PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Acute phase proteins, interleukin 6, and heat shock protein 70 in broiler chickens administered with corticosterone I. Zulkifli,*†1 P. Najafi,* A. J. Nurfarahin,† A. F. Soleimani,* S. Kumari,* A. Anna Aryani,* E. L. O’Reilly,‡ and P. D. Eckersall‡ *Institute of Tropical Agriculture, and †Department of Animal Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; and ‡Institute of Biodiversity, Animal Health and Comparative Medicine, Bearsden Rd, Glasgow, G61 1QH, United Kingdom after cessation of CORT administration for determination of serum levels of CORT, OVT, AGP, CPN, and IL-6. Brain samples (whole cerebrum) were collected to measure HSP 70 density. Although CORT administration significantly increased feed intake, weight gain was significantly depressed. Administration of CORT also increased CORT, OVT, CPN, AGP, IL-6, and HSP 70 expression. Four days following cessation of CORT administration, OVT declined to the basal level but not CPN and AGP. In conclusion, an elevation in CORT can induce an acute-phase response and HSP 70 expression. Thus, APP and HSP 70 may be of value as indicators of stress in poultry.
Key words: acute phase protein, interleukin 6, heat shock protein 70, corticosterone, broiler 2014 Poultry Science 93:1–7 http://dx.doi.org/10.3382/ps.2014-04099
INTRODUCTION
response was noted 72 h after CRH administration. Working with rats, van Gool et al. (1990) demonstrated a relationship among stress, adrenalin, IL-6, and APR, and it is known that corticosteroid has effects on the APP in dogs (Martinez-Subiela et al., 2004; Caldin et al., 2009). The APP in poultry are classified as positive APP because they show an increase in response to challenge. The positive APP include ceruloplasmin (CPN), ovotransferrin (OVT), and α1-acid glycoprotein (AGP). α1-Acid glycoprotein is well known as an immunoregulator affecting T-cell function and providing negative feedback on the APR, whereas ceruloplasmin is documented to have more protective effects by removing oxygen radicals, antihistamine activity, and reversing the hypoferremic state of the APR (Murata et al., 2004). Ovotransferrin is an iron binding protein and could provide antimicrobial properties by sequestering iron, and has been shown to modulate heterophil and macrophage function in chickens (Murata et al., 2004). Acute-phase response may be considered as part of the general physiological stress response, which involved the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic system (Caldin et al., 2009). Although
Acute phase proteins (APP) are a group of blood proteins primarily synthesized in the liver as part of the acute-phase response (APR). The APR is a core part of the innate immune system response to harmful stimuli such as tissue injury, and infection (González et al., 2008; O’Reilly and Eckersall, 2014). The goal of the APR is to reestablish homeostasis. Stresses such as transportation, weaning, mixing with unfamiliar individuals, and housing on slippery floors have been reported to increase serum concentrations of APP in livestock (Alsemgeest et al., 1994; Pineiro et al., 2007; Qiu et al., 2007). Higuchi et al. (1994) reported that APR was stimulated in vitro by addition of glucocorticoids to cultures of bovine liver slices. Cooke et al. (2012) compared APR in steers receiving different doses of bovine corticotropin-releasing hormone (CRH). The hormone-elevated serum level of haptoglobin and peak
©2014 Poultry Science Association Inc. Received April 9, 2014. Accepted September 5, 2014. 1 Corresponding author: [email protected]
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ABSTRACT An experiment was conducted to determine the effect of corticosterone (CORT) administration on serum ovotransferrin (OVT), α1-acid glycoprotein (AGP), ceruloplasmin (CPN), and IL-6 concentrations, and brain heat shock protein (HSP) 70 expression in broiler chickens. From 14 to 20 d of age, equal numbers of birds were subjected to either (i) daily intramuscular injection with CORT in ethanol:saline (1:1, vol/vol) at 6 mg/kg of BW, or (ii) daily intramuscular injection with 0.5 mL ethanol:saline (1:1, vol/vol; control). Blood samples were collected before CORT treatment (14 d old), 3 and 7 d after CORT injections, and 4 d
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MATERIALS AND METHODS Birds, Husbandry, and Housing A total of 128 one-day-old female broiler chicks (Cobb) were obtained from a commercial hatchery and raised in an environmentally controlled room. Ambient temperature on d 1 was set at 32°C and then gradually reduced until 24°C by d 21. Chicks were assigned to groups of 8 to 16 battery cages. Chicks were fed commercial broiler diets. The light regimen was 23L:1D.
Growth Performance Individual BW and feed intake (cage basis) were recorded on d 14, 21, and 24, and feed conversion ratios (feed/gain; FCR) were calculated.
Experimental Treatment From 14 to 20 d of age (doa), equal number of birds were subjected to either (i) daily intramuscular injection with CORT (Tokyo Chemical Industry Co., Tokyo, Japan) in ethanol:saline (1:1, vol/vol) at 6 mg/kg of BW (Dupont et al., 1999), or (ii) daily intramuscular injection with 0.5 mL ethanol:saline (1:1, vol/vol; control). The birds were injected between 0900 to 1030 h. Body weight was recorded daily. At 14 doa, before CORT administration, blood samples were collected from 2 birds per cage (16 chicks per treatment group) via the wing vein for determination of serum levels of CORT, OVT, AGP, CPN, and IL-6. Following blood sampling, chickens were killed by decapitation and brain (whole cerebrum) samples were collected for determination of HSP 70 density (16 chicks per treatment group). Similar sampling procedures were repeated following 3 (17 doa) and 7 (20 doa) d of CORT injection and 4 d (24 doa) following cessation of CORT administration. The sampling on the third and seventh days of CORT administration was carried out 1 h postinjection. All experimental procedures were conducted in accordance with Universiti Putra Malaysia Research Policy on animal care.
Laboratory Analyses The CORT was measured by a commercial ELISA kit (IDS, Boldon, UK). The intra- and interassay variability for this kit were less than 6.7% and less than 7.8%, respectively, and the detection limit was 27 ng/ mL. The AGP concentration was determined by using a commercial ELISA kit specific to chicken (NB-E60049, Life Diagnostics Inc., West Chester, PA). The radial immunodiffusion method, modified from Mancini et al. (1965), was used to measure OVT. Briefly, 1% agarose gel (Sigma A9539) was prepared (0.13 g of agarose in 13 mL of Tris-buffered saline in a water bath at 56°C), and 260 µL of rabbit anti-chicken transferrin antibody (RabMAbs Abcam, Cambridge, MA) was added to the mixture and poured onto a gel membrane (Flow-Mesh, Sigma Aldrich, St. Louis, MO) under room temperature. Nine wells were punched in each gel, and 10 µL of standard or serum samples was loaded in each well. The OVT standards were prepared at 0, 0.078, 0.3125, 1.250, and 5 mg/mL. Gels were incubated in a dark and humid environment for 48 h. Following incubation, the size of the ring around each well was measured and calculated against standards. The concentration of CPN is determined by the rate of formation of a colored product from CPN and the substrate, 1,4-phenylenediamine dihydrochloride, according to the procedure of Martinez-Subiela et al. (2007). Briefly, 20.375 g of sodium acetate trihydrate was dissolved in 250 mL of distilled water and adjusted to pH 6.2 using glacial acetic acid. Then, 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519) was added to the prepared buffer and kept in the dark
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the avian APR during infection and inflammation has been well documented (Chamanza et al., 1999; O’Reilly and Eckersall, 2014), little information is available on the effect of sterile stress (noninfectious stress) on APP in poultry. Activation of APR by nonpathogenic stimulus is still unclear in poultry species. When living organisms are exposed to thermal and nonthermal stressors, the synthesis of most proteins is retarded, but a group of highly conserved proteins known as heat shock proteins (HSP) is rapidly synthesized. In a heat shock cell, the HSP may bind to proteins to protect them from degradation (Soleimani et al., 2012a), or may prevent damaged proteins from immediately precipitating and permanently affecting cell viability (Etches et al., 1995). Heat shock proteins have been shown to play a profound role in regulating protein folding and in coping with proteins affected by heat and other stresses (Gething and Sambrook, 1992). Work in chickens indicated that feed restriction (Zulkifli et al., 2002, 2011; Soleimani et al., 2011), crating and road transportation (Al-Aqil and Zulkifli, 2009), and social isolation (Soleimani et al., 2012a) augmented HSP 70 expression. The HSP response to various stressors suggests that the proteins could be mediated by the hypothalamic-pituitary-adrenal axis. Soleimani et al. (2011) studied the effects of neonatal stress on HSP 70 expression, circulating levels of corticosterone (CORT), and hippocampal glucocorticoid receptor in aged Japanese quail. The authors concluded that reduced hippocampal glucocorticoid receptor in aged birds resulted in marked inability to terminate CORT secretion at the end of stress and to induce HSP 70 expression. Because stress appears to be associated with all the above conditions, the objective of this study was to determine the effect of a stress hormone CORT administration on serum levels of CPN, OVT, AGP, and IL-6, and expression of HSP 70 in broiler chickens. In this study, the administration of CORT was employed to mimic the physiological stress response in broiler chickens.
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Statistical Analysis All data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Blood parameters and feed intake data were analyzed using CORT administration, time, and their interactions as main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. For feed intake, CORT administration was the only main effect in the analysis. When significant effects were found, comparisons among multiple means were made by Duncan’s multiple range test. Statistical significance was considered as P < 0.05.
RESULTS There were significant (P < 0.05) CORT administration × time interactions for all the blood parameters and HSP 70 data. Injection with CORT significantly (P = 0.0015) increased CORT (Figure 1) and depressed (P = 0.0061) the BW (Figure 2A) on d 17, 20, and 24 of age. The birds that received CORT injection had significantly (P = 0.0041) higher feed intake compared with their control counterparts (Figure 2B). The serum APP levels of OVT (Figure 3A) and AGP (Figure 3B) were significantly increased after 3 (P = 0.0002) and 7 (P = 0.0001) d of CORT injection. Four days following termination of CORT administration (d 24), serum level of OVT and AGP significantly decreased.
Figure 1. Time course changes in serum corticosterone concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
However, only OVT returned to the basal level and AGP still remained significantly (P = 0.0130) higher than the control. Interestingly, CPN was increased only after 7 d of CORT administration (Figure 3C). Serum levels of IL-6 (Figure 4) and brain HSP 70 (Figures 5 and 6) were constantly elevated (P = 0.0001) by CORT administration. Following 4 d of injection recovery, although IL-6 and HSP 70 declined significantly, they still remained higher than the control. Prior to CORT administration, the CORT (Figure 1), OVT, AGP, and CPN (Figure 3A, B, and C), IL-6 (Figure 4), and HSP 70 (Figure 5) concentrations of control and treatment group chicks were not significantly (P = 0.3201, 0.6312, 0.5112, 0.7131, 0.4701, and 0.6112, respectively) different.
DISCUSSION As expected, injection with CORT resulted in a significantly higher CORT. Delivery of CORT by daily injections has also been used by Simon (1984) and Buyse et al. (1987). In the present experiment, the significantly suppressed BW gain in the CORT-treated birds when compared with controls is in accordance with those of Lin et al. (2006) and Hull et al. (2007). The lower weight gain in CORT birds could be attributed to enhanced glycogenolysis/gluconeogenesis (Lin et al., 2006) and protein catabolism (Tomas et al., 1984). The effect of CORT treatment on weight gain in poultry is age dependent. Covasa and Forbes (1995) reported that CORT treatment for 5 d depressed weight gain in 2-wkold but not 5-wk-old chickens. Work in Japanese quail showed that reduction in weight gain following CORT treatment was less marked after 3 wk of age (Hull et al., 2007). Bürger et al. (1998) administered piglets with adrenocorticotrophin hormone (ACTH) or cortisol daily
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for a minimum of 45 min. One hundred microliters of the above buffer and 10 µL of samples or standards were added to each microplate well, shaken gently and kept in the dark for 20 min. The absorbance was recorded spectrophotometrically using a microplate reader at 550 nm. Standards were prepared with serial dilution of pig serum of known CPN concentration calibrated against purified CPN (Sigma Chemical Co.) and saline buffer combination to achieve various concentration of 12.75 (20 µL of pig serum + 60 µL of saline buffer), 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 mg/ mL of CPN. The IL-6 was measured by a commercial ELISA kit specific to chicken (NB-E60049, Novateinbio, Cambridge, MA). The standard range was 3.2 to 100 pg/mL, and the detection limit was 0.5 pg/mL. The level of HSP 70 protein expression was determined as previously described (Soleimani et al., 2012b) using SDS-PAGE and Western blotting with some modifications. Briefly, 0.3 g of cerebrum samples was homogenized with 1.5 mL of protein extraction buffer (20 mM Tris, pH 7.5; 0.75 M sodium chloride) and 10 µL/mL of protease inhibitor cocktail (Precision Plus Protein, Bio-Rad, Hercules, CA), and centrifuged at 20,000 × g for 30 min at 4°C. The supernatant were separated and the total protein quantity was measured using bicinchoninic acid protein assay kit (Sigma Chemical Co.). The final brain HSP 70 concentration was calculated as arbitrary unit of band density relative to total protein concentration of each sample.
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for 2 wk. The authors noted a significant increase in C-reactive protein following 7 d of ACTH or cortisol injection. Work in beef steers suggested that intravenous treatment with bovine corticotrophin-releasinghormone elevated plasma CRP at 54 h, whereas plasma haptoglobin concentrations were greater at 54, 66, and 72 h compared with prechallenge values (Cooke and Bohnert, 2011). The authors, however, noted a decline in plasma ceruloplasmin level at 2 and 6 h. A single injection of adrenalin has been shown to increase the level of α-2 macroglobulin, a typical APP in rats (van Gool et al., 1984). Most APP are constitutively present in the serum; however, their concentrations change markedly in the event of inflammation and infection (Petersen et al., 2004). In the present study, significant elevations in OVT and AGP were noted following 4 d (d 17) of CORT treatment. Under normal conditions, AGP exists in low concentrations in serum, also masked by albumin, which makes it difficult to detect (Conner et al., 1990). It can be concluded that CORT administration may induce APR in poultry. Another impor-
Figure 3. Time course changes in serum ovotransferin (A; OVT), α1-acid glycoprotein (B; AGP), and ceruloplasmin (C; CPN) concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
tant finding from this study is confirmation that CPN, OVT, and AGP acted as positive APP in poultry as in mammals. To our knowledge, this is the first study reporting stimulatory effects of CORT treatment on APR in avian species. It is interesting to note that CPN was only affected by CORT treatment on d 20. Thus, despite a dramatic increase in CORT, 4 d of CORT administration and stress attributed to handling and injection did not elicit
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Figure 2. Mean BW (n = 32; A) and feed intake (n = 8; B) of broiler chickens subjected to daily injection of corticosterone for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age) by corticosterone treatment. Means within a treatment group with no common letters (a,b) differ at P < 0.05.
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any change in CPN. It appears that OVT and AGP are more sensitive to stressful stimuli than CPN. The use of AGP as a measure of poultry well-being has been reported. Salamano et al. (2010) reported that hens kept in both conventional and modified battery cages had higher AGP than those in the free-range system. Following 7 d of CORT injection, the serum levels of CPN, OVT, and AGP showed dramatic elevations. Studies in humans on APP such as serum amyloid A and C-reactive protein suggested that the serum levels of these proteins increased markedly within 4 h after inflammatory stimulus and rapid normalizations (Mackiewicz et al., 1987; Gabay and Kushner, 1999). Haptoglobin is characterized by a later increase in serum concentration remaining elevated for up to 2 wk. The significance of elevation in serum levels of APP during stress is in question. The APR response is considered as part of the innate immune response and is observed across all animal species (Cray et al., 2009). The APR will induce a complex systemic reaction to reestablish homeostasis and promote healing (Cray et al., 2009). An increase in serum APP concentrations may be considered as an indicator of intracellular communication, suggesting an increase in the cellular im-
Figure 5. Time course changes in brain heat shock protein 70 (HSP 70) following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
mune response (Cray et al., 2009). However, the APR could be biologically costly. Studies in cattle suggested that higher serum concentrations of APP were associated with poor growth and reproductive performance (Carroll and Forsberg, 2007; Cooke et al., 2009). Thus, the noted lower weight gain of CORT birds compared with controls could be associated with active cellular immune reaction during APR (Klasing et al., 1987). Work with piglets demonstrated a significant decline in plasma levels of C-reactive proteins 7 d after daily injection with ACTH or cortisol was terminated (Bürger et al., 1998). The present findings suggested that despite the elevated CORT, CPN and OVT returned to basal levels following 4 d (d 24) of CORT injection cessation. Although the AGP declined on d 24, the level did not return to the basal value. According to Petersen et al. (2004), the APR was detectable for several days after the stimulus but the kinetics of the response varied according to species involved and on the extent of tissue damage (Kushner and Mackiewicz., 1993). Administration of corticosteroid (prednisolone) to dogs causes an increase in the APP haptoglobin but
Figure 6. Time course changes in heat shock protein 70 density following daily saline (control) or corticosterone (CORT) administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age).
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Figure 4. Time course changes in serum IL-6 concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–d) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
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In conclusion, the changes in serum CPN, OVT, and AGP concentrations following CORT treatment suggested that APP can be used to gauge physiological stress in avian species and are thus another biomarker for well-being. On a cautionary note, however, it is important to establish that the birds are not influenced by other stimuli such as trauma, infection, neoplasia, or inflammation. A combination of APP and HSP 70 may be a more reliable indicator of stress in chickens.
ACKNOWLEDGMENTS This research was funded by the Malaysian Ministry of Science, Technology and Innovation, Putrajaya.
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not in the concentration of CRP (Martinez-Subiela et al. (2004), so there may also be differing effects of corticosterones on APP in chicken. The mechanisms by which nonpathogenic stimuli evoke APR in poultry are not clear. The earlier discussion suggests that elevation in CORT can increase AGP, OVT, and CPN in poultry. Work in rodents suggested a relationship among stress, IL-6, and APP (van Gool et al., 1990; Shini and Kaiser, 2009). Interleukin-6 is a pro-inflammatory signal. Both in vivo and in vitro studies demonstrated that IL-6 is an important mediator in the synthesis of APP (Gauldie et al., 1987; Moshage et al., 1988; Marinkovic et al., 1989). Clinical situations such as burns, endotoxemia, meningitis, and sepsis have been reported to elicit a marked increase in IL-6 (van Gool et al., 1990). All these conditions involved a generalized stress response. The present findings clearly showed that CORT treatment elevated plasma level of IL-6 in chickens. It can be concluded that increase in CORT elicited a pro-inflammatory cytokine response. Similar findings have been reported in beef steers infused with CRH (Cooke and Bohnert, 2011). This is interesting because CORT and cortisol are well known to have anti-inflammatory action (Barnes and Adcock, 1993). According to Higuchi et al. (1994), acute increases in circulating cortisol, such as during a stress challenge, can indirectly stimulate an inflammatory response. During stress, corticosteroids will degrade body tissues to help animals cope with stressors and restore homeostasis. On the contrary, tissue degradation is recognized by the innate immune system as a disruption of homeostasis (Abbas and Lichtman, 2007) and thus leukocytes will synthesize pro-inflammatory cytokines such as IL-6. Injecting rats with adrenalin but not CORT triggered elevation in the plasma IL-6 level (van Gool et al., 1990). It has been documented that both thermal and nonthermal stressors can induce HSP 70 expression in animals (Zulkifli et al., 2002; Al-Aqil and Zulkifli, 2009). Mahmoud et al. (2004) reported a positive correlation of CORT and heart HSP 70 expression in broiler chickens subjected to cyclic heat stress. On the contrary, Deane et al. (1999) showed that daily injection of cortisol into silver sea breams had a negligible effect on liver HSP 70 expression. Soleimani et al. (2012a) also reported that feeding Japanese quail with 30 mg/kg of CORT for 3 d did not affect HSP 70 level in the brain and heart. The findings of the current study demonstrated that daily injection of CORT for 4 and 7 d significantly increased HSP 70 expression in the brain. Thus, the increased HSP 70 density in the brain is directly related to circulating CORT. The inconsistency in findings on corticosteroid treatment and HSP 70 expression could be attributed to variations in age and species or as proposed and elaborated by Jones et al. (2004), it may be explained by reciprocal regulation of glucocorticoid receptor and heat shock factor 1 signaling. The authors defined a dose- and time-dependent response of HSP 70 to glucocorticoid action.
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Moshage, H. J., H. M. J. Roelofs, J. F. van Pelt, B. P. C. Hazenberg, M. A. van Leeuwen, P. C. Limburg, L. A. Aarden, and S. H. Yap. 1988. The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes. Biochem. Biophys. Res. Commun. 155:112–117. Murata, H., N. Shimada, and M. Yoshioka. 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J. 168:28–40. O’Reilly, E. L., and P. D. Eckersall. 2014. Acute phase proteins: A review of their function, behaviour and measurement in chickens. World’s Poult. Sci. J. 70:27–44. Petersen, H. H., J. P. Nielsen, and P. M. H. Heegaard. 2004. Application of acute phase protein measurement in veterinary clinical chemistry. Vet. Res. 35:163–187. Pineiro, M., C. Pineiro, R. Carpintero, J. Morales, F. M. Campbell, P. D. Eckersall, M. J. M. Toussaint, and F. Lampreave. 2007. Characterisation of the pig acute phase protein response to road transport. Vet. J. 173:669–674. Qiu, X., J. D. Arthington, D. G. Riley, C. C. Chase Jr., W. A. Phillips, S. W. Coleman, and T. A. Olson. 2007. Genetic effects on acute phase protein response to the stresses of weaning and transportation in beef calves. J. Anim. Sci. 85:2367–2374. Salamano, G., E. Mellia, M. Tarantola, M. S. Gennero, L. Doglione, and A. Schiavone. 2010. Acute phase proteins and heterophil: lymphocyte ratio in laying hens in different housing systems. Vet. Rec. 167:749–751. Shini, S., and P. Kaiser. 2009. Effects of stress mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes. Stress 12:388–399. Simon, J. 1984. Effects of daily corticosterone injections upon plasma glucose, insulin, uric acid and electrolytes and food intake pattern in the chicken. Diabete Metab. 10:211–217. Soleimani, A. F., I. Zulkifli, M. Hair-Bejo, A. R. Omar, and A. R. Raha. 2012b. The role of heat shock protein 70 in resistance to Salmonella enteritidis in broiler chickens subjected to neonatal feed restriction and thermal stress. Poult. Sci. 91:340–345. Soleimani, A. F., I. Zulkifli, A. R. Omar, and A. R. Raha. 2011. Neonatal feed restriction modulates circulating levels of corticosterone and expression of glucocorticoid receptor and heat shock protein 70 in aged Japanese quail exposed to acute heat stress. Poult. Sci. 90:1427–1434. Soleimani, A. F., I. Zulkifli, A. R. Omar, and A. R. Raha. 2012a. The relationship between adrenocortical function and hsp70 expression in socially isolated Japanese quail. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 161:140–144. Tomas, F. M., A. J. Murray, and L. M. Jones. 1984. Interactive effects of insulin and corticosterone on myofibrillar protein turnover in rats as determined by N tau-methylhistidine excretion. Biochem. J. 220:469–479. van Gool, J. V., W. Boers, M. Sala, and N. C. Ladiges. 1984. Glucocorticoids and catecholamines as mediators of acute-phase proteins, especially rat alpha-macrofoetoprotein. Biochem. J. 220:125–132. van Gool, J., H. van Vugt, M. Helle, and L. A. Aarden. 1990. The relation among stress, adrenalin, interleukin 6, and acute phase proteins in the rat. Clin. Immunol. Immunopathol. 57:200–210. Zulkifli, I., M. T. Che Norma, D. A. Israf, and A. R. Omar. 2002. The effect of early age food restriction on heat shock protein 70 response in heat-stressed female broiler chickens. Br. Poult. Sci. 43:141–145. Zulkifli, I., A. F. Soleimani, M. Khalil, and A. R. Omar. 2011. Inhibition of adrenal steroidogenesis and heat shock protein 70 induction in neonatally feed restricted broiler chickens under heat stress condition. Arch. Geflugelkd. 75:246–252.
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Deane, E. E., S. P. Kelly, C. K. M. Lo, and N. Y. S. Woo. 1999. Effects of GH, prolactin and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba. J. Endocrinol. 161:413–421. Dupont, J., M. Derouet, J. Simon, and M. Taouis. 1999. Corticosterone alters insulin signaling in chicken muscle and liver at different steps. J. Endocrinol. 162:67–76. Etches, R. J., I. M. John, and A. M. V. Gibbins. 1995. Behavioural, physiological, neuroendocrine and molecular responses to heat stress. Pages 31–65 in Poultry Production in Hot Climates. N. J. Daghir, ed. CABI Publishing, Wallingford, UK. Gabay, C., and I. Kushner. 1999. Acute phase proteins and other systemic responses to inflammation. N. Engl. J. Med. 340:448– 454. Gauldie, J., C. Richards, D. Harnish, P. Lansdorp, and H. Baumann. 1987. Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc. Natl. Acad. Sci. USA 84:7251–7255. Gething, M. J., and J. Sambrook. 1992. Protein folding in the cell. Nature 355:33–45. González, F. H. D., F. Tecles, S. Martínez-Subiela, A. Tvarijonaviciute, L. Soler, and J. J. Cerón. 2008. Acute phase protein response in goats. J. Vet. Diagn. Invest. 20:580–584. Higuchi, H., N. Katoh, T. Miyamoto, E. Uchida, A. Yuasa, and K. Takahashi. 1994. Dexamethasone-induced haptoglobin release by calf liver parenchymal cells. Am. J. Vet. Res. 55:1080–1085. Hull, K. L., J. F. Cockrem, J. P. Bridges, E. J. Candy, and C. M. Davidson. 2007. Effects of corticosterone treatment on growth, development, and the corticosterone response to handling in young Japanese quail (Coturnix coturnix japonica). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 148:531–543. Jones, T. J., D. Li, I. Wolf, S. A. Wadekar, S. Periyasamy, and E. R. Snchez. 2004. Enhancement of glucocorticoid receptor-mediated gene expression by constitutively active heat shock factor 1. Mol. Endocrinol. 18:509–520. Klasing, K. C., D. E. Laurin, R. K. Peng, and D. M. Fry. 1987. Immunologically mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin-1. J. Nutr. 117:1629–1637. Kushner, I., and A. Mackiewicz. 1993. Acute Phase Proteins: Molecular Biology, Biochemistry and Clinical Applications. CRC Press, Boca Raton, FL. Lin, H., S. J. Sui, H. C. Jiao, J. Buyse, and E. Decuypere. 2006. Impaired development of broiler chickens by stress mimicked by corticosterone exposure. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 143:400–405. Mackiewicz, A., M. K. Ganapathi, D. Schultz, and I. Kushner. 1987. Monokines regulate glycosylation of acute-phase proteins. J. Exp. Med. 166:253–258. Mahmoud, K. Z., F. W. Edens, E. J. Eisen, and G. B. Havenstein. 2004. Ascorbic acid decreases heat shock protein 70 and plasma corticosterone response in broilers (Gallus gallus domesticus) subjected to cyclic heat stress. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 137:35–42. Mancini, G., A. O. Carbonara, and J. F. Heremans. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235–254. Marinkovic, S., G. P. Jahreis, G. C. Wong, and H. Baumann. 1989. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo. J. Immunol. 142:808–812. Martinez-Subiela, S., P. J. Ginel, and J. J. Ceron. 2004. Effects of different glucocorticoid treatments on serum acute phase proteins in dogs. Vet. Rec. 154:814–817. Martinez-Subiela, S., F. Tecles, and J. J. Ceron. 2007. Comparison of two automated spectrophotometric methods for ceruloplasmin measurement in pigs. Res. Vet. Sci. 83:12–19.
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Key words: acute phase protein, interleukin 6, heat shock protein 70, corticosterone, broiler 2014 Poultry Science 93:1–7 http://dx.doi.org/10.3382/ps.2014-04099
INTRODUCTION
response was noted 72 h after CRH administration. Working with rats, van Gool et al. (1990) demonstrated a relationship among stress, adrenalin, IL-6, and APR, and it is known that corticosteroid has effects on the APP in dogs (Martinez-Subiela et al., 2004; Caldin et al., 2009). The APP in poultry are classified as positive APP because they show an increase in response to challenge. The positive APP include ceruloplasmin (CPN), ovotransferrin (OVT), and α1-acid glycoprotein (AGP). α1-Acid glycoprotein is well known as an immunoregulator affecting T-cell function and providing negative feedback on the APR, whereas ceruloplasmin is documented to have more protective effects by removing oxygen radicals, antihistamine activity, and reversing the hypoferremic state of the APR (Murata et al., 2004). Ovotransferrin is an iron binding protein and could provide antimicrobial properties by sequestering iron, and has been shown to modulate heterophil and macrophage function in chickens (Murata et al., 2004). Acute-phase response may be considered as part of the general physiological stress response, which involved the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic system (Caldin et al., 2009). Although
Acute phase proteins (APP) are a group of blood proteins primarily synthesized in the liver as part of the acute-phase response (APR). The APR is a core part of the innate immune system response to harmful stimuli such as tissue injury, and infection (González et al., 2008; O’Reilly and Eckersall, 2014). The goal of the APR is to reestablish homeostasis. Stresses such as transportation, weaning, mixing with unfamiliar individuals, and housing on slippery floors have been reported to increase serum concentrations of APP in livestock (Alsemgeest et al., 1994; Pineiro et al., 2007; Qiu et al., 2007). Higuchi et al. (1994) reported that APR was stimulated in vitro by addition of glucocorticoids to cultures of bovine liver slices. Cooke et al. (2012) compared APR in steers receiving different doses of bovine corticotropin-releasing hormone (CRH). The hormone-elevated serum level of haptoglobin and peak
©2014 Poultry Science Association Inc. Received April 9, 2014. Accepted September 5, 2014. 1 Corresponding author: [email protected]
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ABSTRACT An experiment was conducted to determine the effect of corticosterone (CORT) administration on serum ovotransferrin (OVT), α1-acid glycoprotein (AGP), ceruloplasmin (CPN), and IL-6 concentrations, and brain heat shock protein (HSP) 70 expression in broiler chickens. From 14 to 20 d of age, equal numbers of birds were subjected to either (i) daily intramuscular injection with CORT in ethanol:saline (1:1, vol/vol) at 6 mg/kg of BW, or (ii) daily intramuscular injection with 0.5 mL ethanol:saline (1:1, vol/vol; control). Blood samples were collected before CORT treatment (14 d old), 3 and 7 d after CORT injections, and 4 d
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MATERIALS AND METHODS Birds, Husbandry, and Housing A total of 128 one-day-old female broiler chicks (Cobb) were obtained from a commercial hatchery and raised in an environmentally controlled room. Ambient temperature on d 1 was set at 32°C and then gradually reduced until 24°C by d 21. Chicks were assigned to groups of 8 to 16 battery cages. Chicks were fed commercial broiler diets. The light regimen was 23L:1D.
Growth Performance Individual BW and feed intake (cage basis) were recorded on d 14, 21, and 24, and feed conversion ratios (feed/gain; FCR) were calculated.
Experimental Treatment From 14 to 20 d of age (doa), equal number of birds were subjected to either (i) daily intramuscular injection with CORT (Tokyo Chemical Industry Co., Tokyo, Japan) in ethanol:saline (1:1, vol/vol) at 6 mg/kg of BW (Dupont et al., 1999), or (ii) daily intramuscular injection with 0.5 mL ethanol:saline (1:1, vol/vol; control). The birds were injected between 0900 to 1030 h. Body weight was recorded daily. At 14 doa, before CORT administration, blood samples were collected from 2 birds per cage (16 chicks per treatment group) via the wing vein for determination of serum levels of CORT, OVT, AGP, CPN, and IL-6. Following blood sampling, chickens were killed by decapitation and brain (whole cerebrum) samples were collected for determination of HSP 70 density (16 chicks per treatment group). Similar sampling procedures were repeated following 3 (17 doa) and 7 (20 doa) d of CORT injection and 4 d (24 doa) following cessation of CORT administration. The sampling on the third and seventh days of CORT administration was carried out 1 h postinjection. All experimental procedures were conducted in accordance with Universiti Putra Malaysia Research Policy on animal care.
Laboratory Analyses The CORT was measured by a commercial ELISA kit (IDS, Boldon, UK). The intra- and interassay variability for this kit were less than 6.7% and less than 7.8%, respectively, and the detection limit was 27 ng/ mL. The AGP concentration was determined by using a commercial ELISA kit specific to chicken (NB-E60049, Life Diagnostics Inc., West Chester, PA). The radial immunodiffusion method, modified from Mancini et al. (1965), was used to measure OVT. Briefly, 1% agarose gel (Sigma A9539) was prepared (0.13 g of agarose in 13 mL of Tris-buffered saline in a water bath at 56°C), and 260 µL of rabbit anti-chicken transferrin antibody (RabMAbs Abcam, Cambridge, MA) was added to the mixture and poured onto a gel membrane (Flow-Mesh, Sigma Aldrich, St. Louis, MO) under room temperature. Nine wells were punched in each gel, and 10 µL of standard or serum samples was loaded in each well. The OVT standards were prepared at 0, 0.078, 0.3125, 1.250, and 5 mg/mL. Gels were incubated in a dark and humid environment for 48 h. Following incubation, the size of the ring around each well was measured and calculated against standards. The concentration of CPN is determined by the rate of formation of a colored product from CPN and the substrate, 1,4-phenylenediamine dihydrochloride, according to the procedure of Martinez-Subiela et al. (2007). Briefly, 20.375 g of sodium acetate trihydrate was dissolved in 250 mL of distilled water and adjusted to pH 6.2 using glacial acetic acid. Then, 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519) was added to the prepared buffer and kept in the dark
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the avian APR during infection and inflammation has been well documented (Chamanza et al., 1999; O’Reilly and Eckersall, 2014), little information is available on the effect of sterile stress (noninfectious stress) on APP in poultry. Activation of APR by nonpathogenic stimulus is still unclear in poultry species. When living organisms are exposed to thermal and nonthermal stressors, the synthesis of most proteins is retarded, but a group of highly conserved proteins known as heat shock proteins (HSP) is rapidly synthesized. In a heat shock cell, the HSP may bind to proteins to protect them from degradation (Soleimani et al., 2012a), or may prevent damaged proteins from immediately precipitating and permanently affecting cell viability (Etches et al., 1995). Heat shock proteins have been shown to play a profound role in regulating protein folding and in coping with proteins affected by heat and other stresses (Gething and Sambrook, 1992). Work in chickens indicated that feed restriction (Zulkifli et al., 2002, 2011; Soleimani et al., 2011), crating and road transportation (Al-Aqil and Zulkifli, 2009), and social isolation (Soleimani et al., 2012a) augmented HSP 70 expression. The HSP response to various stressors suggests that the proteins could be mediated by the hypothalamic-pituitary-adrenal axis. Soleimani et al. (2011) studied the effects of neonatal stress on HSP 70 expression, circulating levels of corticosterone (CORT), and hippocampal glucocorticoid receptor in aged Japanese quail. The authors concluded that reduced hippocampal glucocorticoid receptor in aged birds resulted in marked inability to terminate CORT secretion at the end of stress and to induce HSP 70 expression. Because stress appears to be associated with all the above conditions, the objective of this study was to determine the effect of a stress hormone CORT administration on serum levels of CPN, OVT, AGP, and IL-6, and expression of HSP 70 in broiler chickens. In this study, the administration of CORT was employed to mimic the physiological stress response in broiler chickens.
BROILER CHICKENS ADMINISTERED CORTICOSTERONE
Statistical Analysis All data were subjected to ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Blood parameters and feed intake data were analyzed using CORT administration, time, and their interactions as main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. For feed intake, CORT administration was the only main effect in the analysis. When significant effects were found, comparisons among multiple means were made by Duncan’s multiple range test. Statistical significance was considered as P < 0.05.
RESULTS There were significant (P < 0.05) CORT administration × time interactions for all the blood parameters and HSP 70 data. Injection with CORT significantly (P = 0.0015) increased CORT (Figure 1) and depressed (P = 0.0061) the BW (Figure 2A) on d 17, 20, and 24 of age. The birds that received CORT injection had significantly (P = 0.0041) higher feed intake compared with their control counterparts (Figure 2B). The serum APP levels of OVT (Figure 3A) and AGP (Figure 3B) were significantly increased after 3 (P = 0.0002) and 7 (P = 0.0001) d of CORT injection. Four days following termination of CORT administration (d 24), serum level of OVT and AGP significantly decreased.
Figure 1. Time course changes in serum corticosterone concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
However, only OVT returned to the basal level and AGP still remained significantly (P = 0.0130) higher than the control. Interestingly, CPN was increased only after 7 d of CORT administration (Figure 3C). Serum levels of IL-6 (Figure 4) and brain HSP 70 (Figures 5 and 6) were constantly elevated (P = 0.0001) by CORT administration. Following 4 d of injection recovery, although IL-6 and HSP 70 declined significantly, they still remained higher than the control. Prior to CORT administration, the CORT (Figure 1), OVT, AGP, and CPN (Figure 3A, B, and C), IL-6 (Figure 4), and HSP 70 (Figure 5) concentrations of control and treatment group chicks were not significantly (P = 0.3201, 0.6312, 0.5112, 0.7131, 0.4701, and 0.6112, respectively) different.
DISCUSSION As expected, injection with CORT resulted in a significantly higher CORT. Delivery of CORT by daily injections has also been used by Simon (1984) and Buyse et al. (1987). In the present experiment, the significantly suppressed BW gain in the CORT-treated birds when compared with controls is in accordance with those of Lin et al. (2006) and Hull et al. (2007). The lower weight gain in CORT birds could be attributed to enhanced glycogenolysis/gluconeogenesis (Lin et al., 2006) and protein catabolism (Tomas et al., 1984). The effect of CORT treatment on weight gain in poultry is age dependent. Covasa and Forbes (1995) reported that CORT treatment for 5 d depressed weight gain in 2-wkold but not 5-wk-old chickens. Work in Japanese quail showed that reduction in weight gain following CORT treatment was less marked after 3 wk of age (Hull et al., 2007). Bürger et al. (1998) administered piglets with adrenocorticotrophin hormone (ACTH) or cortisol daily
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for a minimum of 45 min. One hundred microliters of the above buffer and 10 µL of samples or standards were added to each microplate well, shaken gently and kept in the dark for 20 min. The absorbance was recorded spectrophotometrically using a microplate reader at 550 nm. Standards were prepared with serial dilution of pig serum of known CPN concentration calibrated against purified CPN (Sigma Chemical Co.) and saline buffer combination to achieve various concentration of 12.75 (20 µL of pig serum + 60 µL of saline buffer), 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 mg/ mL of CPN. The IL-6 was measured by a commercial ELISA kit specific to chicken (NB-E60049, Novateinbio, Cambridge, MA). The standard range was 3.2 to 100 pg/mL, and the detection limit was 0.5 pg/mL. The level of HSP 70 protein expression was determined as previously described (Soleimani et al., 2012b) using SDS-PAGE and Western blotting with some modifications. Briefly, 0.3 g of cerebrum samples was homogenized with 1.5 mL of protein extraction buffer (20 mM Tris, pH 7.5; 0.75 M sodium chloride) and 10 µL/mL of protease inhibitor cocktail (Precision Plus Protein, Bio-Rad, Hercules, CA), and centrifuged at 20,000 × g for 30 min at 4°C. The supernatant were separated and the total protein quantity was measured using bicinchoninic acid protein assay kit (Sigma Chemical Co.). The final brain HSP 70 concentration was calculated as arbitrary unit of band density relative to total protein concentration of each sample.
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for 2 wk. The authors noted a significant increase in C-reactive protein following 7 d of ACTH or cortisol injection. Work in beef steers suggested that intravenous treatment with bovine corticotrophin-releasinghormone elevated plasma CRP at 54 h, whereas plasma haptoglobin concentrations were greater at 54, 66, and 72 h compared with prechallenge values (Cooke and Bohnert, 2011). The authors, however, noted a decline in plasma ceruloplasmin level at 2 and 6 h. A single injection of adrenalin has been shown to increase the level of α-2 macroglobulin, a typical APP in rats (van Gool et al., 1984). Most APP are constitutively present in the serum; however, their concentrations change markedly in the event of inflammation and infection (Petersen et al., 2004). In the present study, significant elevations in OVT and AGP were noted following 4 d (d 17) of CORT treatment. Under normal conditions, AGP exists in low concentrations in serum, also masked by albumin, which makes it difficult to detect (Conner et al., 1990). It can be concluded that CORT administration may induce APR in poultry. Another impor-
Figure 3. Time course changes in serum ovotransferin (A; OVT), α1-acid glycoprotein (B; AGP), and ceruloplasmin (C; CPN) concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
tant finding from this study is confirmation that CPN, OVT, and AGP acted as positive APP in poultry as in mammals. To our knowledge, this is the first study reporting stimulatory effects of CORT treatment on APR in avian species. It is interesting to note that CPN was only affected by CORT treatment on d 20. Thus, despite a dramatic increase in CORT, 4 d of CORT administration and stress attributed to handling and injection did not elicit
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Figure 2. Mean BW (n = 32; A) and feed intake (n = 8; B) of broiler chickens subjected to daily injection of corticosterone for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age) by corticosterone treatment. Means within a treatment group with no common letters (a,b) differ at P < 0.05.
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any change in CPN. It appears that OVT and AGP are more sensitive to stressful stimuli than CPN. The use of AGP as a measure of poultry well-being has been reported. Salamano et al. (2010) reported that hens kept in both conventional and modified battery cages had higher AGP than those in the free-range system. Following 7 d of CORT injection, the serum levels of CPN, OVT, and AGP showed dramatic elevations. Studies in humans on APP such as serum amyloid A and C-reactive protein suggested that the serum levels of these proteins increased markedly within 4 h after inflammatory stimulus and rapid normalizations (Mackiewicz et al., 1987; Gabay and Kushner, 1999). Haptoglobin is characterized by a later increase in serum concentration remaining elevated for up to 2 wk. The significance of elevation in serum levels of APP during stress is in question. The APR response is considered as part of the innate immune response and is observed across all animal species (Cray et al., 2009). The APR will induce a complex systemic reaction to reestablish homeostasis and promote healing (Cray et al., 2009). An increase in serum APP concentrations may be considered as an indicator of intracellular communication, suggesting an increase in the cellular im-
Figure 5. Time course changes in brain heat shock protein 70 (HSP 70) following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–c) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
mune response (Cray et al., 2009). However, the APR could be biologically costly. Studies in cattle suggested that higher serum concentrations of APP were associated with poor growth and reproductive performance (Carroll and Forsberg, 2007; Cooke et al., 2009). Thus, the noted lower weight gain of CORT birds compared with controls could be associated with active cellular immune reaction during APR (Klasing et al., 1987). Work with piglets demonstrated a significant decline in plasma levels of C-reactive proteins 7 d after daily injection with ACTH or cortisol was terminated (Bürger et al., 1998). The present findings suggested that despite the elevated CORT, CPN and OVT returned to basal levels following 4 d (d 24) of CORT injection cessation. Although the AGP declined on d 24, the level did not return to the basal value. According to Petersen et al. (2004), the APR was detectable for several days after the stimulus but the kinetics of the response varied according to species involved and on the extent of tissue damage (Kushner and Mackiewicz., 1993). Administration of corticosteroid (prednisolone) to dogs causes an increase in the APP haptoglobin but
Figure 6. Time course changes in heat shock protein 70 density following daily saline (control) or corticosterone (CORT) administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age).
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Figure 4. Time course changes in serum IL-6 concentrations following daily saline (control) or corticosterone administration for d 0 (14 d of age), d 3 (17 d of age), d 7 (20 d of age), and d 11 (24 d of age). Means (n = 16) within a treatment group with no common letters (a–d) differ at P < 0.05. *Significant difference between treatment groups (P < 0.05).
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In conclusion, the changes in serum CPN, OVT, and AGP concentrations following CORT treatment suggested that APP can be used to gauge physiological stress in avian species and are thus another biomarker for well-being. On a cautionary note, however, it is important to establish that the birds are not influenced by other stimuli such as trauma, infection, neoplasia, or inflammation. A combination of APP and HSP 70 may be a more reliable indicator of stress in chickens.
ACKNOWLEDGMENTS This research was funded by the Malaysian Ministry of Science, Technology and Innovation, Putrajaya.
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not in the concentration of CRP (Martinez-Subiela et al. (2004), so there may also be differing effects of corticosterones on APP in chicken. The mechanisms by which nonpathogenic stimuli evoke APR in poultry are not clear. The earlier discussion suggests that elevation in CORT can increase AGP, OVT, and CPN in poultry. Work in rodents suggested a relationship among stress, IL-6, and APP (van Gool et al., 1990; Shini and Kaiser, 2009). Interleukin-6 is a pro-inflammatory signal. Both in vivo and in vitro studies demonstrated that IL-6 is an important mediator in the synthesis of APP (Gauldie et al., 1987; Moshage et al., 1988; Marinkovic et al., 1989). Clinical situations such as burns, endotoxemia, meningitis, and sepsis have been reported to elicit a marked increase in IL-6 (van Gool et al., 1990). All these conditions involved a generalized stress response. The present findings clearly showed that CORT treatment elevated plasma level of IL-6 in chickens. It can be concluded that increase in CORT elicited a pro-inflammatory cytokine response. Similar findings have been reported in beef steers infused with CRH (Cooke and Bohnert, 2011). This is interesting because CORT and cortisol are well known to have anti-inflammatory action (Barnes and Adcock, 1993). According to Higuchi et al. (1994), acute increases in circulating cortisol, such as during a stress challenge, can indirectly stimulate an inflammatory response. During stress, corticosteroids will degrade body tissues to help animals cope with stressors and restore homeostasis. On the contrary, tissue degradation is recognized by the innate immune system as a disruption of homeostasis (Abbas and Lichtman, 2007) and thus leukocytes will synthesize pro-inflammatory cytokines such as IL-6. Injecting rats with adrenalin but not CORT triggered elevation in the plasma IL-6 level (van Gool et al., 1990). It has been documented that both thermal and nonthermal stressors can induce HSP 70 expression in animals (Zulkifli et al., 2002; Al-Aqil and Zulkifli, 2009). Mahmoud et al. (2004) reported a positive correlation of CORT and heart HSP 70 expression in broiler chickens subjected to cyclic heat stress. On the contrary, Deane et al. (1999) showed that daily injection of cortisol into silver sea breams had a negligible effect on liver HSP 70 expression. Soleimani et al. (2012a) also reported that feeding Japanese quail with 30 mg/kg of CORT for 3 d did not affect HSP 70 level in the brain and heart. The findings of the current study demonstrated that daily injection of CORT for 4 and 7 d significantly increased HSP 70 expression in the brain. Thus, the increased HSP 70 density in the brain is directly related to circulating CORT. The inconsistency in findings on corticosteroid treatment and HSP 70 expression could be attributed to variations in age and species or as proposed and elaborated by Jones et al. (2004), it may be explained by reciprocal regulation of glucocorticoid receptor and heat shock factor 1 signaling. The authors defined a dose- and time-dependent response of HSP 70 to glucocorticoid action.
BROILER CHICKENS ADMINISTERED CORTICOSTERONE
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