Inhibition of corticosterone synthesis and its effect on acute phase proteins, heat shock protein 70, and interleukin-6 in broiler chickens subjected to feed restriction

Inhibition of corticosterone synthesis and its effect on acute phase proteins, heat shock protein... Abstract The aim of the current study was to elucidate whether inhibition of corticosterone (CORT) synthesis could modify stress response to feed deprivation and its possible interactions with feed restriction in the neonatal period in broiler chickens. Equal numbers of broiler chicks were subjected to either 60% feed restriction (60FR) or ad libitum (AL) on d 4, 5, and 6. On day 7, blood CORT, acute phase proteins (APP), interleukin-6 (IL-6) levels, and brain heat shock protein 70 (HSP70) expression were determined. On d 35, chickens in each early age feeding regimen were subjected to one of the following treatments: (i) ad libitum feeding (ALF), (ii) 24 h feed deprivation (SFR), or (iii) 24 h feed deprivation with intramuscular injection of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) at 100 mg/kg BW (SFR+DDT). The effect of SFR on CORT, APP, IL-6, and HSP 70 were determined on d 36. The results showed that subjecting chicks to 60FR significantly elevated CORT and brain HSP70 concentration compared to the AL group on d 7. The early feeding regimen had no significant effect on CORT, alpha-1 acid glycoprotein (AGP), ovotransferrin (OVT), ceruoplasmin (CP), IL-6, or brain HSP70 on d 36. The CORT, AGP, OVT, CP, IL-6, and brain HSP70 expression of SFR birds following 24 h of feed deprivation (d 36) were significantly higher than their ALF and SFR+DDT counterparts. Both ALF and SFR+DDT birds had similar values. Stress attributed to feed deprivation without concurrent increase in CORT had a negligible effect on serum levels of APP and IL-6 and brain HSP70 expression. INTRODUCTION When birds are exposed to noxious stimuli the hypothalamic-pituitary-adrenal axis (HPA) will be evoked and leads to an increase in the synthesis and release of adrenal glucocorticoids (mainly corticosterone, CORT) into the bloodstream. Elevation in circulating levels of glucocorticoids in response to stresses is crucial to restore homeostasis (Sapolsky et al., 2000). Zulkifli et al. (1994a,b, 2000) demonstrated that stress attributed to early age feed restriction enhanced stress tolerance later in life when compared to that fed ad libitum throughout. However, stressful experiences during the neonatal stage without simultaneous increases in the synthesis and release of CORT may not assist a bird in response to subsequent stressors (Zulkifli et al., 1994a). Thus, stress-induced increases in the synthesis of CORT may be critical to prepare a bird in coping with subsequent physiological disruptions (Soleimani et al., 2011; Zulkifli et al., 2011, 2012). The underlying mechanism of neonatal stress and enhancement of coping ability later in life in poultry has been associated with the ability to express heat shock protein 70 (HSP70) (Zulkifli et al., 2002). It is well documented that HSP play a profound role in modifying physiological stress response and in acquisition of stress tolerance (Kregel, 2002). Acute phase proteins (APP) are serum proteins primarily synthesized by hepatocytes as part of the acute phase response (APR). The APR is part of the early defense or innate immune system, which is elicited by a diversity of challenges such as inflammation, bacterial infection, endotoxin exposure, and tissue injury (Murata et al., 2004; O’Reilly and Eckersall, 2014). The APR results in a complex systemic reaction with the goal of re-establishing homeostasis and promoting healing. In poultry, stress caused by feed deprivation increased serum levels of APP ovotransferrin (OVT), ceruoplasmin (CP), and alpha-1 acid glycoprotein (AGP) (Najafi et al., 2016). The mechanism of APP elicitation in response to noxious stimuli is not fully understood. Murata et al. (2004) reported that pro-inflammatory cytokines such as interleukin-6 (IL-6) stimulated APP synthesis in the liver. They suggested that activation of the HPA axis by stressors may trigger IL-6 production and subsequently the release of APP into the bloodstream. Zulkifli et al. (2014) showed significant elevations in OVT, AGP, CP, plasma IL-6, and brain HSP70 expression in chickens treated with exogenous CORT. Hence, it appears that circulating CORT modulates the elicitation of HSP70, APP, and IL-6 responses. On the contrary, Soleimani et al. (2011) reported that feeding Japanese quail with 30 mg/kg CORT did not affect HSP70 expression in the brain or heart. Similarly, Deane et al. (1999) showed that daily injection of cortisol into silver sea breams had a negligible effect on liver HSP70 expression. Murata et al. (2004), in their review, postulated that stress signal, independent of the HPA axis, also may induce macrophages to produce IL-6 and thereby elevate hepatic production of APP. It appears that the interrelationship between CORT, as the main endocrine response to stress, and the other physiological stress responses, such as HSP 70, APP, and IL-6, is still not clear. Moreover, the possible interaction of such a relationship with neonatal exposure to stress also warrants further investigation. Therefore, the objectives of this study were: (i) to determine the effect of 24 h feed restriction in neonatal chicks on CORT, HSP70, APP, and IL-6, (ii) to elucidate whether inhibition of CORT synthesis could modify stress response to feed deprivation in 36-day-old chickens, and (iii) to evaluate whether stress during the neonatal stage may modify the impact of inhibiting CORT synthesis on stress response in broiler chickens subjected to 24 h of feed deprivation later in life. MATERIALS AND METHODS Birds, Husbandry, and Housing All experimental procedures were conducted in accordance with the University of Putra Malaysia Research Policy on animal care. A total of 192 one-day-old female Cobb 500 broiler chicks was obtained from a local hatchery. At one d of age (d 1), the chicks were weighed and randomly allocated into groups of 8 to 24 battery cages with wire floors 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. The chicks were fed commercial broiler diets. The lighting regimen was 18L:6D. Experimental Treatment On d 4, 5 and 6, 12 cages of birds were assigned to 60% feed restriction (60FR), while the other 12 cages were fed ad libitum (AL). The feed restricted group received 60% of the feed intake of the AL group on the previous day. On d 7 at 08:00 h (before feeding), 12 birds (one per cage) from each feeding regimen were randomly chosen and removed with minimum disturbance to flock mates. Immediately following the capture, birds were decapitated and blood samples were collected for serum separation. The serum samples were stored at -80°C awaiting analysis for CORT, OVT, AGP, CP, and IL-6. Following the blood sampling, brain (whole cerebrum) samples were collected for determination of HSP expression. On d 35, birds in AL and 60FR groups were subjected to either of the following treatments (4 cages per group per treatment): (i) ad libitum feeding (ALF), (ii) 24 h of feed deprivation (SFR), or (iii) 24 h feed deprivation with intramuscular injection of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (Sigma Aldrich Chemical Co., Saint Louis, MO) dissolved in corn oil at 100 mg/kg BW (Adamsson et al., 2009) (SFR+DDT). The birds were injected at 20:00 h, which was 12 h before starting the feeding regimen (Gross, 1990). The adrenal blocking effect of DDT in chickens has been reported previously (Colmano and Gross, 1971; Srebocan et al., 1971; Gross and Chickering, 1987; Gross, 1990; Jonsson et al., 1994). The chemical, inhibits conversion of deoxycorticosterone to CORT via inhibiting 11β-hydroxylase activity (Kenaga, 1966). The DDT dosage used in the present study was based on a pilot study in our laboratory. Dosage of 100 mg/kg BW was the most optimal level to block the synthesis of CORT in broiler chickens subjected to feed deprivation for 24 hours. The ALF and SFR groups were injected intramuscularly with 0.5 mL corn oil. Similar sampling procedures (12 birds per treatment group) as described earlier were repeated following the 24 h (d 36) feed deprivation. CORT and IL-6 Assays Plasma CORT was determined using a commercial high-sensitivity EIA kit (AC-15F1, IDS, Boldon, UK). The intra- and inter-assay variabilities were less than 6.7% and 7.8%, respectively; and the detection limit was 27 ng/mL. The protocol of analysis was according to the manufacturer's recommendations. 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 samples of different d and treatments were analyzed mixed in the same batches. APP Assay AGP concentration was determined by using a commercial ELISA kit specific to chicken (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 TBS 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-MeshTM, Sigma Aldrich, St. Louis, MO) at room temperature. Nine wells were punched in each gel, and 10 μl of standard or serum samples were loaded in each well. OVT standards (albumin from chicken egg white, Sigma Aldrich, St. Lois, MO) 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 hours. Following incubation, the size of the ring around each well was measured and calculated against standards. The concentration of CP is determined by the rate of formation of a colored product from CP 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 were dissolved in 250 mL distilled water and adjusted to pH 6.2 using glacial acetic acid. 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519) were added to the prepared buffer and kept in the dark for a minimum of 45 minutes. One hundred μl 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 minutes. The absorbance was recorded spectrophotometrically using a microplate reader at 550 nm. Standards were prepared with serial dilution of pig serum of known CP concentration calibrated against purified CP (Sigma Chemical Co St. Louis, MO) and saline buffer combination to achieve various concentrations of 12.75 (20 μl pig serum + 60 μl saline buffer), 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 mg/mL CP. SDS-PAGE and Immunoblot Analysis for HSP70 Expression The levels of HSP70 protein expression were determined as previously described (Soleimani et al., 2012) with some modification. Briefly, brain samples (0.3 g, whole cerebrum) were homogenized with 1.5 mL of protein extraction buffer (20 Mm Tris, pH 7.5; 0.75 M sodium chloride) and 10 μl/ml protease inhibitor cocktail (P8340, Sigma Chemical Co., St. Louis) and centrifuged at 20,000 g for 30 min at 4°C. The supernatant was separated, and the total protein was measured using a bicinchoninic acid protein assay kit (B9643, Sigma Chemical Co., St. Louis, MO). Total protein (25 μg) was loaded and separated on 10% polyacrylamide gels containing SDS. Gels were electrophoresed at 120 V until the tracking dye reached the base of the gel, and the fractionated proteins were transferred to polyvinylidene difluoride membranes (MSI, Westborough, MA) using a trans-blot semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA). The membranes were incubated for 1 h with 5 mL of blocking buffer containing monoclonal mouse antibody (ab6535, Abcam, Cambridge, MA) against HSP70 in a 1:20,000 dilution. The membranes were washed 3 times (5 min each) with 10 mL of cold tris-buffered saline Tween 20 and incubated in a horseradish peroxidase conjugated rabbit anti-mouse secondary antibody for 30 min in a 1:40,000 dilution (ab6728, Abcam, Cambridge, MA). Membranes were washed again and HSP70 protein bands were visualized colorimetrically with the DAB Substrate System (E885, AMRESCO LLC, Solon, OH). The protein size was confirmed using Precision Plus Protein™ Dual Color Standard (Bio-Rad, Hercules, CA). The final HSP70 concentration was calculated as an arbitrary unit of band density relative to total protein concentration of each sample. Statistical Analysis Data were subjected to ANOVA using the GLM procedure of SAS (SAS, 2005). Early feeding regimen (AL and 60FR) was the only main effect to analyze data measured on d 7. Data for traits measured on d 36 were analyzed using early feeding regimen (AL and 60FR), late feeding regimen (ALF, SFR, and SFR+DDT), and their interactions as main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. When significant effects were found, comparisons among multiple means were modeled by Duncan's multiple-range test. Statistical significance is considered as P < 0.05. RESULTS Subjecting chicks to 60FR significantly elevated CORT (P < 0.001) and brain HSP70 (P = 0.0036) concentration when compared to the AL group on d 7. There were no significant effects of early age feeding regimen on AGP, OVT, CP, or IL-6 (P = 0.3481; 0.1384; 0.6173; 0.1053, respectively) (Table 1). The early feeding regimen had no significant effect on CORT, AGP, OVT, CP, IL-6, or brain HSP70 concentration on d 36 (P = 0.6307, 0.6928, 0.8406, 0.2376, 0.2376, 0.0536, 0.5922, respectively). The CORT, AGP, OVT, CP, IL-6, and brain HSP70 expression of SFR birds following 24 h (d 36) of feed deprivation were significantly higher than their ALF and SFR+DDT counterparts (Table 2). Both ALF and SFR+DDT birds had similar values for all the traits measured (Table 2). There was no significant interaction for any of the traits measured on d 36. A representative blot image of HSP70 expression is illustrated in Figure 1. The gel image representing the effect of early and late feeding regimens on serum OVT level is presented in Figure 2. Figure 1. View largeDownload slide Representative blot image of brain heat shock protein 70 (HSP70) expression as affected by early feeding regimen (AL vs. 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 1. View largeDownload slide Representative blot image of brain heat shock protein 70 (HSP70) expression as affected by early feeding regimen (AL vs. 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 2. View largeDownload slide Representative gel image of serum ovotransferrin (OVT) as affected by early feeding regimen (AL vs 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 2. View largeDownload slide Representative gel image of serum ovotransferrin (OVT) as affected by early feeding regimen (AL vs 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Table 1. Effect of early feeding regimen on mean (±SEM) levels of serum corticosterone (CORT) (ng/mL), α1-acid glycoprotein (AGP) (mg/mL), ceruloplasmin (CP) (mg/mL), ovotransferrin (OVT) (mg/mL) concentrations, interleukin 6 (IL-6), and brain heat shock protein 70 (HSP70) expression in broiler chickens at 7 d of age. Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1               AL  0.68 ± 0.37b  0.89 ± 0.78  0.18 ± 0.08  0.11 ± 0.03  0.12 ± 0.11  1.12 ± 0.45b   60FR  1.61 ± 0.49a  1.14 ± 0.41  0.31 ± 0.25  0.12 ± 0.08  0.21 ± 0.12  2.47 ± 1.35a  Analysis of variance  Probabilities   Feeding regimen  <.0001  0.3481  0.1384  0.6173  0.1053  0.0036  Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1               AL  0.68 ± 0.37b  0.89 ± 0.78  0.18 ± 0.08  0.11 ± 0.03  0.12 ± 0.11  1.12 ± 0.45b   60FR  1.61 ± 0.49a  1.14 ± 0.41  0.31 ± 0.25  0.12 ± 0.08  0.21 ± 0.12  2.47 ± 1.35a  Analysis of variance  Probabilities   Feeding regimen  <.0001  0.3481  0.1384  0.6173  0.1053  0.0036  a,bMeans within a column with no common letters differ at P < 0.05. 1AL = fed ad libitum; 60FR = 60% of fed ad libitum. n = 12. View Large Table 2. Effect of early and late feeding regimen on mean (±SEM) levels of serum corticosterone (CORT) (ng/mL), α1-acid glycoprotein (AGP) (mg/mL), ceruloplasmin (CP) (mg/mL), ovotransferrin (OVT) (mg/mL) concentrations, interleukin 6 (IL-6), and brain heat shock protein 70 (HSP70) expression in broiler chickens at 36 d of age. Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1   AL  0.98 ± 0.14  1.48 ± 0.25  0.52 ± 0.08  0.31 ± 0.04  0.98 ± 0.12  2.04 ± 0.28   60FR  1.04 ± 0.15  1.42 ± 0.27  0.52 ± 0.07  0.33 ± 0.05  1.21 ± 0.16  2.15 ± 0.32  Late feeding regimen2   ALF  0.61 ± 0.07b  0.78 ± 0.08b  0.28 ± 0.01b  0.15 ± 0.07b  0.67 ± 0.09b  1.13 ± 0.05b   SFR  1.86 ± 0.15a  2.89 ± 0.16a  1.03 ± 0.08a  0.59 ± 0.03a  1.94 ± 0.13a  3.92 ± 0.28a   SFR+DDT  0.58 ± 0.05b  0.67 ± 0.05b  0.27 ± 0.02b  0.21 ± 0.01b  0.68 ± 0.06b  1.23 ± 0.11b  Analysis of variance  Probabilities   Early feeding regimen  0.6307  0.6928  0.8406  0.2376  0.0536  0.5922   Late feeding regimen  <.0001  <.0001  <.0001  <.0001  <.0001  <.0001   Early feeding regimen × Late feeding regimen  0.6939  0.3071  0.8853  0.0586  0.1115  0.6174  Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1   AL  0.98 ± 0.14  1.48 ± 0.25  0.52 ± 0.08  0.31 ± 0.04  0.98 ± 0.12  2.04 ± 0.28   60FR  1.04 ± 0.15  1.42 ± 0.27  0.52 ± 0.07  0.33 ± 0.05  1.21 ± 0.16  2.15 ± 0.32  Late feeding regimen2   ALF  0.61 ± 0.07b  0.78 ± 0.08b  0.28 ± 0.01b  0.15 ± 0.07b  0.67 ± 0.09b  1.13 ± 0.05b   SFR  1.86 ± 0.15a  2.89 ± 0.16a  1.03 ± 0.08a  0.59 ± 0.03a  1.94 ± 0.13a  3.92 ± 0.28a   SFR+DDT  0.58 ± 0.05b  0.67 ± 0.05b  0.27 ± 0.02b  0.21 ± 0.01b  0.68 ± 0.06b  1.23 ± 0.11b  Analysis of variance  Probabilities   Early feeding regimen  0.6307  0.6928  0.8406  0.2376  0.0536  0.5922   Late feeding regimen  <.0001  <.0001  <.0001  <.0001  <.0001  <.0001   Early feeding regimen × Late feeding regimen  0.6939  0.3071  0.8853  0.0586  0.1115  0.6174  a,bMeans within a column-subgroup with no common letters differ at P < 0.05. 1AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. 2ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. View Large DISCUSSION As expected, 60FR resulted in significantly higher CORT and brain HSP70 expression on d 7 (Table 1). These results confirmed those of Soleimani et al. (2011) that neonatal feed restriction increased brain HSP70 expression and CORT. We noted that 60FR did not affect AGP, OVT, or CP in chicks on d 7. In agreement, Najafi et al. (2015) reported that feed restriction at 75, 60, 45, and 30% of ad libitum intake from d 28 to 42 d of age had negligible effect on AGP, OVT, and CP in broiler breeder pullets. Zulkifli et al. (1995) reported that White Plymouth Rock chicks subjected to neonatal feed restriction had lower heterophil to lymphocyte ratios (HLR) but similar CORT following 24 h feed deprivation at 36 d of age when compared to those fed ad libitum during the neonatal stage. The authors concluded that stresses early in life can evoke long-lasting changes in the physiological response to a stimulus. In the present study, the neonatal feed restriction did not modify CORT, OVT, AGP, CP, or IL-6 responses to 24 h of feed deprivation in 36-day-old broiler chickens. There is no clear explanation for the discrepancies, although they could be associated with the differences in breeds of chickens used in both studies. Zulkifli et al. (1994a) demonstrated that neonatal feed restriction improved stress tolerance in 43-day-old non-dwarf White Plymouth Rocks but not in their dwarf counterparts. Physiological stress reaction to deprivation also varied according to breed. Working with Light Sussex, Freeman et al. (1984) concluded that depriving food, water, or both for 24 h had negligible effect on CORT. On the contrary, Zulkifli et al. (1995) showed that 24 h of feed deprivation elevated HLR in 47-week-old White Leghorns. The present findings indicated that 24 h of feed deprivation was stressful to broiler chickens. The SFR chickens had higher CORT, HLR, OVT, AGP, CP, IL-6, and brain HSP70 expression than their ALF counterparts. On the contrary, Najafi et al. (2016) reported elevations in OVT, AGP, and CP only after 30 h of feed deprivation. There is no clear explanation for the discrepancy, although it may be associated with the variations in the ages of the experimental chickens. Najafi et al. (2016) used broiler chicks of 22 d of age, whereas we used 36-day-old chicks. Thus, APP could be used to gauge physiological stress in avian species and are thus another biomarker for well being. Alterations in serum levels of APP following fasting suggested the role of the proteins in restoring homeostasis in animals subjected to non-inflammatory, psychophysical stressors (Cray et al., 2009). Il-6 is a pro-inflammatory cytokine, having a key role in inflammatory responses via activation and regulation of other stimulated cells and tissues (Wigley and Kaiser, 2003). Zulkifli et al. (2014) demonstrated that administration of exogenous CORT elicited plasma IL-6 activity in chickens. The present findings indicated that stress associated with 24 h feed deprivation may increase serum IL-6 level. In vivo and in vitro findings suggested that IL-6 is an important mediator in the synthesis of APP (Moshage et al., 1988; Marinkovic et al., 1989). van Gool et al. (1990) and Shini and Kaiser (2009) suggested a relationship among stress, IL-6, and APP in rodents. The preceding discussion suggests that stress may stimulate the macrophages and Kupfer cells to release IL-6, which elicits the hepatocytes to synthesize APP. Murata et al. (2004), in their review, suggested that induction and regulation of APP during stress are mediated through the HPA axis and IL-6. The authors also postulated that internal and external challenges can directly evoke the production of IL-6. It appears that APP reaction can be elicited by stress without evoking adrenal cortical activity. In the present study, administration of DDT blocked CORT synthesis and suppressed IL-6 and APP responses following the 24 h feed deprivation. It appears that stress without concomitant increase in the synthesis and liberation of CORT may not trigger APP response in avian species. There is, however, a question as to whether suppression of IL-6 response is attributed to the direct effect of DDT or the secondary effect of inhibition of adrenal cortical activity. Because administration of exogenous CORT elevated IL-6 (Zulkifli et al., 2014) in poultry, it is possible that the suppressed IL-6 reaction in DDT-treated birds is a consequence of inhibition of CORT. Interestingly, although stress may elicit IL-6, the cytokine also has been reported to increase the release of ACTH in the pituitary, and the hormone stimulates glucocorticoid synthesis in the adrenal cortex (Heinrich et al., 1998). Sapolsky et al. (2000) proposed that the modulating actions of glucocorticoids can be permissive, suppressive, stimulating, and preparative, and, thus, animals with impaired ability to synthesize and liberate glucocorticoids may not be able to cope with stress as well as their normal counterparts. In the present study, our results are consistent with the stimulating role of glucocorticoids in which inhibition of the CORT by DDT, eliminated the APP, IL-6, and HSP70 synthesis after feed deprivation. The relationship between CORT and expression of HSP70 has been inconsistent in poultry. Mahmoud et al. (2004) reported a positive correlation between CORT and heart HSP70 expression in broiler chickens subjected to cyclic heat stress. On the contrary, Soleimani et al. (2012) reported that feeding Japanese quail with 30 mg/kg CORT for 3 d did not affect HSP70 level in the brain or heart. Zulkifli et al. (2014) demonstrated that daily injection of CORT for 4 and 7 d significantly increased HSP70 expression in the brain. The present findings clearly suggested that failure to increase CORT during perturbation of homeostasis may result in retardation of brain HSP70 expression. In conclusion, under the conditions of this study, feed deprivation for 24 h elevated CORT, IL-6, AGP, OVT, CP, and brain HSP70 expression in broilers. However, following injection of DDT as the CORT synthesis inhibitor, 24 h feed deprivation had negligible effects on APP and brain HSP70 expression. On a cautionary note, however, DDT also may influence the endocrine system in other ways (Grassle and Biessmann, 1982), and some of the effects observed in our study may have been associated with those changes. Acknowledgements This research was funded by the Malaysian Ministry of Science, Technology, and Innovation. REFERENCES Adamsson A., Salonen V., Paranko J., Toppari J.. 2009. Effects of maternal exposure to di-isononylphthalate (DINP) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p΄-DDE) on steroidogenesis in the fetal rat testis and adrenal gland. Reprod. Toxicol.  28: 66– 74. Google Scholar CrossRef Search ADS PubMed  Colmano G., Gross W. B.. 1971. Effect of metyrapone and DDD on infectious diseases. Poult. Sci.  50: 850– 854. Google Scholar CrossRef Search ADS PubMed  Cray C., Zaias J., Altman N. H.. 2009. Acute phase response in animals: A review. Comp. Med.  59: 517. Google Scholar PubMed  Deane E. E., Kelly S. P., Lo C. K., Woo N. Y.. 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. Google Scholar CrossRef Search ADS PubMed  Freeman B. M., Manning A. C. C., Flack I. H.. 1984. Changes in plasma corticosterone concentrations in the water-deprived fowl, Gallus domesticus. Comp. Biochem. Physiol. A Physiol.  79: 457– 458. Google Scholar CrossRef Search ADS   Grassle B., Biessmann A.. 1982. Effects of DDT, polychlorinated biphenyls and thiouracil on circulating thyroid hormones, thyroid histology and eggshell quality in Japanese quail (Coturnix coturnix japonica). Chem. Biol. Interact.  42: 371– 377. Google Scholar CrossRef Search ADS PubMed  Gross W. B. 1990. Effect of adrenal blocking chemicals on the responses of chickens and turkeys to environmental stressors and ACTH. Avian. Pathol.  19: 295– 304. Google Scholar CrossRef Search ADS PubMed  Gross W. B., Chickering W.. 1987. Effects of fasting, water deprivation and adrenal blocking chemical on resistance to Escherichia coli challenge. Poult. Sci.  66: 270– 272. Google Scholar CrossRef Search ADS PubMed  Heinrich P. C., Horn F., Graeve L., Dittrich E., Kerr I., Müller-Newen G., Grötzinger J., Wollmer A.. 1998. Interleukin-6 and related cytokines: effect on the acute phase reaction. Eur. J. Nutr.  37: 43– 49. Jonsson C. J., Lund B. O., Brunstrom B., Brandt I.. 1994. Toxicity and irreversible binding of two DDT metabolites-3-methylsulfone-DDE and o,p΄-DDD-in adrenal interrenal cells in birds. Environ. Toxicol. Chem.  13: 1303– 1310. Kenaga E. E. 1966. Commercial and experimental organic insecticides. Bull. Entomol. Soc. Am.  12: 161– 217. Kregel K. 2002. Heat shock proteins: Modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol.  92: 2177– 2186. Google Scholar CrossRef Search ADS PubMed  Mahmoud K. Z., Edens F. W., Eisen E. J., Havenstein G. B.. 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. A Mol. Integr. Physiol.  137: 35– 42. Google Scholar CrossRef Search ADS   Mancini G. A., Carbonara A. T., Heremans J. F.. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry  2: 235IN235– 254IN236. Google Scholar CrossRef Search ADS   Marinkovic S., Jahreis G. P., Wong G. G., Baumann H.. 1989. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo. J. Immunol.  142: 808– 812. Google Scholar PubMed  Martinez-Subiela S., Tecles F., Ceron J.. 2007. Comparison of two automated spectrophotometric methods for ceruloplasmin measurement in pigs. Res. Vet. Sci.  83: 12– 19. Google Scholar CrossRef Search ADS PubMed  Moshage H. J., Roelofs H. M. J., Van Pelt J. F., Hazenberg B. P. C., Van Leeuwen M. A., Limburg P. C., Aarden L. A., Yap S. H.. 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. Google Scholar CrossRef Search ADS PubMed  Murata H., Shimada N., Yoshioka M.. 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J.  168: 28– 40. Google Scholar CrossRef Search ADS PubMed  Najafi P., Zulkifli I., Soleimani A. F., Goh Y. M.. 2016. Acute phase proteins response to feed deprivation in broiler chickens. Poult. Sci.  95: 760– 763. Google Scholar CrossRef Search ADS PubMed  Najafi P., Zulkifli I., Soleimani A. F., Kashiani P.. 2015. The effect of different degrees of feed restriction on heat shock protein 70, acute phase proteins, and other blood parameters in female broiler breeders. Poult. Sci.  94: 2322– 2329. Google Scholar CrossRef Search ADS PubMed  O’Reilly E. L., Eckersall P. D.. 2014. Acute phase proteins: A review of their function, behaviour and measurement in chickens. Worlds Poult. Sci. J.  70: 27– 44. Google Scholar CrossRef Search ADS   Sapolsky R. M., Romero L. M., Munck A. U.. 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev.  21: 55– 89. Google Scholar PubMed  SAS. 2005. SAS/STAT software, version 9.2 . SAS Inst. Inc., Cary, NC, USA. Shini S., Kaiser P.. 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. Google Scholar CrossRef Search ADS PubMed  Srebocan V., Gotal J. P., Adamovic V., Sokic B., Delak M.. 1971. Effect of technical grade DDT and p,p΄-DDT on adrenocortical function in chicks. Poult. Sci.  50: 1271– 1278. Google Scholar CrossRef Search ADS   Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 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. Google Scholar CrossRef Search ADS PubMed  Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 2012. The relationship between adrenocortical function and Hsp70 expression in socially isolated Japanese quail. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol.  161: 140– 144. Google Scholar CrossRef Search ADS PubMed  van Gool J., van Vugt H., Helle M., Aarden L. A.. 1990. The relation among stress, adrenalin, interleukin 6 and acute phase proteins in the rat. Clin. Immunol. Immunopathol.  57: 200– 210. Google Scholar CrossRef Search ADS PubMed  Wigley P., Kaiser P.. 2003. Avian cytokines in health and disease. Braz. J. Poult. Sci.  5: 1– 14. Zulkifli I., Soleimani A. F., Khalil M., Omar A. R., Raha A. R.. 2011. Inhibition of adrenal steroidogenesis and heat shock protein 70 induction in neonatally feed restricted broiler chickens under heat stress condition. Arch. Geflügelk.  75: 246– 252. Zulkifli I., Che Norma M. T., Israf D. A., Omar A. R.. 2000. The effect of early age feed restriction on subsequent response to high environmental temperatures in female broiler chickens. Poult. Sci.  79: 1401– 1407. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Dunnington E. A., Gross W. B., Siegel P. B.. 1994a. Food restriction early or later in life and its effect on adaptability, disease resistance, and immunocompetence of heat-stressed dwarf and nondwarf chickens. Br. Poult. Sci.  35: 203– 213. Google Scholar CrossRef Search ADS   Zulkifli I., Dunnington E. A., Gross W. B., Siegel P. B.. 1994b. Inhibition of adrenal steroidogenesis, food restriction and acclimation to high ambient temperatures in chickens. Br. Poult. Sci.  35: 417– 426. Google Scholar CrossRef Search ADS   Zulkifli I., Dunnington E. A., Siegel P. B.. 1995. Age and psychogenic factors in response to food deprivation and refeeding in White Leghorn chickens. Eur. Poult. Sci.  59: 175– 181. Zulkifli I., Najafi P., Nurfarahin A. J., Soleimani A. F., Kumari S., Aryani A. A., O’Reilly E. L., Eckersall P. D.. 2014. Acute phase proteins, interleukin 6, and heat shock protein 70 in broiler chickens administered with corticosterone. Poult. Sci.  93: 3112– 3118. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Norma M. C., Israf D. A., Omar A. R.. 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. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Inhibition of corticosterone synthesis and its effect on acute phase proteins, heat shock protein 70, and interleukin-6 in broiler chickens subjected to feed restriction

Loading next page...
 
/lp/ou_press/inhibition-of-corticosterone-synthesis-and-its-effect-on-acute-phase-47WOVAzkHy
Publisher
Oxford University Press
Copyright
© 2018 Poultry Science Association Inc.
ISSN
0032-5791
eISSN
1525-3171
D.O.I.
10.3382/ps/pex364
Publisher site
See Article on Publisher Site

Abstract

Abstract The aim of the current study was to elucidate whether inhibition of corticosterone (CORT) synthesis could modify stress response to feed deprivation and its possible interactions with feed restriction in the neonatal period in broiler chickens. Equal numbers of broiler chicks were subjected to either 60% feed restriction (60FR) or ad libitum (AL) on d 4, 5, and 6. On day 7, blood CORT, acute phase proteins (APP), interleukin-6 (IL-6) levels, and brain heat shock protein 70 (HSP70) expression were determined. On d 35, chickens in each early age feeding regimen were subjected to one of the following treatments: (i) ad libitum feeding (ALF), (ii) 24 h feed deprivation (SFR), or (iii) 24 h feed deprivation with intramuscular injection of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) at 100 mg/kg BW (SFR+DDT). The effect of SFR on CORT, APP, IL-6, and HSP 70 were determined on d 36. The results showed that subjecting chicks to 60FR significantly elevated CORT and brain HSP70 concentration compared to the AL group on d 7. The early feeding regimen had no significant effect on CORT, alpha-1 acid glycoprotein (AGP), ovotransferrin (OVT), ceruoplasmin (CP), IL-6, or brain HSP70 on d 36. The CORT, AGP, OVT, CP, IL-6, and brain HSP70 expression of SFR birds following 24 h of feed deprivation (d 36) were significantly higher than their ALF and SFR+DDT counterparts. Both ALF and SFR+DDT birds had similar values. Stress attributed to feed deprivation without concurrent increase in CORT had a negligible effect on serum levels of APP and IL-6 and brain HSP70 expression. INTRODUCTION When birds are exposed to noxious stimuli the hypothalamic-pituitary-adrenal axis (HPA) will be evoked and leads to an increase in the synthesis and release of adrenal glucocorticoids (mainly corticosterone, CORT) into the bloodstream. Elevation in circulating levels of glucocorticoids in response to stresses is crucial to restore homeostasis (Sapolsky et al., 2000). Zulkifli et al. (1994a,b, 2000) demonstrated that stress attributed to early age feed restriction enhanced stress tolerance later in life when compared to that fed ad libitum throughout. However, stressful experiences during the neonatal stage without simultaneous increases in the synthesis and release of CORT may not assist a bird in response to subsequent stressors (Zulkifli et al., 1994a). Thus, stress-induced increases in the synthesis of CORT may be critical to prepare a bird in coping with subsequent physiological disruptions (Soleimani et al., 2011; Zulkifli et al., 2011, 2012). The underlying mechanism of neonatal stress and enhancement of coping ability later in life in poultry has been associated with the ability to express heat shock protein 70 (HSP70) (Zulkifli et al., 2002). It is well documented that HSP play a profound role in modifying physiological stress response and in acquisition of stress tolerance (Kregel, 2002). Acute phase proteins (APP) are serum proteins primarily synthesized by hepatocytes as part of the acute phase response (APR). The APR is part of the early defense or innate immune system, which is elicited by a diversity of challenges such as inflammation, bacterial infection, endotoxin exposure, and tissue injury (Murata et al., 2004; O’Reilly and Eckersall, 2014). The APR results in a complex systemic reaction with the goal of re-establishing homeostasis and promoting healing. In poultry, stress caused by feed deprivation increased serum levels of APP ovotransferrin (OVT), ceruoplasmin (CP), and alpha-1 acid glycoprotein (AGP) (Najafi et al., 2016). The mechanism of APP elicitation in response to noxious stimuli is not fully understood. Murata et al. (2004) reported that pro-inflammatory cytokines such as interleukin-6 (IL-6) stimulated APP synthesis in the liver. They suggested that activation of the HPA axis by stressors may trigger IL-6 production and subsequently the release of APP into the bloodstream. Zulkifli et al. (2014) showed significant elevations in OVT, AGP, CP, plasma IL-6, and brain HSP70 expression in chickens treated with exogenous CORT. Hence, it appears that circulating CORT modulates the elicitation of HSP70, APP, and IL-6 responses. On the contrary, Soleimani et al. (2011) reported that feeding Japanese quail with 30 mg/kg CORT did not affect HSP70 expression in the brain or heart. Similarly, Deane et al. (1999) showed that daily injection of cortisol into silver sea breams had a negligible effect on liver HSP70 expression. Murata et al. (2004), in their review, postulated that stress signal, independent of the HPA axis, also may induce macrophages to produce IL-6 and thereby elevate hepatic production of APP. It appears that the interrelationship between CORT, as the main endocrine response to stress, and the other physiological stress responses, such as HSP 70, APP, and IL-6, is still not clear. Moreover, the possible interaction of such a relationship with neonatal exposure to stress also warrants further investigation. Therefore, the objectives of this study were: (i) to determine the effect of 24 h feed restriction in neonatal chicks on CORT, HSP70, APP, and IL-6, (ii) to elucidate whether inhibition of CORT synthesis could modify stress response to feed deprivation in 36-day-old chickens, and (iii) to evaluate whether stress during the neonatal stage may modify the impact of inhibiting CORT synthesis on stress response in broiler chickens subjected to 24 h of feed deprivation later in life. MATERIALS AND METHODS Birds, Husbandry, and Housing All experimental procedures were conducted in accordance with the University of Putra Malaysia Research Policy on animal care. A total of 192 one-day-old female Cobb 500 broiler chicks was obtained from a local hatchery. At one d of age (d 1), the chicks were weighed and randomly allocated into groups of 8 to 24 battery cages with wire floors 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. The chicks were fed commercial broiler diets. The lighting regimen was 18L:6D. Experimental Treatment On d 4, 5 and 6, 12 cages of birds were assigned to 60% feed restriction (60FR), while the other 12 cages were fed ad libitum (AL). The feed restricted group received 60% of the feed intake of the AL group on the previous day. On d 7 at 08:00 h (before feeding), 12 birds (one per cage) from each feeding regimen were randomly chosen and removed with minimum disturbance to flock mates. Immediately following the capture, birds were decapitated and blood samples were collected for serum separation. The serum samples were stored at -80°C awaiting analysis for CORT, OVT, AGP, CP, and IL-6. Following the blood sampling, brain (whole cerebrum) samples were collected for determination of HSP expression. On d 35, birds in AL and 60FR groups were subjected to either of the following treatments (4 cages per group per treatment): (i) ad libitum feeding (ALF), (ii) 24 h of feed deprivation (SFR), or (iii) 24 h feed deprivation with intramuscular injection of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (Sigma Aldrich Chemical Co., Saint Louis, MO) dissolved in corn oil at 100 mg/kg BW (Adamsson et al., 2009) (SFR+DDT). The birds were injected at 20:00 h, which was 12 h before starting the feeding regimen (Gross, 1990). The adrenal blocking effect of DDT in chickens has been reported previously (Colmano and Gross, 1971; Srebocan et al., 1971; Gross and Chickering, 1987; Gross, 1990; Jonsson et al., 1994). The chemical, inhibits conversion of deoxycorticosterone to CORT via inhibiting 11β-hydroxylase activity (Kenaga, 1966). The DDT dosage used in the present study was based on a pilot study in our laboratory. Dosage of 100 mg/kg BW was the most optimal level to block the synthesis of CORT in broiler chickens subjected to feed deprivation for 24 hours. The ALF and SFR groups were injected intramuscularly with 0.5 mL corn oil. Similar sampling procedures (12 birds per treatment group) as described earlier were repeated following the 24 h (d 36) feed deprivation. CORT and IL-6 Assays Plasma CORT was determined using a commercial high-sensitivity EIA kit (AC-15F1, IDS, Boldon, UK). The intra- and inter-assay variabilities were less than 6.7% and 7.8%, respectively; and the detection limit was 27 ng/mL. The protocol of analysis was according to the manufacturer's recommendations. 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 samples of different d and treatments were analyzed mixed in the same batches. APP Assay AGP concentration was determined by using a commercial ELISA kit specific to chicken (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 TBS 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-MeshTM, Sigma Aldrich, St. Louis, MO) at room temperature. Nine wells were punched in each gel, and 10 μl of standard or serum samples were loaded in each well. OVT standards (albumin from chicken egg white, Sigma Aldrich, St. Lois, MO) 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 hours. Following incubation, the size of the ring around each well was measured and calculated against standards. The concentration of CP is determined by the rate of formation of a colored product from CP 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 were dissolved in 250 mL distilled water and adjusted to pH 6.2 using glacial acetic acid. 0.615 g of 1,4-phenylenediamine dihydrochloride (Sigma P1519) were added to the prepared buffer and kept in the dark for a minimum of 45 minutes. One hundred μl 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 minutes. The absorbance was recorded spectrophotometrically using a microplate reader at 550 nm. Standards were prepared with serial dilution of pig serum of known CP concentration calibrated against purified CP (Sigma Chemical Co St. Louis, MO) and saline buffer combination to achieve various concentrations of 12.75 (20 μl pig serum + 60 μl saline buffer), 6.375, 3.1875, 1.59375, 0.79608, 0.39804, 0.199, and 0.099 mg/mL CP. SDS-PAGE and Immunoblot Analysis for HSP70 Expression The levels of HSP70 protein expression were determined as previously described (Soleimani et al., 2012) with some modification. Briefly, brain samples (0.3 g, whole cerebrum) were homogenized with 1.5 mL of protein extraction buffer (20 Mm Tris, pH 7.5; 0.75 M sodium chloride) and 10 μl/ml protease inhibitor cocktail (P8340, Sigma Chemical Co., St. Louis) and centrifuged at 20,000 g for 30 min at 4°C. The supernatant was separated, and the total protein was measured using a bicinchoninic acid protein assay kit (B9643, Sigma Chemical Co., St. Louis, MO). Total protein (25 μg) was loaded and separated on 10% polyacrylamide gels containing SDS. Gels were electrophoresed at 120 V until the tracking dye reached the base of the gel, and the fractionated proteins were transferred to polyvinylidene difluoride membranes (MSI, Westborough, MA) using a trans-blot semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA). The membranes were incubated for 1 h with 5 mL of blocking buffer containing monoclonal mouse antibody (ab6535, Abcam, Cambridge, MA) against HSP70 in a 1:20,000 dilution. The membranes were washed 3 times (5 min each) with 10 mL of cold tris-buffered saline Tween 20 and incubated in a horseradish peroxidase conjugated rabbit anti-mouse secondary antibody for 30 min in a 1:40,000 dilution (ab6728, Abcam, Cambridge, MA). Membranes were washed again and HSP70 protein bands were visualized colorimetrically with the DAB Substrate System (E885, AMRESCO LLC, Solon, OH). The protein size was confirmed using Precision Plus Protein™ Dual Color Standard (Bio-Rad, Hercules, CA). The final HSP70 concentration was calculated as an arbitrary unit of band density relative to total protein concentration of each sample. Statistical Analysis Data were subjected to ANOVA using the GLM procedure of SAS (SAS, 2005). Early feeding regimen (AL and 60FR) was the only main effect to analyze data measured on d 7. Data for traits measured on d 36 were analyzed using early feeding regimen (AL and 60FR), late feeding regimen (ALF, SFR, and SFR+DDT), and their interactions as main effects. When interactions between main effects were significant, comparisons were made within each experimental variable. When significant effects were found, comparisons among multiple means were modeled by Duncan's multiple-range test. Statistical significance is considered as P < 0.05. RESULTS Subjecting chicks to 60FR significantly elevated CORT (P < 0.001) and brain HSP70 (P = 0.0036) concentration when compared to the AL group on d 7. There were no significant effects of early age feeding regimen on AGP, OVT, CP, or IL-6 (P = 0.3481; 0.1384; 0.6173; 0.1053, respectively) (Table 1). The early feeding regimen had no significant effect on CORT, AGP, OVT, CP, IL-6, or brain HSP70 concentration on d 36 (P = 0.6307, 0.6928, 0.8406, 0.2376, 0.2376, 0.0536, 0.5922, respectively). The CORT, AGP, OVT, CP, IL-6, and brain HSP70 expression of SFR birds following 24 h (d 36) of feed deprivation were significantly higher than their ALF and SFR+DDT counterparts (Table 2). Both ALF and SFR+DDT birds had similar values for all the traits measured (Table 2). There was no significant interaction for any of the traits measured on d 36. A representative blot image of HSP70 expression is illustrated in Figure 1. The gel image representing the effect of early and late feeding regimens on serum OVT level is presented in Figure 2. Figure 1. View largeDownload slide Representative blot image of brain heat shock protein 70 (HSP70) expression as affected by early feeding regimen (AL vs. 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 1. View largeDownload slide Representative blot image of brain heat shock protein 70 (HSP70) expression as affected by early feeding regimen (AL vs. 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 2. View largeDownload slide Representative gel image of serum ovotransferrin (OVT) as affected by early feeding regimen (AL vs 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Figure 2. View largeDownload slide Representative gel image of serum ovotransferrin (OVT) as affected by early feeding regimen (AL vs 60 FR) and late feeding regimen (ALF vs. SFR vs. SFR+DDT) in broiler chickens at 36 d of age. AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. **: P < 0.01. n = 12. Table 1. Effect of early feeding regimen on mean (±SEM) levels of serum corticosterone (CORT) (ng/mL), α1-acid glycoprotein (AGP) (mg/mL), ceruloplasmin (CP) (mg/mL), ovotransferrin (OVT) (mg/mL) concentrations, interleukin 6 (IL-6), and brain heat shock protein 70 (HSP70) expression in broiler chickens at 7 d of age. Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1               AL  0.68 ± 0.37b  0.89 ± 0.78  0.18 ± 0.08  0.11 ± 0.03  0.12 ± 0.11  1.12 ± 0.45b   60FR  1.61 ± 0.49a  1.14 ± 0.41  0.31 ± 0.25  0.12 ± 0.08  0.21 ± 0.12  2.47 ± 1.35a  Analysis of variance  Probabilities   Feeding regimen  <.0001  0.3481  0.1384  0.6173  0.1053  0.0036  Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1               AL  0.68 ± 0.37b  0.89 ± 0.78  0.18 ± 0.08  0.11 ± 0.03  0.12 ± 0.11  1.12 ± 0.45b   60FR  1.61 ± 0.49a  1.14 ± 0.41  0.31 ± 0.25  0.12 ± 0.08  0.21 ± 0.12  2.47 ± 1.35a  Analysis of variance  Probabilities   Feeding regimen  <.0001  0.3481  0.1384  0.6173  0.1053  0.0036  a,bMeans within a column with no common letters differ at P < 0.05. 1AL = fed ad libitum; 60FR = 60% of fed ad libitum. n = 12. View Large Table 2. Effect of early and late feeding regimen on mean (±SEM) levels of serum corticosterone (CORT) (ng/mL), α1-acid glycoprotein (AGP) (mg/mL), ceruloplasmin (CP) (mg/mL), ovotransferrin (OVT) (mg/mL) concentrations, interleukin 6 (IL-6), and brain heat shock protein 70 (HSP70) expression in broiler chickens at 36 d of age. Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1   AL  0.98 ± 0.14  1.48 ± 0.25  0.52 ± 0.08  0.31 ± 0.04  0.98 ± 0.12  2.04 ± 0.28   60FR  1.04 ± 0.15  1.42 ± 0.27  0.52 ± 0.07  0.33 ± 0.05  1.21 ± 0.16  2.15 ± 0.32  Late feeding regimen2   ALF  0.61 ± 0.07b  0.78 ± 0.08b  0.28 ± 0.01b  0.15 ± 0.07b  0.67 ± 0.09b  1.13 ± 0.05b   SFR  1.86 ± 0.15a  2.89 ± 0.16a  1.03 ± 0.08a  0.59 ± 0.03a  1.94 ± 0.13a  3.92 ± 0.28a   SFR+DDT  0.58 ± 0.05b  0.67 ± 0.05b  0.27 ± 0.02b  0.21 ± 0.01b  0.68 ± 0.06b  1.23 ± 0.11b  Analysis of variance  Probabilities   Early feeding regimen  0.6307  0.6928  0.8406  0.2376  0.0536  0.5922   Late feeding regimen  <.0001  <.0001  <.0001  <.0001  <.0001  <.0001   Early feeding regimen × Late feeding regimen  0.6939  0.3071  0.8853  0.0586  0.1115  0.6174  Item  CORT  AGP  OVT  CP  IL-6  HSP70  Early feeding regimen1   AL  0.98 ± 0.14  1.48 ± 0.25  0.52 ± 0.08  0.31 ± 0.04  0.98 ± 0.12  2.04 ± 0.28   60FR  1.04 ± 0.15  1.42 ± 0.27  0.52 ± 0.07  0.33 ± 0.05  1.21 ± 0.16  2.15 ± 0.32  Late feeding regimen2   ALF  0.61 ± 0.07b  0.78 ± 0.08b  0.28 ± 0.01b  0.15 ± 0.07b  0.67 ± 0.09b  1.13 ± 0.05b   SFR  1.86 ± 0.15a  2.89 ± 0.16a  1.03 ± 0.08a  0.59 ± 0.03a  1.94 ± 0.13a  3.92 ± 0.28a   SFR+DDT  0.58 ± 0.05b  0.67 ± 0.05b  0.27 ± 0.02b  0.21 ± 0.01b  0.68 ± 0.06b  1.23 ± 0.11b  Analysis of variance  Probabilities   Early feeding regimen  0.6307  0.6928  0.8406  0.2376  0.0536  0.5922   Late feeding regimen  <.0001  <.0001  <.0001  <.0001  <.0001  <.0001   Early feeding regimen × Late feeding regimen  0.6939  0.3071  0.8853  0.0586  0.1115  0.6174  a,bMeans within a column-subgroup with no common letters differ at P < 0.05. 1AL = ad libitum feeding; 60FR = 60% of ad libitum feed intake. 2ALF = ad libitum feeding; SFR = 24 h feed restriction; SFR+DDT = 24 h feed restriction with intramuscular injection of DDT. View Large DISCUSSION As expected, 60FR resulted in significantly higher CORT and brain HSP70 expression on d 7 (Table 1). These results confirmed those of Soleimani et al. (2011) that neonatal feed restriction increased brain HSP70 expression and CORT. We noted that 60FR did not affect AGP, OVT, or CP in chicks on d 7. In agreement, Najafi et al. (2015) reported that feed restriction at 75, 60, 45, and 30% of ad libitum intake from d 28 to 42 d of age had negligible effect on AGP, OVT, and CP in broiler breeder pullets. Zulkifli et al. (1995) reported that White Plymouth Rock chicks subjected to neonatal feed restriction had lower heterophil to lymphocyte ratios (HLR) but similar CORT following 24 h feed deprivation at 36 d of age when compared to those fed ad libitum during the neonatal stage. The authors concluded that stresses early in life can evoke long-lasting changes in the physiological response to a stimulus. In the present study, the neonatal feed restriction did not modify CORT, OVT, AGP, CP, or IL-6 responses to 24 h of feed deprivation in 36-day-old broiler chickens. There is no clear explanation for the discrepancies, although they could be associated with the differences in breeds of chickens used in both studies. Zulkifli et al. (1994a) demonstrated that neonatal feed restriction improved stress tolerance in 43-day-old non-dwarf White Plymouth Rocks but not in their dwarf counterparts. Physiological stress reaction to deprivation also varied according to breed. Working with Light Sussex, Freeman et al. (1984) concluded that depriving food, water, or both for 24 h had negligible effect on CORT. On the contrary, Zulkifli et al. (1995) showed that 24 h of feed deprivation elevated HLR in 47-week-old White Leghorns. The present findings indicated that 24 h of feed deprivation was stressful to broiler chickens. The SFR chickens had higher CORT, HLR, OVT, AGP, CP, IL-6, and brain HSP70 expression than their ALF counterparts. On the contrary, Najafi et al. (2016) reported elevations in OVT, AGP, and CP only after 30 h of feed deprivation. There is no clear explanation for the discrepancy, although it may be associated with the variations in the ages of the experimental chickens. Najafi et al. (2016) used broiler chicks of 22 d of age, whereas we used 36-day-old chicks. Thus, APP could be used to gauge physiological stress in avian species and are thus another biomarker for well being. Alterations in serum levels of APP following fasting suggested the role of the proteins in restoring homeostasis in animals subjected to non-inflammatory, psychophysical stressors (Cray et al., 2009). Il-6 is a pro-inflammatory cytokine, having a key role in inflammatory responses via activation and regulation of other stimulated cells and tissues (Wigley and Kaiser, 2003). Zulkifli et al. (2014) demonstrated that administration of exogenous CORT elicited plasma IL-6 activity in chickens. The present findings indicated that stress associated with 24 h feed deprivation may increase serum IL-6 level. In vivo and in vitro findings suggested that IL-6 is an important mediator in the synthesis of APP (Moshage et al., 1988; Marinkovic et al., 1989). van Gool et al. (1990) and Shini and Kaiser (2009) suggested a relationship among stress, IL-6, and APP in rodents. The preceding discussion suggests that stress may stimulate the macrophages and Kupfer cells to release IL-6, which elicits the hepatocytes to synthesize APP. Murata et al. (2004), in their review, suggested that induction and regulation of APP during stress are mediated through the HPA axis and IL-6. The authors also postulated that internal and external challenges can directly evoke the production of IL-6. It appears that APP reaction can be elicited by stress without evoking adrenal cortical activity. In the present study, administration of DDT blocked CORT synthesis and suppressed IL-6 and APP responses following the 24 h feed deprivation. It appears that stress without concomitant increase in the synthesis and liberation of CORT may not trigger APP response in avian species. There is, however, a question as to whether suppression of IL-6 response is attributed to the direct effect of DDT or the secondary effect of inhibition of adrenal cortical activity. Because administration of exogenous CORT elevated IL-6 (Zulkifli et al., 2014) in poultry, it is possible that the suppressed IL-6 reaction in DDT-treated birds is a consequence of inhibition of CORT. Interestingly, although stress may elicit IL-6, the cytokine also has been reported to increase the release of ACTH in the pituitary, and the hormone stimulates glucocorticoid synthesis in the adrenal cortex (Heinrich et al., 1998). Sapolsky et al. (2000) proposed that the modulating actions of glucocorticoids can be permissive, suppressive, stimulating, and preparative, and, thus, animals with impaired ability to synthesize and liberate glucocorticoids may not be able to cope with stress as well as their normal counterparts. In the present study, our results are consistent with the stimulating role of glucocorticoids in which inhibition of the CORT by DDT, eliminated the APP, IL-6, and HSP70 synthesis after feed deprivation. The relationship between CORT and expression of HSP70 has been inconsistent in poultry. Mahmoud et al. (2004) reported a positive correlation between CORT and heart HSP70 expression in broiler chickens subjected to cyclic heat stress. On the contrary, Soleimani et al. (2012) reported that feeding Japanese quail with 30 mg/kg CORT for 3 d did not affect HSP70 level in the brain or heart. Zulkifli et al. (2014) demonstrated that daily injection of CORT for 4 and 7 d significantly increased HSP70 expression in the brain. The present findings clearly suggested that failure to increase CORT during perturbation of homeostasis may result in retardation of brain HSP70 expression. In conclusion, under the conditions of this study, feed deprivation for 24 h elevated CORT, IL-6, AGP, OVT, CP, and brain HSP70 expression in broilers. However, following injection of DDT as the CORT synthesis inhibitor, 24 h feed deprivation had negligible effects on APP and brain HSP70 expression. On a cautionary note, however, DDT also may influence the endocrine system in other ways (Grassle and Biessmann, 1982), and some of the effects observed in our study may have been associated with those changes. Acknowledgements This research was funded by the Malaysian Ministry of Science, Technology, and Innovation. REFERENCES Adamsson A., Salonen V., Paranko J., Toppari J.. 2009. Effects of maternal exposure to di-isononylphthalate (DINP) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p΄-DDE) on steroidogenesis in the fetal rat testis and adrenal gland. Reprod. Toxicol.  28: 66– 74. Google Scholar CrossRef Search ADS PubMed  Colmano G., Gross W. B.. 1971. Effect of metyrapone and DDD on infectious diseases. Poult. Sci.  50: 850– 854. Google Scholar CrossRef Search ADS PubMed  Cray C., Zaias J., Altman N. H.. 2009. Acute phase response in animals: A review. Comp. Med.  59: 517. Google Scholar PubMed  Deane E. E., Kelly S. P., Lo C. K., Woo N. Y.. 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. Google Scholar CrossRef Search ADS PubMed  Freeman B. M., Manning A. C. C., Flack I. H.. 1984. Changes in plasma corticosterone concentrations in the water-deprived fowl, Gallus domesticus. Comp. Biochem. Physiol. A Physiol.  79: 457– 458. Google Scholar CrossRef Search ADS   Grassle B., Biessmann A.. 1982. Effects of DDT, polychlorinated biphenyls and thiouracil on circulating thyroid hormones, thyroid histology and eggshell quality in Japanese quail (Coturnix coturnix japonica). Chem. Biol. Interact.  42: 371– 377. Google Scholar CrossRef Search ADS PubMed  Gross W. B. 1990. Effect of adrenal blocking chemicals on the responses of chickens and turkeys to environmental stressors and ACTH. Avian. Pathol.  19: 295– 304. Google Scholar CrossRef Search ADS PubMed  Gross W. B., Chickering W.. 1987. Effects of fasting, water deprivation and adrenal blocking chemical on resistance to Escherichia coli challenge. Poult. Sci.  66: 270– 272. Google Scholar CrossRef Search ADS PubMed  Heinrich P. C., Horn F., Graeve L., Dittrich E., Kerr I., Müller-Newen G., Grötzinger J., Wollmer A.. 1998. Interleukin-6 and related cytokines: effect on the acute phase reaction. Eur. J. Nutr.  37: 43– 49. Jonsson C. J., Lund B. O., Brunstrom B., Brandt I.. 1994. Toxicity and irreversible binding of two DDT metabolites-3-methylsulfone-DDE and o,p΄-DDD-in adrenal interrenal cells in birds. Environ. Toxicol. Chem.  13: 1303– 1310. Kenaga E. E. 1966. Commercial and experimental organic insecticides. Bull. Entomol. Soc. Am.  12: 161– 217. Kregel K. 2002. Heat shock proteins: Modifying factors in physiological stress responses and acquired thermotolerance. J. Appl. Physiol.  92: 2177– 2186. Google Scholar CrossRef Search ADS PubMed  Mahmoud K. Z., Edens F. W., Eisen E. J., Havenstein G. B.. 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. A Mol. Integr. Physiol.  137: 35– 42. Google Scholar CrossRef Search ADS   Mancini G. A., Carbonara A. T., Heremans J. F.. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry  2: 235IN235– 254IN236. Google Scholar CrossRef Search ADS   Marinkovic S., Jahreis G. P., Wong G. G., Baumann H.. 1989. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo. J. Immunol.  142: 808– 812. Google Scholar PubMed  Martinez-Subiela S., Tecles F., Ceron J.. 2007. Comparison of two automated spectrophotometric methods for ceruloplasmin measurement in pigs. Res. Vet. Sci.  83: 12– 19. Google Scholar CrossRef Search ADS PubMed  Moshage H. J., Roelofs H. M. J., Van Pelt J. F., Hazenberg B. P. C., Van Leeuwen M. A., Limburg P. C., Aarden L. A., Yap S. H.. 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. Google Scholar CrossRef Search ADS PubMed  Murata H., Shimada N., Yoshioka M.. 2004. Current research on acute phase proteins in veterinary diagnosis: An overview. Vet. J.  168: 28– 40. Google Scholar CrossRef Search ADS PubMed  Najafi P., Zulkifli I., Soleimani A. F., Goh Y. M.. 2016. Acute phase proteins response to feed deprivation in broiler chickens. Poult. Sci.  95: 760– 763. Google Scholar CrossRef Search ADS PubMed  Najafi P., Zulkifli I., Soleimani A. F., Kashiani P.. 2015. The effect of different degrees of feed restriction on heat shock protein 70, acute phase proteins, and other blood parameters in female broiler breeders. Poult. Sci.  94: 2322– 2329. Google Scholar CrossRef Search ADS PubMed  O’Reilly E. L., Eckersall P. D.. 2014. Acute phase proteins: A review of their function, behaviour and measurement in chickens. Worlds Poult. Sci. J.  70: 27– 44. Google Scholar CrossRef Search ADS   Sapolsky R. M., Romero L. M., Munck A. U.. 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev.  21: 55– 89. Google Scholar PubMed  SAS. 2005. SAS/STAT software, version 9.2 . SAS Inst. Inc., Cary, NC, USA. Shini S., Kaiser P.. 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. Google Scholar CrossRef Search ADS PubMed  Srebocan V., Gotal J. P., Adamovic V., Sokic B., Delak M.. 1971. Effect of technical grade DDT and p,p΄-DDT on adrenocortical function in chicks. Poult. Sci.  50: 1271– 1278. Google Scholar CrossRef Search ADS   Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 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. Google Scholar CrossRef Search ADS PubMed  Soleimani A. F., Zulkifli I., Omar A. R., Raha A. R.. 2012. The relationship between adrenocortical function and Hsp70 expression in socially isolated Japanese quail. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol.  161: 140– 144. Google Scholar CrossRef Search ADS PubMed  van Gool J., van Vugt H., Helle M., Aarden L. A.. 1990. The relation among stress, adrenalin, interleukin 6 and acute phase proteins in the rat. Clin. Immunol. Immunopathol.  57: 200– 210. Google Scholar CrossRef Search ADS PubMed  Wigley P., Kaiser P.. 2003. Avian cytokines in health and disease. Braz. J. Poult. Sci.  5: 1– 14. Zulkifli I., Soleimani A. F., Khalil M., Omar A. R., Raha A. R.. 2011. Inhibition of adrenal steroidogenesis and heat shock protein 70 induction in neonatally feed restricted broiler chickens under heat stress condition. Arch. Geflügelk.  75: 246– 252. Zulkifli I., Che Norma M. T., Israf D. A., Omar A. R.. 2000. The effect of early age feed restriction on subsequent response to high environmental temperatures in female broiler chickens. Poult. Sci.  79: 1401– 1407. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Dunnington E. A., Gross W. B., Siegel P. B.. 1994a. Food restriction early or later in life and its effect on adaptability, disease resistance, and immunocompetence of heat-stressed dwarf and nondwarf chickens. Br. Poult. Sci.  35: 203– 213. Google Scholar CrossRef Search ADS   Zulkifli I., Dunnington E. A., Gross W. B., Siegel P. B.. 1994b. Inhibition of adrenal steroidogenesis, food restriction and acclimation to high ambient temperatures in chickens. Br. Poult. Sci.  35: 417– 426. Google Scholar CrossRef Search ADS   Zulkifli I., Dunnington E. A., Siegel P. B.. 1995. Age and psychogenic factors in response to food deprivation and refeeding in White Leghorn chickens. Eur. Poult. Sci.  59: 175– 181. Zulkifli I., Najafi P., Nurfarahin A. J., Soleimani A. F., Kumari S., Aryani A. A., O’Reilly E. L., Eckersall P. D.. 2014. Acute phase proteins, interleukin 6, and heat shock protein 70 in broiler chickens administered with corticosterone. Poult. Sci.  93: 3112– 3118. Google Scholar CrossRef Search ADS PubMed  Zulkifli I., Norma M. C., Israf D. A., Omar A. R.. 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. Google Scholar CrossRef Search ADS PubMed  © 2018 Poultry Science Association Inc.

Journal

Poultry ScienceOxford University Press

Published: Apr 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve Freelancer

DeepDyve Pro

Price
FREE
$49/month

$360/year
Save searches from Google Scholar, PubMed
Create lists to organize your research
Export lists, citations
Access to DeepDyve database
Abstract access only
Unlimited access to over
18 million full-text articles
Print
20 pages/month
PDF Discount
20% off