weighed. Blood samples were centrifuged at 5,000 × g at 4◦ C and for 10 min, and aliquots were transferred to microfuge tubes. Sera were kept on ice and protected from light to avoid oxidation during sampling and then stored at −75◦ C for determination of Cr, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ -glutamyl transferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), glucose, cholesterol, protein, albumin, globulin, and creatine concentrations.

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SUPPLEMENTAL CHROMIUM FORM AND HEAT-DISTRESSED BROILER

Breast muscle and liver samples were removed and stored at −75◦ C for determination of Cr concentration as well as GLUT-4, Nrf2 and NF-κB expressions in muscle and Cr concentration and GLUT-2 expression in the liver.

Laboratory Analyses Feed samples were analyzed for crude protein (#988.05), ether extract (#932.06), crude fiber (#962.09), crude ash (#936.07), Ca (#968.08), and P (#965.17) in triplicates (AOAC, 1990). For determination of Cr concentration, 0.290 to 0.305 g feed, muscle, and liver samples, as well as 0.5 mL serum samples were first digested with 5 mL concentrated HNO3 in a Microwave Digestion System (Berghoff, Eningen, Germany) for 30 minutes. The specimens were subjected to a graphite furnace atomic absorption spectrophotometer (AAS, PerkinElmer, Analyst 800, Norwalk, CT). Serum AST, ALT, GGT, LDH, CK, glucose, cholesterol, protein, albumin, globulin, and creatine concentrations were measured using Biochemistry Test 9 kits (Samsung LABGEO, IVR-PT05, Seoul, Korea) on auto-analyzer (Samsung LABGEOPT10 ). To determine expressions of hepatic and muscular proteins in Western blot analysis, samples were homogenized in phosphate buffered saline (PBS) with a protease inhibitor cocktail (Calbiochem, San Diego, CA). The sample (20 μg of protein per lane) was mixed with sample buffer, boiled for 5 min, and separated by sodium dodecyl sulphate-polyacrylamide (12%) gel electrophoresis under denaturing conditions, and then electroblotted onto nitrocellulose membrane (Schleicher and Schuell Inc., Keene, NH). Nitrocellulose blots were washed in PBS and blocked with 1% bovine serum albumin in PBS for 1 h prior to application of the primary antibodies (Nrf2, NF-κB, GLUT-2, GLUT-4; Abcam, Cambridge, UK). Primary antibody was previously diluted (1:1000) in the same buffer containing 0.05% Tween-20. The nitrocellulose membrane was incubated overnight at 4◦ C with protein antibody. The immunoreaction was continued with the secondary goat antirabbit horseradish-peroxidase-conjugated antibody after washing for 2 h at room temperature the next day. Specific binding was detected using diaminobenzidine and H2 O2 as substrates. Protein load was controlled using a monoclonal mouse antibody against β actin antibody (A5316; Sigma, St. Louis, MO). Protein levels were quantified densitometrically (Figure 1, upper panel) using an image analysis system (Image J; National Institute of Health, Bethesda, MD).

Statistical Analyses In a 2 × 3 factorially arranged treatment within a completely randomized design experiment, data were analyzed by 2-way ANOVA using the PROC GLM

3

procedure (SAS, 2002). The linear model to test the effect of treatments on response variables was as follows: Yijk = μ + ETi + DSj + (ETxDS)ij + eijk , where Y = response variable, μ = population mean, ET = environmental temperature, DS = dietary supplement, and e = residual error [N (σ , μ; 0, 1)]. Statistical contrasts were constructed in the model to attain the effects of supplemental Cr (C vs. Ave = CrPic and CrHis) and Cr source (CrPic vs. CrHis), and statistical significance was considered at P ≤ 0.05.

RESULTS Performance Variables The adverse effect of HS on performance variables was evident (Table 2), as reflected by decreased feed intake (−12.1%) and weight gain (−21.1%) and worsened FCR (+11.2%) and increased abdominal fat deposition (+32.8%) (P < 0.0001 for all). Performance variables for broilers fed the diets enriched with Cr were superior to those for broilers fed the C diet. Feed intake and weight gain were greatest, and FCR and abdominal fat were the least in broilers fed the CrHis diet, followed by the CrPic diet. Positive effects of supplemental Cr on performance variables were more notable under the HS condition than under the TN condition, being superior for CrHis to CrPic. Relative increases in feed intake for broilers fed the CrPic and CrHis diets as compared to those fed the C diet were 0.7 and 1.0% under the TN condition and 2.3 and 5.4% under the HS condition, respectively (P < 0.02). Relative increases in gain (P < 0.001) were 0.7 and 2.6% under the TN condition and 7.0 and 12.9% under the HS condition in broilers fed the CrPic and CrHis diets as compared to those fed the C diet, respectively. Feeding the CrPic and CrHis diets, as compared to the C diet, resulted in greater improvement in FCR (P < 0.04) under the HS condition (−4.3 and −6.8%) than under the TN condition (−0.01 and −1.1%). Under both conditions, diets enriched with Cr decreased abdominal fat at a similar level (P < 0.34).

Tissue Chromium Levels Broilers exposed to the HS condition had remarkably lower serum (−38.7%), breast muscle (−35.3%), and liver (−30.5%) Cr concentrations than those reared under the TN condition (P < 0.0001 for all; Table 3). Supplemental Cr increased serum, breast muscle, and liver Cr concentrations as compared to the C group (P < 0.0001 for all). Although the increase in serum Cr concentration for the CrHis group was higher than that for the CrPic group (P < 0.05), increases in breast muscle and liver Cr concentrations did not differ by the Cr source. The supplemental Cr effect on serum and breast muscle Cr concentrations did not vary by the environment.


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NF-κB NF-

GLUT4

GLUT2

β-actin

β-actin -actin Control

CrPic

0

CrHis

Control

CrPic

CrHis

Control

HS

TN TN

CrPic

0

CrHis

Control

TN

HS

(B)

150

Control CrPic CrHis

100

50

0

Muscle NF - κB, per cent of contr ol

Muscle Nr f2, per cent of contr ol

(A)

TN

250

Control CrPic CrHis

200 150 100 50 0

HS

TN

HS

(D)

150

Control CrPic CrHis

100

50

0

Liver G LUT2, per cent of contr ol

Muscle G LUT4, per cent of contr ol

(C)

TN

CrHis

HS

TN

HS

CrPic

150

Control CrPic CrHis

100

50

0

HS

TN

HS

Figure 1. Effects of different chromium chelates on expressions of Nrf2 (A), NF-κ B (B), and GLUT-4 (C) in muscle and GLUT-2 (D) in the liver of broilers reared under thermoneutral (TN) and heat stress (HS) environments. The probability value for the main effects of environmental temperature and dietary supplementation as well as environmental temperature by dietary supplementation interaction was less than 0.0001 for muscular transcription factors and hepatic and muscular glucose transporters. CrPic = chromium picolinate; CrHis = chromium histidinate.

Table 2. Effects of different chromium chelates on performance in broilers reared under heat stress. Response variables∗

Groups Environment (E)

Supplement (S)

Thermoneutral Heat stress

Thermoneutral Heat stress ANOVA E S ExS

Control CrPic CrHis Control CrPic CrHis Control CrPic CrHis

Feed intake (g) 3669 3224 3396 3444 3499 3648 3673 3686 3143 3215 3313 0.0001 0.0001 0.02

± ± ± ± ± ± ± ± ± ± ±

16 13 42c 40b 36a 17 29 35 14 14 17

Weight gain (g)

FCR

Abdominal fat (%)

2059 ± 13 1.79 ± 0.01 1.28 ± 1625 ± 14 1.99 ± 0.01 1.70 ± 1781 ± 43c 1.93 ± 0.03b 1.65 ± 1841 ± 36b 1.88 ± 0.02a,b 1.48 ± 1904 ± 34a 1.85 ± 0.02a 1.33 ± 2037 ± 21 1.79 ± 0.02 1.43 ± 2052 ± 13 1.79 ± 0.02 1.28 ± 2089 ± 29 1.77 ± 0.03 1.13 ± 1524 ± 15 2.07 ± 0.02 1.88 ± 1631 ± 19 1.98 ± 0.02 1.68 ± 1720 ± 14 1.93 ± 0.02 1.54 ± ——————————– P < ——————————– 0.0001 0.0001 0.0001 0.0001 0.004 0.0001 0.001 0.04 0.34

0.02 0.02 0.04c 0.03b 0.04a 0.01 0.02 0.02 0.02 0.02 0.02

∗ Data are ± SEM. Data with uncommon superscripts (a–c) in rows (main effect of supplements) differ (P < 0.05). FCR = feed conversion ratio (feed consumed, g:weight gained, g).


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SUPPLEMENTAL CHROMIUM FORM AND HEAT-DISTRESSED BROILER Table 3. Effects of different chromium chelates on tissue chromium levels in broilers reared under heat stress. Response variables∗


Supplement (S)




ANOVA E S ExS

Serum Cr (mg/L) 1.99 1.22 1.25 1.68 1.87 1.67 2.02 2.27 0.84 1.34 1.47 0.0001 0.0001 0.79

± ± ± ± ± ± ± ± ± ± ±

Breast muscle Cr (mg/kg)

Liver Cr (mg/kg)

0.06 2.01 ± 0.06 2.39 ± 0.04 1.30 ± 0.04 1.66 ± 1.44 ± 0.08b 1.74 ± 0.08c 0.07b 1.72 ± 0.08a 2.00 ± 0.08a 1.80 ± 0.09a 2.11 ± 0.07 1.77 ± 0.09 1.92 ± 0.10 2.07 ± 0.08 2.33 ± 0.07 2.18 ± 0.10 2.47 ± 0.06 1.11 ± 0.06 1.55 ± 0.03 1.37 ± 0.07 1.68 ± 0.03 1.42 ± 0.07 1.75 ± ——————————– P < ——————————– 0.0001 0.0001 0.0001 0.0001 0.80 0.03

0.06 0.03 0.04b 0.07a 0.09a 0.05 0.07 0.12 0.04 0.04 0.06

∗ Data are ± SEM. Data with uncommon superscripts (a–c) in rows (main effect of supplements) differ (P < 0.05). CrPic = chromium picolinate; CrHis = chromium histidinate.

However, increase in liver Cr concentration in response to supplemental Cr under the TN condition (21.4 and 28.6% for the CrPic and CrHis diets, respectively) was higher than under the HS condition (8.4 and 12.9% for the CrPic and CrHis diets, respectively) (P < 0.03).

Serum Metabolites and Enzymes The HS condition caused increases in serum glucose (1.58 folds), cholesterol (1.17 folds), and creatine (1.19 folds) concentrations (P < 0.0001 for all), while not affecting serum total protein, albumin, or globulin concentrations (Table 4). Supplemental Cr decreased serum glucose and cholesterol concentrations (P < 0.0001 for both), but did not alter serum total protein, albumin, globulin, or creatine concentrations. Cr as CrHis was superior to CrPic in reducing serum cholesterol concentration (P < 0.05), but equivalent in reducing serum glucose concentration. Except for serum glucose concentration, ET by DS interaction effects on metabolic parameters were insignificant. Cr supplementation decreased serum glucose concentration in comparison with the C group at a greater extent under the HS condition than under the TN condition, CrHis being superior to CrPic (P < 0.04). The effects of different Cr chelates on concentrations of serum enzymes are shown in Table 5. Exposure to HS caused variable increases in concentrations of serum enzymes (3.5% in AST, P < 0.10; 12.9% in ALT, P < 0.0001; 6.6% in GGT, P < 0.004; 28.3% in LDH, P < 0.0001; and 13.2% in CK, P < 0.0001). Except for LDH (P < 0.0001), other enzymes were unresponsive to dietary Cr supplementation. Decreases in serum LDH concentrations were 10.2 and 16.8% in broilers fed the CrPic and CrHis diets, respectively (P < 0.05), as compared to those fed the C diet. There were no effects of ET by DS interaction on concentrations of serum enzymes.

Hepatic and Muscular Transcription Factors and Glucose Transporters (GLUT) Exposure to HS decreased expression of hepatic GLUT-2 by 39.0%, muscular Nrf2 by 37.4%, and GLUT-4 by 42.9% and increased expression of muscular NF-κB by 69.0% (Figure 1; P < 0.0001 for all). Supplemental Cr increased expression of hepatic GLUT-2 (1.32x) as well as muscular Nrf2 and GLUT-4 (1.27x, for both) and decreased expression of muscular NF-κB (0.70x) (Figure 1; P < 0.0001 for all), CrHis being superior to CrPic (P < 0.05 for all). These trends in hepatic and muscular transcription factors and glucose transporters were more notable under the HS condition than under the TN condition. Efficacy of CrHis on these parameters under the HS condition almost doubled that of CrPic (Figure 1; P < 0.0001 for all).

DISCUSSION In addition to causing economic losses associated with poor performance and product quality, suppressing immune potency, and increasing the susceptibility to infectious diseases, HS is a welfare problem in broiler production. Poor performance (Table 2) in response to exposure to HS is in agreement with a number of previous experiments involving various poultry species (Cupo and Donaldson, 1987; Geraert et al., 1996; Orhan et al., 2012). Moreover, HS aggravates Cr mobilization from tissues and also increases Cr excretion and decreases Cr retention (Geraert et al., 1996; Sahin et al., 2002), which may lead to its deficiency, suggesting that the Cr requirement may increase (Hayirli, 2005). Broilers exposed to HS had lower serum, liver, and breast muscle Cr concentrations than those reared under the TN condition (Table 3). Supplemental Cr compensates body Cr reserves and alleviates the adverse effects of


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Table 4. Effects of different chromium chelates on metabolic parameters in broilers reared under heat stress. Response variables∗

Groups

Environment (E) Supplement (S) Glucose (mg/dL) Cholesterol (mg/dL) Protein (g/dL) Albumin (g/dL) Globulin (g/dL) Creatine (mg/dL) Thermoneutral Heat stress



ANOVA E S ExS

219 345 300 277 268 229 215 212 372 339 324 0.0001 0.0001 0.04

± ± ± ± ± ± ± ± ± ± ±

3 106 ± 1 6.10 ± 0.12 2.03 ± 0.02 4.08 ± 0.12 0.27 ± 0.01 5 124 ± 1 5.84 ± 0.12 2.02 ± 0.02 3.82 ± 0.12 0.32 ± 0.01 12a 122 ± 2a 5.91 ± 0.12 2.03 ± 0.02 3.88 ± 0.12 0.31 ± 0.01 10b 114 ± 2b 5.99 ± 0.16 2.02 ± 0.02 3.97 ± 0.17 0.29 ± 0.01 10b 109 ± 2c 6.01 ± 0.15 2.02 ± 0.03 3.99 ± 0.16 0.29 ± 0.01 5 111 ± 2 6.01 ± 0.16 2.04 ± 0.04 3.80 ± 0.16 0.28 ± 0.01 3 106 ± 2 6.16 ± 0.24 2.01 ± 0.02 3.80 ± 0.25 0.27 ± 0.01 4 100 ± 2 6.15 ± 0.22 2.03 ± 0.04 3.86 ± 0.23 0.26 ± 0.01 8 133 ± 2 5.82 ± 0.19 2.02 ± 0.03 3.97 ± 0.19 0.33 ± 0.01 6 122 ± 2 5.83 ± 0.22 2.04 ± 0.03 4.14 ± 0.22 0.31 ± 0.01 8 117 ± 2 5.86 ± 0.22 2.01 ± 0.04 4.12 ± 0.22 0.31 ± 0.01 ——————————————————– P < ——————————————————0.0001 0.12 0.81 0.14 0.0001 0.0001 0.89 0.97 0.88 0.19 0.25 0.94 0.70 0.91 0.89

∗ Data are ± SEM. Data with uncommon superscripts (a–c) in rows (main effect of supplements) differ (P < 0.05). CrPic = chromium picolinate; CrHis = chromium histidinate.

Table 5. Effects of different chromium chelates on serum enzymes in broilers reared under heat stress. Response variables∗


Supplement (S)


Thermoneutral Heat stress ANOVA E S ExS


AST (U/L) 199 206 204 203 201 201 200 197 206 206 205

± ± ± ± ± ± ± ± ± ± ±

0.10 0.82 0.95

2 3 4 3 3 5 4 3 6 5 4

ALT (U/L)

GGT (U/L)

LDH (IU/L)

1.86 ± 0.04 24.1 ± 0.4 1605 ± 37 2.10 ± 0.03 25.7 ± 0.3 2059 ± 47 2.00 ± 0.05 25.1 ± 30.5 2013 ± 56a 2.00 ± 0.04 25.0 ± 0.5 1808 ± 49b 1.95 ± 0.05 24.7 ± 0.4 1675 ± 71c 1.89 ± 0.06 23.8 ± 0.7 1779 ± 55 1.87 ± 0.05 24.7 ± 0.7 1599 ± 51 1.82 ± 0.08 23.9 ± 0.6 1437 ± 62 2.10 ± 0.06 26.3 ± 0.6 2247 ± 62 2.13 ± 0.04 25.2 ± 0.7 2017 ± 53 2.08 ± 0.04 25.5 ± 0.5 1913 ± 103 ——————————— P < ——————————– 0.0001 0.004 0.0001 0.63 0.83 0.0001 0.91 0.30 0.89

CK (U/L) 2067 2340 2221 2212 2178 2106 2059 2036 2335 2365 2321

± ± ± ± ± ± ± ± ± ± ±

48 42 57 56 66 76 74 99 78 69 75

0.0001 0.85 0.88

∗ Data are ± SEM. Data with uncommon superscripts (a–c) in rows (main effect of supplements) differ (P < 0.05). CrPic = chromium picolinate; CrHis = chromium histidinate; AST = aspartate-aminotransferase; ALT = alanine-amino transferase; GGT = γ -glutamil transferase; LDH = lactate dehydrogenase; CK = creatine kinase.

HS (Steele and Rosebrough, 1981; Sahin et al., 2010; Toghyani et al., 2012). Cr has been chelated with yeast, picolinate, nicotinate, propionate, and methionine (Hayirli, 2005). Efficacy of Cr in the organic complex is superior to Cr in inorganic form (Hayirli, 2005; Suksombat and Kanchanatawee, 2005; Sahin et al., 2010). Both Cr sources alleviated performance parameters, especially under the HS condition, as compared to the C group, CrHis being more effective than CrPic (Table 2). The superiority of CrHis to CrPic could be related to the fact that it is a stable complex structure, and histidinate allows greater absorbability and bioavailability (Anderson et al., 2004). Supplemental Cr, especially in organic chelates, was shown to alleviate the adverse effects of HS on performance in broilers (Sands and Smith, 1999; Ghazi et al., 2012; Toghyani et al., 2012). Reducing abdominal fat in the poultry sector is desirable. Cr improves insulin action, leading to improvements in carbohydrate, protein, and lipid metabolism,

as reflected by decreased nonesterified fatty acids, fastened serum triglyceride removal, and uptake of glucose for lipogenesis in the liver (Lien et al., 1999). These are accompanied by increasing protein accretion (Hossain et al., 1998; Debski et al., 2004; Ahmed et al., 2005), allowing higher leanness (Hossain et al., 1998; Debski et al., 2004; Choct et al., 2005). Growth promoting effects of supplemental Cr are related to upregulation of expressions of skeletal muscle protein (Zha et al., 2009; Pan et al., 2013). Cr plays a role in the regulation of metabolism, especially under stress conditions in poultry (Kim et al., 1996). Stress is accompanied by catabolic profile, because it induces proteolysis, lipolysis, and glycogenolysis (Donkoh, 1989). Although there was no significant alteration in serum protein concentration, concentrations of glucose and cholesterol (Table 4), as well as enzymes (Table 5), were higher for broilers reared under the HS environment than those reared under the TN environment (Table 4).


SUPPLEMENTAL CHROMIUM FORM AND HEAT-DISTRESSED BROILER

The membrane electron transport system is harmed by reactive oxygen species that arise due to HS (Mujahid et al., 2005). They negatively affect the function and integrity of the cells, resulting in disruption of oxidative metabolism, transcription, translation, and RNA processing (Iwagami, 1996). Furthermore, these changes within the cell membranes activate the expression of transcription factors such as Nrf2 and NF-κB (Nelson et al., 2004). Nrf2 is an important cytoprotective transcription factor and expresses phase II detoxifying antioxidant enzymes (e.g., glutamate-cysteine ligase, heme oxygenase, glutathione S-transferase, uridine diphosphate-glucuronosyltransferase) that are involved in inhibition of oxidative stress (Kim et al., 2010). NFκβ is responsible for controlling the DNA transcription and generates a series of events related to activation of some genes when entering the nucleus upon expression (Surh and Na, 2008). Both Cr sources were effective in augmenting expression of NF-κB and Nrf2 in breast muscle. Inhibitory effects on NF-κB expression and stimulatory effects on Nrf2 for CrHis were more notable than CrPic under the HS environment (Figure 1). Orhan et al. (2012) also reported reversal of oxidative stress as reflected by similar changes in expression of hepatic nuclear transcription factors in quails supplemented with either CrHis or CrPic under the HS condition. Glucose is a major metabolic fuel for cellular energy metabolism (Zierler, 1999). Because it is a hydrophilic molecule, a transporter protein is needed for its internalization prior to metabolism (Joost and Thorens, 2001). Among GLUT, only 4 (GLUT-1, 2, 3, and 8) are expressed in chickens (White et al., 1991; Wang et al., 1994). In chicken, GLUT-2 is expressed mainly in liver and kidney tissues (Kono et al., 2005). Studies revealing GLUT expression in poultry species exposed to HS are limited. Broilers reared under the HS condition had a lower hepatic GLUT-2 expression, which was significantly increased by supplemental CrHis at a greater extent than by supplemental CrPic (Figure 1). In conclusion, supplemental Cr alleviates the adverse effects of HS on broiler performance parameters associated with oxidative stress, as reflected by the reduced severity of catabolic profile through modulating the expressions of GLUT-2 in the liver, as well as GLUT-4, NF-κB, and Nrf2 in muscle. The positive effect of supplemental Cr was more notable when Cr was chelated with histidinate than when Cr was chelated with picolinate. CrHis can be a part of a potential protective nutritional management practice in alleviating the metabolic profile to prevent heat stress-related depression in performance.

ACKNOWLEDGMENTS The study was funded by the Small and Medium Business Development and Support Administration of Turkey (KOSGEB) and also in part by the Turkish Academy of Sciences (Ankara, Turkey). The authors

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thank Nutrition 21 (NY) for providing chromium picolinate and chromium histidinate. Thanks are extended to ˙ Farmavet International (Istanbul, Turkey) for donating vitamin-mineral premixes and reconstituting premixes with Cr chelates. Conflict of interest: The authors declare that there are no conflicts of interest.

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