This article was downloaded by: [Selcuk Universitesi] On: 01 February 2015, At: 22:03 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails through reducing oxidative stress and inhibiting heat-shock protein expression a
b
b
c
b
d
F. Akdemir , N. Sahin , C. Orhan , M. Tuzcu , K. Sahin & A. Hayirli a
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Dicle University, Diyarbakir b
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, Elazig c
Department of Biology, Faculty of Science, Firat University, Elazig, Turkey
d
Click for updates
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey Accepted author version posted online: 27 Jan 2015.
To cite this article: F. Akdemir, N. Sahin, C. Orhan, M. Tuzcu, K. Sahin & A. Hayirli (2015): Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails through reducing oxidative stress and inhibiting heat-shock protein expression, British Poultry Science, DOI: 10.1080/00071668.2015.1008992 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1008992
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2015.1008992
ip t
CBPS-2014-230 Ed. Kjaer, December 2014;
cr
M an
us
through reducing oxidative stress and inhibiting heat-shock protein expression
F. AKDEMIR1, N. SAHIN2, C. ORHAN2, M. TUZCU3, K. SAHIN2 AND A. HAYIRLI4
1
pt e
d
Running title: Suppression of expression of hepatic NF-κB and HSPs in heatstress
ce
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine,
Dicle University, Diyarbakir, 2Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, Elazig, 3Department of Biology, Faculty of
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails
Science, Firat University, Elazig, Turkey,and 4Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey
Correspondence to: Dr. Kazim Sahin, Department of Animal Nutrition, Faculty of Veterinary Medicine, Firat University, 23119 Elazig, Turkey. Email: [email protected]
Accepted for publication 8th December 2014
Abstract. 1. An experiment was conducted to evaluate the effects of a histidine complex of chromium (CrHis) on egg production, lipid peroxidation and the expression of hepatic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and heat-shock proteins (HSPs) in Japanese quails (Coturnix coturnix japonica) exposed to heat stress (HS). 2. A total of 180 5-week-old female quails were reared either at 22oC for 24 h/d
ip t
(thermoneutral, TN) or 34oC for 8 h/d (heat stress, HS) for 12 weeks. Birds in both
environments were randomly given 1 of 3 diets: basal diet and basal diet added with 400 or
cr
us
the end of trial were analysed to determine concentrations of cholesterol and malondialdehyte (MDA) and expressions of transcription and heat shock proteins.
M an
3. Exposure to HS caused reductions in feed intake (-8.1%) and egg production (-15.8%), elevations in serum (14.8%) and egg yolk (29.0%) cholesterol concentrations, decreases in serum (113%) and egg yolk (73.0%) MDA concentrations and increases in the expressions of hepatic NF-κB (52.3%) and HSPs (averaging 53.6%).
pt e
d
4. The effects of increasing supplemental CrHis on the response variables were more notable in the HS environment than in the TN environment. There were considerable improvements in feed intake and egg production, decreases in serum and egg yolk cholesterol concentrations
ce
and suppressions in the expressions of hepatic nuclear protein and HSPs in response to increasing supplemental CrHis concentration in the diet of quails reared under the HS envirionment.
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
800 µg of elemental Cr as CrHis per kg of diet. Blood, egg yolk and liver samples collected at
5. In conclusion, supplemental CrHis improves productivity through alleviating oxidative stress and modulating the expressions of hepatic NF-κB and HSPs in heat-stressed quails. INTRODUCTION
Heat stress (HS) adversely affects survival (Bogin et al., 1996), performance parameters (feed intake, egg production, weight gain and feed efficiency) and nutrient digestibility (Wolfenson et al., 1979; Austic, 1985; Siegel, 1995; Yalcin et al., 2001; Sahin and Kucuk, 2003) as well 2
as product quality (Smith, 1974) in poultry species. HS is also associated with deprivation of tissue vitamin and micromineral reserves and excessive generation of reactive oxygen species (ROS) resulting from oxidative stress (Halliwell and Gutteridge, 1989; Siegel, 1995; Sahin and Kucuk, 2003). Thus, replenishment of these reserves via supplementation of vitamins and minerals through feed and water is a rational strategy to overcome the adverse consequences
ip t
in poultry production at high environmental temperature (El Husseiny and Creger, 1981; Bollengier-Lee et al., 1998; Sahin and Kucuk, 2003).
cr
us
by which HS adversely affect productivity and wellbeing. Nuclear factor κ-light-chainenhancer of activated B cells (NF-κB) is a member of the transcription factors that is known
M an
as an antiapoptotic transcription factor (Gilmore, 2006). It is responsible for regulating the transfer of genetic information from DNA to mRNA and controlling DNA transcription (Latchman, 1997). NF-κB is normally presented in the cytosol of the cells in an inactive form and enters the nucleus in response to stimuli, including ROS, to activate the expression of
pt e
d
specific genes (Nelson et al., 2004; Gilmore, 2006). Heat shock proteins (HSP), also known as stress proteins, are expressed in different cell types and involved in many promoter pathways (Lindquist and Craig, 1988; Hightower,
ce
1991). These cytoprotective proteins act as molecular chaperones for other cellular proteins (Lindquist and Craig, 1988; Wang and Edens, 1998). HSPs play a significant role in cell proliferation, differentiation, oncogenesis and in protein folding, unfolding, assembling,
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Recent studies have focused on molecular biology to elucidate underlying mechanisms
disassembling and translocation (Pelham, 1986; Murukami et al., 1988). A number of stimuli including acidosis, hypoxia, energy depletion, ischemia- reperfusion and reactive oxygen or nitrogen species as well as HS are known to induce HSPs expression (Burdon, 1986; Kregel, 2002; Mahmoud et al., 2003). Chromium (Cr) is involved in carbohydrate, lipid and protein metabolism through activating certain enzymes in poultry (Anderson, 1994) as well as being an active component 3
of a glucose tolerance factor that enhances insulin actions (Schwartz and Mertz, 1957). As a strong and effective antioxidant (Lindemann, 1996), Cr also alleviates productivity and metabolism decline in poultry exposed to HS (Preuss et al., 1997; Anderson et al., 2001; Sahin et al., 2002; Sahin et al., 2009). These effects could partially be related to replenishment of body Cr reserves (Liu et al., 1998; Sahin et al., 2003, 2004). Moreover, the
ip t
form of supplemental Cr is important to amplify its effects. For example, chromium-
histidinate (CrHis), an organic Cr form, is shown to be absorbed about 20-30 fold more
cr
us
supplemental Cr at increasing dietary concentrations could help to elucidate the role of Cr in heat stressed birds (Amatya et al., 2004; Sahin et al., 2010; Ebrahimzadeh et al., 2013; Ghazi
M an
et al., 2012; Zhang and Kim, 2014). Therefore, this experiment was conducted to investigate the effects of CrHis supplementation on performance, oxidative status and the expression of NF-κB and HSPs in Japanese quails (Coturnix coturnix japonica) exposed to HS. MATERIALS AND METHODS
pt e
d
Animals, diets and experimental design
The birds were managed according to animal welfare regulations at the Veterinary Control and Research Institute of Elazig, Turkey. A total of 180, 5week old female Japanese quails
ce
were housed in wire cages in temperature-controlled rooms at either 22oC for 24 h/d (Termoneutral, TN) or 34oC for 8 h/d followed by 22°C for 16 h/d (Heat Stress, HS). All quails were hatched from the same group of the parent stock on the same day (Insanay AY
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
efficiently than the inorganic form (CrCl 3 ) (Mowat, 1994). The use of organic forms of
Kanatli Hayvan Uretim Paz. Tic. Inc., Elazig, Turkey). Birds at both environmental temperatures (ET) were randomly fed on one of three
diets: basal diet or the basal diet with 400 or 800 µg/kh diet of elemental Cr as CrHis (Nutrition 21, NY, USA) for a period of 12 weeks. Ingredients and chemical compositions of the basal diet are shown in the Table. Each of the 6 factorially arranged groups (2 x 3) was replicated in 10 cages, each (20 x 20 cm) containing 3 quails. Feed and fresh water were 4
offered ad libitum throughout the experiment. Birds were subjected to a light regimen of 16L: Table near here
8D. Data and sample collection
Feed consumption was measured weekly and egg yield was recorded daily during the experimental period. At the end of the experiment, one bird from each cage chosen randomly
ip t
was slaughtered (10 birds/group) by decapitation, and then scalded and hand-plucked. Prior to slaughtering, blood samples were collected into additive-free blood tubes and centrifuged at
cr
us
(MDA) and cholesterol. The liver was removed and stored at -80°C for determination of the expressions of NF-κB and HSPs (Hsp60, Hsp70 and Hsp90). One egg from each cage was
M an
selected randomly (10 eggs/group) and egg yolks were separated from albumen for measurement of MDA and cholesterol concentrations. Laboratory analyses
Feed samples collected monthly were analysed for crude protein (#988.05), ether extract
pt e
d
(#932.06), crude fibre (#962.09), crude ash (#936.07), Ca (#968.08) and P (#965.17) in triplicates (AOAC, 1990). Energy and amino acid (methionine and lysine) contents were calculated from tabular values listed for the feedstuffs (Jurgens, 1996).
ce
To determine Cr content, 300 mg feed samples were first digested with 5 ml
concentrated HNO 3 in a Microwave Digestion System (Berghoff, Eningen, Germany) for 30 min. Analysis of elemental Cr was conducted using a graphite furnace atomic absorption
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
3000 g for 10 min. Sera were harvested and stored at -20°C for analysis of malondialdehyde
spectrophotometer (Perkin-Elmer, Analyst 800, Norwalk, CT, USA).
Cholesterol and malondialdehyde Serum and egg yolk cholesterol concentrations were measured using an auto-analyser (Olympus AU-600, Diamond Diagnostics, Holliston, MA, USA). Serum and egg yolk MDA concentrations were measured as described previously (Karatepe, 2004) using a fully 5
automatic high performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) equipped with a pump (LC-20AD), a diode array detector (SPD-M10A), a column oven (CTO-10ASVP), an autosampler (SIL-20A), a degasser unit (DGU-20A5), inertsil ODS-3 C 18 column (250 x 4.6 mm, 5 µm) and a computer with LC solution Software. Transcription and heat stress proteins
ip t
Liver tissue was homogenised in 1:10 (w/v) in 10 mM Tris-HCl buffer at pH 7.4, containing 0.1 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and 5 µM soybean soluble powder
cr
us
000 g at 4ºC for 30 min, and the supernatants were transferred into fresh tubes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample buffer containing 2% β-
M an
mercaptoethanol was added to the supernatants. Equal amounts of protein (20 µg) were electrophoresed and subsequently transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). Nitrocellulose blots were washed twice for 5 min in phosphatebuffered saline (PBS) and blocked with 1% bovine serum albumin in PBS for 1 h prior to the
pt e
d
application of primary antibody. Chicken antibodies against Hsp60 (ab46798), Hsp70 (ab47455), Hsp90 (ab1429) and NF-κB (ab16502) (Abcam, Cambridge, UK) were diluted (1:1000) in the same buffer containing 0.05% Tween-20. The nitrocellulose membrane was
ce
incubated overnight at 4ºC with protein antibody. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for NF-κB and HSPs (Abcam). Specific binding was detected using diaminobenzidine and hydrogen peroxide as substrates.
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
(Sigma, St. Louis, MO, USA) as trypsin inhibitor. Tissue homogenates were centrifuged at 15
Protein loading was controlled using a monoclonal mouse antibody against β-actin antibody (A5316; Sigma). Samples were analysed in 4 replicates and protein concentrations were determined densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA). Statistical analyses
6
Performance parameters (feed intake and egg production), oxidative stress biomarkers (MDA and cholesterol) and protein expressions (NF-κB, Hsp60, Hsp70 and Hsp90) were analysed by 2-way ANOVA using the PROC GLM procedure (SAS, 2002). The linear model to elucidate group effects was as follows: yijk = µ + Ei + Sj + (E*S)ij + eijk, where y = response variable; µ = population mean; E = environmental temperature, i=1,2; S = CrHis supplementation,
ip t
j=1,2,3; and e = residual error [N (σ, µ; 0, 1)]. The polynomial contrast option was computed
us
RESULTS Performance parameters
M an
The effects of ET and supplemental CrHis on feed intake and egg production are shown in Figure 1. Exposure to HS caused 8.1 and 15.8% reductions in feed intake (P < 0.001; Figure 1A) and egg production (P < 0.0001; Figure 1B), respectively. There were linear increases in both feed intake and egg production as supplemental Cr increased from 0 to 800 μg/kg in both
pt e
d
environments (P < 0.0001, Figure 1). The magnitude of these increases were more notable in the HS environment than in the TN environment compared with the unsupplemented quails. There were 1.8 and 8.2% increases in feed intake (P < 0.001; Figure 1A) and 2.5 and 15.5%
ce
in egg production (P < 0.0001; Figure 1B) in quails reared in the TN and HS environments, Figure 1 near here
respectively.
Lipid peroxidation
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Statistical significance was considered at P ≤ 0.05.
cr
to evaluate the CrHis supplementation concentration-response relationship (SAS, 2002).
Exposure to HS caused 14.8 and 29.0% elevations in cholesterol concentrations in serum (P
0.05); (C) serum MDA (P < 0.001; pooled SEM = 0.09) and (D) egg yolk MDA (P < 0.001;
B
16 14 12 10
0
400 Supplemental Cr as CrHis, mg/kg
800
ed
1.4
400 800
Supplemental Cr as CrHis, mg/kg
1.0
0.6 Egg yolk MDA, nmol/g
C
20
1
3
t
rip
2
sc
an u
M
0
pt
0.2
Ac ce
Serum MDA, nmol/ml
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
1.8 4
D
0
400
Supplemental Cr as CrHis, mg/kg
800
Figure 3. Effects of environmental temperature (ET) [white bars; TN = thermoneutral (22oC for 24 h/d) and grey bars; HS = heat stress (34oC for 8
rip
t
h/d followed by 22oC for 16 h/d)] and dietary Cr supplementation as chromium histidinate (CrHis) (without line, 0 µg/kg; vertical line, 400 µg/kg;
sc
proteins (HSP). Data are means of 4 replicates and expressed as relative to control (%). A representative blot is shown. (A) The environmental
an u
temperature by CrHis interaction on hepatic NF-κB (P < 0.001; pooled SEM = 4.5); (B) HSP60 (P < 0.001; pooled SEM = 5.3); (C) HSP70 (P < 0.001; pooled SEM = 4.8) and (D) HSP90 (P < 0.001; pooled SEM = 6.1) expression. ANOVA, degree of freedom was 1, 2, 2 and 55 for ET, CrHis,
ed
200
A
120 80 40
TN
Ac ce
pt
160
HSP60, % of the control
M
ET*CrHis and error term, respectively.
NF-κB, % of the control
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
horizontal line, 800 µg/kg) on the expression of hepatic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and heat-shock
HS
21
200
B
160 120 80 40
TN
HS
ed
160
HS
an u
M
TN
pt
C
120
80
HSP90, % of the control
40
Ac ce
HSP70, % of the control
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
200
22
80
40
t
rip
120
sc
200
160
D
TN HS
Table. Ingredients and nutrient composition of the basal diet1 Ingredient
Amount (g/kg) 550.3
Soyabean meal
281.7
Soyabean oil
47.8
Sodium chloride
3.3
DL-methionine
2.1
ip t
Maize
Limestone
94.3
Dicalcium phosphate
17.0
cr
3.5
Nutrient composition (g/kg, dry matter basis)
us
Metabolisable energy, MJ/kg3 Calcium Phosphorus Methionine3 Lysine3 Chromium, mg/kg
175.9 39.1 6.1 4.0 10.7 1.68
Chromium (400 or 800 µg/kg) was added to the basal diet as Cr-histidinate (Nutrition 21, NY, USA) at the
pt e
expense of maize. 2
11.9
d
1
M an
Crude protein
Per kilogram contained: retinyl acetate, 1.8 mg; cholecalciferol, 0.025 md; dl-tocopheryl acetate, 1.25 mg;
menadione sodium bisulfite, 2,5 mg; thiamine-hydrochloride, 1.5 mg; riboflavin, 3 mg; niacin, 12.5 mg; dpantothenic acid, 5 mg; pyridoxine hydrochloride, 2.5 mg; vitamin B 12 , 0,0075 mg; folic acid, 0.25 mg; choline
ce
chloride, 125 mg; Mn (MnSO 4 -H 2 O), 50 mg; Fe (FeSO 4 -7H 2 O), 30 mg; Zn (ZnO), 30 mg; Cu (CuSO 4 -5H 2 O), 5 mg; Co (CoCl 2 -6H 2 O), 0,1 mg; I (KI), 0,4 mg; Se (Na 2 SeO 3 ), 0,15 mg. 3
Calculated value according to tabular values listed for the feed ingredients (Jurgens, 1996).
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Vitamin-Mineral premix2
20
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails through reducing oxidative stress and inhibiting heat-shock protein expression a
b
b
c
b
d
F. Akdemir , N. Sahin , C. Orhan , M. Tuzcu , K. Sahin & A. Hayirli a
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Dicle University, Diyarbakir b
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, Elazig c
Department of Biology, Faculty of Science, Firat University, Elazig, Turkey
d
Click for updates
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey Accepted author version posted online: 27 Jan 2015.
To cite this article: F. Akdemir, N. Sahin, C. Orhan, M. Tuzcu, K. Sahin & A. Hayirli (2015): Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails through reducing oxidative stress and inhibiting heat-shock protein expression, British Poultry Science, DOI: 10.1080/00071668.2015.1008992 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1008992
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Publisher: Taylor & Francis & British Poultry Science Ltd Journal: British Poultry Science DOI: 10.1080/00071668.2015.1008992
ip t
CBPS-2014-230 Ed. Kjaer, December 2014;
cr
M an
us
through reducing oxidative stress and inhibiting heat-shock protein expression
F. AKDEMIR1, N. SAHIN2, C. ORHAN2, M. TUZCU3, K. SAHIN2 AND A. HAYIRLI4
1
pt e
d
Running title: Suppression of expression of hepatic NF-κB and HSPs in heatstress
ce
Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine,
Dicle University, Diyarbakir, 2Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Firat University, Elazig, 3Department of Biology, Faculty of
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Chromium-histidinate ameliorates productivity in heat-stressed Japanese quails
Science, Firat University, Elazig, Turkey,and 4Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey
Correspondence to: Dr. Kazim Sahin, Department of Animal Nutrition, Faculty of Veterinary Medicine, Firat University, 23119 Elazig, Turkey. Email: [email protected]
Accepted for publication 8th December 2014
Abstract. 1. An experiment was conducted to evaluate the effects of a histidine complex of chromium (CrHis) on egg production, lipid peroxidation and the expression of hepatic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and heat-shock proteins (HSPs) in Japanese quails (Coturnix coturnix japonica) exposed to heat stress (HS). 2. A total of 180 5-week-old female quails were reared either at 22oC for 24 h/d
ip t
(thermoneutral, TN) or 34oC for 8 h/d (heat stress, HS) for 12 weeks. Birds in both
environments were randomly given 1 of 3 diets: basal diet and basal diet added with 400 or
cr
us
the end of trial were analysed to determine concentrations of cholesterol and malondialdehyte (MDA) and expressions of transcription and heat shock proteins.
M an
3. Exposure to HS caused reductions in feed intake (-8.1%) and egg production (-15.8%), elevations in serum (14.8%) and egg yolk (29.0%) cholesterol concentrations, decreases in serum (113%) and egg yolk (73.0%) MDA concentrations and increases in the expressions of hepatic NF-κB (52.3%) and HSPs (averaging 53.6%).
pt e
d
4. The effects of increasing supplemental CrHis on the response variables were more notable in the HS environment than in the TN environment. There were considerable improvements in feed intake and egg production, decreases in serum and egg yolk cholesterol concentrations
ce
and suppressions in the expressions of hepatic nuclear protein and HSPs in response to increasing supplemental CrHis concentration in the diet of quails reared under the HS envirionment.
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
800 µg of elemental Cr as CrHis per kg of diet. Blood, egg yolk and liver samples collected at
5. In conclusion, supplemental CrHis improves productivity through alleviating oxidative stress and modulating the expressions of hepatic NF-κB and HSPs in heat-stressed quails. INTRODUCTION
Heat stress (HS) adversely affects survival (Bogin et al., 1996), performance parameters (feed intake, egg production, weight gain and feed efficiency) and nutrient digestibility (Wolfenson et al., 1979; Austic, 1985; Siegel, 1995; Yalcin et al., 2001; Sahin and Kucuk, 2003) as well 2
as product quality (Smith, 1974) in poultry species. HS is also associated with deprivation of tissue vitamin and micromineral reserves and excessive generation of reactive oxygen species (ROS) resulting from oxidative stress (Halliwell and Gutteridge, 1989; Siegel, 1995; Sahin and Kucuk, 2003). Thus, replenishment of these reserves via supplementation of vitamins and minerals through feed and water is a rational strategy to overcome the adverse consequences
ip t
in poultry production at high environmental temperature (El Husseiny and Creger, 1981; Bollengier-Lee et al., 1998; Sahin and Kucuk, 2003).
cr
us
by which HS adversely affect productivity and wellbeing. Nuclear factor κ-light-chainenhancer of activated B cells (NF-κB) is a member of the transcription factors that is known
M an
as an antiapoptotic transcription factor (Gilmore, 2006). It is responsible for regulating the transfer of genetic information from DNA to mRNA and controlling DNA transcription (Latchman, 1997). NF-κB is normally presented in the cytosol of the cells in an inactive form and enters the nucleus in response to stimuli, including ROS, to activate the expression of
pt e
d
specific genes (Nelson et al., 2004; Gilmore, 2006). Heat shock proteins (HSP), also known as stress proteins, are expressed in different cell types and involved in many promoter pathways (Lindquist and Craig, 1988; Hightower,
ce
1991). These cytoprotective proteins act as molecular chaperones for other cellular proteins (Lindquist and Craig, 1988; Wang and Edens, 1998). HSPs play a significant role in cell proliferation, differentiation, oncogenesis and in protein folding, unfolding, assembling,
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Recent studies have focused on molecular biology to elucidate underlying mechanisms
disassembling and translocation (Pelham, 1986; Murukami et al., 1988). A number of stimuli including acidosis, hypoxia, energy depletion, ischemia- reperfusion and reactive oxygen or nitrogen species as well as HS are known to induce HSPs expression (Burdon, 1986; Kregel, 2002; Mahmoud et al., 2003). Chromium (Cr) is involved in carbohydrate, lipid and protein metabolism through activating certain enzymes in poultry (Anderson, 1994) as well as being an active component 3
of a glucose tolerance factor that enhances insulin actions (Schwartz and Mertz, 1957). As a strong and effective antioxidant (Lindemann, 1996), Cr also alleviates productivity and metabolism decline in poultry exposed to HS (Preuss et al., 1997; Anderson et al., 2001; Sahin et al., 2002; Sahin et al., 2009). These effects could partially be related to replenishment of body Cr reserves (Liu et al., 1998; Sahin et al., 2003, 2004). Moreover, the
ip t
form of supplemental Cr is important to amplify its effects. For example, chromium-
histidinate (CrHis), an organic Cr form, is shown to be absorbed about 20-30 fold more
cr
us
supplemental Cr at increasing dietary concentrations could help to elucidate the role of Cr in heat stressed birds (Amatya et al., 2004; Sahin et al., 2010; Ebrahimzadeh et al., 2013; Ghazi
M an
et al., 2012; Zhang and Kim, 2014). Therefore, this experiment was conducted to investigate the effects of CrHis supplementation on performance, oxidative status and the expression of NF-κB and HSPs in Japanese quails (Coturnix coturnix japonica) exposed to HS. MATERIALS AND METHODS
pt e
d
Animals, diets and experimental design
The birds were managed according to animal welfare regulations at the Veterinary Control and Research Institute of Elazig, Turkey. A total of 180, 5week old female Japanese quails
ce
were housed in wire cages in temperature-controlled rooms at either 22oC for 24 h/d (Termoneutral, TN) or 34oC for 8 h/d followed by 22°C for 16 h/d (Heat Stress, HS). All quails were hatched from the same group of the parent stock on the same day (Insanay AY
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
efficiently than the inorganic form (CrCl 3 ) (Mowat, 1994). The use of organic forms of
Kanatli Hayvan Uretim Paz. Tic. Inc., Elazig, Turkey). Birds at both environmental temperatures (ET) were randomly fed on one of three
diets: basal diet or the basal diet with 400 or 800 µg/kh diet of elemental Cr as CrHis (Nutrition 21, NY, USA) for a period of 12 weeks. Ingredients and chemical compositions of the basal diet are shown in the Table. Each of the 6 factorially arranged groups (2 x 3) was replicated in 10 cages, each (20 x 20 cm) containing 3 quails. Feed and fresh water were 4
offered ad libitum throughout the experiment. Birds were subjected to a light regimen of 16L: Table near here
8D. Data and sample collection
Feed consumption was measured weekly and egg yield was recorded daily during the experimental period. At the end of the experiment, one bird from each cage chosen randomly
ip t
was slaughtered (10 birds/group) by decapitation, and then scalded and hand-plucked. Prior to slaughtering, blood samples were collected into additive-free blood tubes and centrifuged at
cr
us
(MDA) and cholesterol. The liver was removed and stored at -80°C for determination of the expressions of NF-κB and HSPs (Hsp60, Hsp70 and Hsp90). One egg from each cage was
M an
selected randomly (10 eggs/group) and egg yolks were separated from albumen for measurement of MDA and cholesterol concentrations. Laboratory analyses
Feed samples collected monthly were analysed for crude protein (#988.05), ether extract
pt e
d
(#932.06), crude fibre (#962.09), crude ash (#936.07), Ca (#968.08) and P (#965.17) in triplicates (AOAC, 1990). Energy and amino acid (methionine and lysine) contents were calculated from tabular values listed for the feedstuffs (Jurgens, 1996).
ce
To determine Cr content, 300 mg feed samples were first digested with 5 ml
concentrated HNO 3 in a Microwave Digestion System (Berghoff, Eningen, Germany) for 30 min. Analysis of elemental Cr was conducted using a graphite furnace atomic absorption
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
3000 g for 10 min. Sera were harvested and stored at -20°C for analysis of malondialdehyde
spectrophotometer (Perkin-Elmer, Analyst 800, Norwalk, CT, USA).
Cholesterol and malondialdehyde Serum and egg yolk cholesterol concentrations were measured using an auto-analyser (Olympus AU-600, Diamond Diagnostics, Holliston, MA, USA). Serum and egg yolk MDA concentrations were measured as described previously (Karatepe, 2004) using a fully 5
automatic high performance liquid chromatography (HPLC; Shimadzu, Kyoto, Japan) equipped with a pump (LC-20AD), a diode array detector (SPD-M10A), a column oven (CTO-10ASVP), an autosampler (SIL-20A), a degasser unit (DGU-20A5), inertsil ODS-3 C 18 column (250 x 4.6 mm, 5 µm) and a computer with LC solution Software. Transcription and heat stress proteins
ip t
Liver tissue was homogenised in 1:10 (w/v) in 10 mM Tris-HCl buffer at pH 7.4, containing 0.1 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride and 5 µM soybean soluble powder
cr
us
000 g at 4ºC for 30 min, and the supernatants were transferred into fresh tubes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample buffer containing 2% β-
M an
mercaptoethanol was added to the supernatants. Equal amounts of protein (20 µg) were electrophoresed and subsequently transferred to nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). Nitrocellulose blots were washed twice for 5 min in phosphatebuffered saline (PBS) and blocked with 1% bovine serum albumin in PBS for 1 h prior to the
pt e
d
application of primary antibody. Chicken antibodies against Hsp60 (ab46798), Hsp70 (ab47455), Hsp90 (ab1429) and NF-κB (ab16502) (Abcam, Cambridge, UK) were diluted (1:1000) in the same buffer containing 0.05% Tween-20. The nitrocellulose membrane was
ce
incubated overnight at 4ºC with protein antibody. The blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG for NF-κB and HSPs (Abcam). Specific binding was detected using diaminobenzidine and hydrogen peroxide as substrates.
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
(Sigma, St. Louis, MO, USA) as trypsin inhibitor. Tissue homogenates were centrifuged at 15
Protein loading was controlled using a monoclonal mouse antibody against β-actin antibody (A5316; Sigma). Samples were analysed in 4 replicates and protein concentrations were determined densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, MD, USA). Statistical analyses
6
Performance parameters (feed intake and egg production), oxidative stress biomarkers (MDA and cholesterol) and protein expressions (NF-κB, Hsp60, Hsp70 and Hsp90) were analysed by 2-way ANOVA using the PROC GLM procedure (SAS, 2002). The linear model to elucidate group effects was as follows: yijk = µ + Ei + Sj + (E*S)ij + eijk, where y = response variable; µ = population mean; E = environmental temperature, i=1,2; S = CrHis supplementation,
ip t
j=1,2,3; and e = residual error [N (σ, µ; 0, 1)]. The polynomial contrast option was computed
us
RESULTS Performance parameters
M an
The effects of ET and supplemental CrHis on feed intake and egg production are shown in Figure 1. Exposure to HS caused 8.1 and 15.8% reductions in feed intake (P < 0.001; Figure 1A) and egg production (P < 0.0001; Figure 1B), respectively. There were linear increases in both feed intake and egg production as supplemental Cr increased from 0 to 800 μg/kg in both
pt e
d
environments (P < 0.0001, Figure 1). The magnitude of these increases were more notable in the HS environment than in the TN environment compared with the unsupplemented quails. There were 1.8 and 8.2% increases in feed intake (P < 0.001; Figure 1A) and 2.5 and 15.5%
ce
in egg production (P < 0.0001; Figure 1B) in quails reared in the TN and HS environments, Figure 1 near here
respectively.
Lipid peroxidation
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Statistical significance was considered at P ≤ 0.05.
cr
to evaluate the CrHis supplementation concentration-response relationship (SAS, 2002).
Exposure to HS caused 14.8 and 29.0% elevations in cholesterol concentrations in serum (P
0.05); (C) serum MDA (P < 0.001; pooled SEM = 0.09) and (D) egg yolk MDA (P < 0.001;
B
16 14 12 10
0
400 Supplemental Cr as CrHis, mg/kg
800
ed
1.4
400 800
Supplemental Cr as CrHis, mg/kg
1.0
0.6 Egg yolk MDA, nmol/g
C
20
1
3
t
rip
2
sc
an u
M
0
pt
0.2
Ac ce
Serum MDA, nmol/ml
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
1.8 4
D
0
400
Supplemental Cr as CrHis, mg/kg
800
Figure 3. Effects of environmental temperature (ET) [white bars; TN = thermoneutral (22oC for 24 h/d) and grey bars; HS = heat stress (34oC for 8
rip
t
h/d followed by 22oC for 16 h/d)] and dietary Cr supplementation as chromium histidinate (CrHis) (without line, 0 µg/kg; vertical line, 400 µg/kg;
sc
proteins (HSP). Data are means of 4 replicates and expressed as relative to control (%). A representative blot is shown. (A) The environmental
an u
temperature by CrHis interaction on hepatic NF-κB (P < 0.001; pooled SEM = 4.5); (B) HSP60 (P < 0.001; pooled SEM = 5.3); (C) HSP70 (P < 0.001; pooled SEM = 4.8) and (D) HSP90 (P < 0.001; pooled SEM = 6.1) expression. ANOVA, degree of freedom was 1, 2, 2 and 55 for ET, CrHis,
ed
200
A
120 80 40
TN
Ac ce
pt
160
HSP60, % of the control
M
ET*CrHis and error term, respectively.
NF-κB, % of the control
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
horizontal line, 800 µg/kg) on the expression of hepatic nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and heat-shock
HS
21
200
B
160 120 80 40
TN
HS
ed
160
HS
an u
M
TN
pt
C
120
80
HSP90, % of the control
40
Ac ce
HSP70, % of the control
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
200
22
80
40
t
rip
120
sc
200
160
D
TN HS
Table. Ingredients and nutrient composition of the basal diet1 Ingredient
Amount (g/kg) 550.3
Soyabean meal
281.7
Soyabean oil
47.8
Sodium chloride
3.3
DL-methionine
2.1
ip t
Maize
Limestone
94.3
Dicalcium phosphate
17.0
cr
3.5
Nutrient composition (g/kg, dry matter basis)
us
Metabolisable energy, MJ/kg3 Calcium Phosphorus Methionine3 Lysine3 Chromium, mg/kg
175.9 39.1 6.1 4.0 10.7 1.68
Chromium (400 or 800 µg/kg) was added to the basal diet as Cr-histidinate (Nutrition 21, NY, USA) at the
pt e
expense of maize. 2
11.9
d
1
M an
Crude protein
Per kilogram contained: retinyl acetate, 1.8 mg; cholecalciferol, 0.025 md; dl-tocopheryl acetate, 1.25 mg;
menadione sodium bisulfite, 2,5 mg; thiamine-hydrochloride, 1.5 mg; riboflavin, 3 mg; niacin, 12.5 mg; dpantothenic acid, 5 mg; pyridoxine hydrochloride, 2.5 mg; vitamin B 12 , 0,0075 mg; folic acid, 0.25 mg; choline
ce
chloride, 125 mg; Mn (MnSO 4 -H 2 O), 50 mg; Fe (FeSO 4 -7H 2 O), 30 mg; Zn (ZnO), 30 mg; Cu (CuSO 4 -5H 2 O), 5 mg; Co (CoCl 2 -6H 2 O), 0,1 mg; I (KI), 0,4 mg; Se (Na 2 SeO 3 ), 0,15 mg. 3
Calculated value according to tabular values listed for the feed ingredients (Jurgens, 1996).
Ac
Downloaded by [Selcuk Universitesi] at 22:03 01 February 2015
Vitamin-Mineral premix2
20
