Introduction

Many studies from tropical and semitropical regions have been concerned with the economic losses caused by heat stress in livestock industries. In the USA alone, the annual losses attributed to heat stress across livestock animal classes average 2.4 billion dollar with 128 million dollar from the poultry industry (St-Pierre et al. 2003). Heat stress results from a negative balance between the quantity of energy flowing from the bird’s body to its surrounding environment and the quantity of heat energy produced by the bird (Lara and Rostagno 2013). It has been suggested that modern broiler genotypes produce more body heat due to their greater metabolic activity which increase the heat load on such animals (Deeb and Cahaner 2002).

It has been shown that broiler chickens subjected to chronic heat stress had markedly reduced final body weight, feed consumption, and feed efficiency (Cooper and Washburn 1998; Niu et al. 2009; Attia et al. 2011; Imik et al. 2012; Ghazi et al. 2012; Sohail et al. 2012; Quinteiro-Filho et al. 2012). The low performance during heat stress is usually accompanied by an increase in mortality and a rapid drop in the net yield of commercial broiler chickens (Geraert et al. 1996; Yalçin et al. 1997; Syafwan et al. 2011). In addition, the continuous exposure to elevated temperatures appears to adversely affect the inflammatory and immune responses in chickens (Niu et al. 2009; Quinteiro-Filho et al. 2012; Ohtsu et al. 2015). Namely, plasma corticosterone and heterophil/lymphocyte ratio are increased in broiler chickens under heat stress (Khajavi et al. 2003; Quinteiro-Filho et al. 2012). Also, exposure of chickens to extreme heat stress induced a decrease in serum immunoglobulin IgG and IgM levels (Park et al. 2013), antibody response to sheep red blood cells (SRBC) (Niu et al. 2009), and T cell proliferation in the blood (Khajavi et al. 2003). Other reports suggested that immune stress can reallocate nutrients to immune response (Feng et al. 2012) and thus negatively affect the utilization of energy in broilers, resulting in the decreased growth performance (Hu et al. 2011; Liu et al. 2014).

It is well-known that protein synthesis in eukaryotes is a key factor for normal cell function and optimum production performance. Cells exposed to elevated temperature showed an inhibition of protein synthesis through alterations in the phosphorylation state of many components of the translational process (Kregel 2002; Garriga et al. 2006; Sukarieh et al. 2009). One group of these components consists of the peptide-chain eukaryotic elongation factors such as eEF2. The inhibition of eEF2 by phosphorylation of its specific kinase is the best characterized mechanism controlling the rate of elongation (Dorovkov et al. 2002). It was found that eEF2 phosphorylation decreases the global protein translation rate, thereby contributing to the overall decrease of protein synthesis (Taha et al. 2013; Gismondi et al. 2014).

Researches on broiler chickens exposed to high environmental temperatures have focused on describing the immune response to heat stress to be directly or indirectly correlated with growth rate and feed consumption. However, the physiological and biological reasons behind the impaired performance of broilers during heat stress remain poorly understood. To our knowledge, the hypothesis that heat stress suppresses growth performance and immune function via affecting the protein synthesis steps in chicken tissues was not reported previously. Therefore, the objective of the current study was to investigate the consequences of chronic heat stress in broiler chickens on changes in leukocyte protein synthesis and the deterioration in the immune function that lead to the observed low performance in stressed chickens.

Materials and methods

Birds’ management and experimental design

A total of 300 one-day-old male broiler chicks (Cobb500™) were purchased from a local hatchery and were raised in a floor house, with ad libitum access to water and feed that met NRC (1994) recommendations. The birds received 24 h of light (24 L: 0D) during the first 3 days of age, and then chicks were exposed to 23 L: 1D. A brooding temperature was maintained at 33 °C for the first 3 days of age, and then it was decreased to 30 °C for the rest of the first week. The temperature was reduced by 3 °C per week until it reached 24 °C at 21 days of age. All birds were vaccinated according to hygiene program of poultry housing of Cairo University.

At 21 days of age, broiler chicks were randomly allocated into two identical environmental chambers (5 replicates of 30 chicks each). The two chambers were fully cleaned before initiation of the experiment and contained the same conditions of size, ventilation, humidity, temperature, light intensity, and light schedule. From 21 to 42 days of age, broilers in each of the two chambers received a different heat treatment according to Mashaly et al. (2004). Broilers in the first chamber were exposed to 24 °C and 50% RH with a heat index of 25 °C, representing normal heat index (control group), while broilers in the second chamber were exposed to 35 °C and 50% RH with heat index of 41 °C, representing chronic heat stress (HS group). At 42 days of age, blood samples were obtained from the brachial vein of birds in each group using heparinized tubes (#367874, BD Co., Ltd., Franklin Lakes, NJ, USA) to measure heterophil/lymphocyte (H/L) ratio, plasma corticosterone concentration, plasma immunoglobulin IgG and IgM concentration, and antibody titer against sheep red blood cells (SRBC). In addition, leukocytes were isolated from blood samples to assay the lymphocyte proliferation, protein synthesis, and the phosphorylation level of eukaryotic elongation factor 2 (eEF2). Each bird in the group was sampled only once, and each sample was used for only one assay. Also, the production performance of each group, as described below, was recorded, including the average body weight gain, feed consumption, feed conversion ratio, and total mortality during the treatment.

Broiler production performance

Individual body weights were recorded at the start of the treatment (21 days of age) and at the end of the treatment (42 days of age) for each replicate in each group (5 replicates per group, 20 birds each). The average body weight gain (kg/bird), the total feed consumption (kg/bird), and feed conversion ratio (feed/gain) in each replicate in each group were obtained for the entire treatment period (21–42 days of age). Dead birds were weighed and the weight was included in calculating the feed conversion ratio. The total percent mortality was calculated as a percentage of the number of total dead birds, throughout the treatment period, over the number of birds at the start of the treatment.

Stress indicators

Two blood samples from each replicate in each group (10 samples per group; 3 mL each) were assigned to measure the H/L ratio and plasma corticosterone concentration as indicators for stress induced by the elevated temperature. In brief, 5 μL of blood was used to make a smear on a clean glass slide. The smears were fixed with methyl alcohol and stained with Wright-Giemsa (Sigma Chemical Co., St. Louis, MO) after dried. For each slide, a total of 100 cells were counted using a light microscope. The number of cells in the two slides was averaged and the H/L ratio was then calculated. After that, blood samples were centrifuged at 2000×g at 4 °C for 10 min, and then plasma was decanted into new tubes and stored at −20 °C. Plasma corticosterone concentrations were determined in duplicates using specific chicken corticosterone ELISA kits (MyBioSource, San Diego, CA, USA). The intra- and inter-assay coefficient of variations was <8 and <10%, respectively. The analytical sensitivity of the assay was less than 0.0625 ng/mL, and the dynamic range of the assay was 0.5–20 ng/mL.

Immune function

Plasma IgG and IgM concentration

At 42 days of age, 10 blood samples were collected from broiler chickens (2 chicks per replicate × 5 replicates), centrifuged, and plasma was separated to measure the immunoglobulin IgG and IgM concentrations, using chicken IgG or IgM ELISA quantitation kits (Bethyl Laboratories, Montgomery, TX) as described elsewhere (Kang and Kim 2016). Briefly, 96-well microplates were coated for 1 h with coating buffer containing capture antibody (goat anti-chicken IgG or IgM affinity purified) and were washed three times with washing solution. The wells were incubated with blocking solution for 30 min and then rinsed three times with washing solution. The chicken reference plasma was used to do standard curve, whereas plasma samples were diluted at 1:1000 in the sample-conjugate diluent and were incubated in wells for 60 min, and then the wells were washed five times with washing solution. Detection antibody horseradish peroxidase (goat anti-chicken IgG or IgM) was added and incubated for 15 min (IgM) or 30 min (IgG). Afterward, the measurement of the optical density values was performed at 450 nm using ELISA microtiter plate reader (model 550 Microplate Reader, Bio-Rad), and plasma immunoglobulin (IgG or IgM) concentration was calculated using the broiler chicken reference plasma absorbance.

Antibody titer against SRBC

Ten chicks from each group (two chicks per replicate × five replicates) were intravenously injected with 0.5 mL of 5% saline suspension of SRBC at 35 days of age. One week following the injection (42 days), blood samples were collected and the antibody production against SRBC was quantified by microhemagglutination assay (Trout et al. 1996). Antibody values were expressed as log2 of the reciprocal of the highest dilution where visible agglutination was observed.

Lymphocyte proliferation assay

The lymphocyte proliferation assay was measured in 10 blood samples for each group as described by Zhang and Guo (2008). Briefly, leukocytes were separated from blood using histopaque-1077 (Sigma Chemical Co., St. Louis, MO). Leukocytes were plated at 5 × 105 cells per well, and 50 μL of Concanavalin-A (Con-A, Sigma Chemical Co., St. Louis, MO) at 45 μg/mL was added. Cells were then incubated at 41 °C in a humidified atmosphere of 5% CO2. Sixty-eight hours later, 15 μL of 5 mg/mL 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) was added to each well, followed by further 4 h incubation. Subsequently, 100 μL of 10% sodium dodecyl sulfates dissolved in 0.04 M HCl solution was added to each single well to lyse the cells and solubilize the MTT crystals. Finally, the plates were read using ELISA reader (model 550 Microplate Reader, Bio-Rad) at 570 nm to obtain the optical density reading, and the stimulation index for leukocyte proliferation was calculated as follows (SI = OD570experimental group/OD570blank control).

Protein synthesis and eEF2 phosphorylation assay in leukocytes

At 42 days of age, 10 birds from each group were used to determine protein synthesis as described by Gordon et al. (2014). Briefly, birds were injected with 0.1 mL of saline which contained [35S] EasyTag Express protein labeling mix (11.0 mCi/mL; NEN, Boston, MA, USA). Fifteen minutes later, blood samples were collected and leukocytes were isolated and homogenized to measure protein synthesis. Cell lysate was applied to absorbent glass microfiber filters (Sigma-Aldrich), and protein was precipitated in the filter with 10% trichloroacetic acid (TCA) followed by washing three times with 5% TCA. Filters were then dried and protein was solubilized followed by scintillation counting (1209 Rackbeta, LKB Instruments, Gaithersburg, MD 20877). Global rates of protein synthesis were estimated by the incorporation of [35S] methionine and [35S] cysteine into TCA-precipitated protein.

The total and phosphorylation levels of eukaryotic elongation factor 2 (T-eEF2 and P-eEF2, respectively) were measured using immunoblotting technique as described by Kelleher et al. (2013) with some modification. In brief, 10 blood samples were obtained from birds of each group at 42 days of age. The leukocytes were isolated and homogenized in lysis buffer with protease inhibitor cocktail (# P8340; Sigma, St. Louis, MO). Samples were boiled for 5 min and centrifuged by 10,000×g at 4 °C. Total cell lysates (150 μg/lane) were electrophoresed in 10% SDS polyacrylamide and then were electroblotted to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked in 5% nonfat milk and probed with primary antibodies against T-eEF2 and P-eEF2 at threonine 56 (Thr56) site (Cell Signaling Technology, MA, USA). Horseradish peroxidase-labeled goat anti-rabbit-IgG (1:2000; Santa Cruz) was used for detection with enhanced chemiluminescent reagents (ECL; Amersham, Little Chalfont, Buckinghamshire, England). The level of phosphorylated eEF2 was expressed relatively to its total amount.

Statistical analysis

Statistical analyses were performed using SPSS 16.0 software package (SPSS Inc., Chicago, USA, 2007). A Student’s t test was performed to compare the differences between the control and HS broiler groups in all data of protein synthesis, eEF2 phosphorylation, stress indicators, immune function, and production performance. Results were represented as mean ± standard error of the mean. A P value of less than 0.05 was considered significant.

Results

Leukocyte protein synthesis and eEF2 phosphorylation

The protein synthesis and the eEF2 Thr56 phosphorylation level in leukocytes of broilers exposed to chronic heat stress during 21–42 days of age are shown in Fig. 1. Compared with the broilers in the control group, those in the HS group demonstrated a significant (P < 0.05) decrease in leukocyte protein synthesis (108.6 vs. 173.8 DPM/μg for HS vs. control group, Fig. 1a). In contrast, the phosphorylation level of eEF2 in leukocytes of broilers that were exposed to chronic heat stress was significantly higher than that in control broilers (1.20 vs. 0.44 P-eEF2 in HS vs. control group, P < 0.05, Fig. 1b).

Fig. 1
figure 1

The leukocytes protein synthesis (a) and relative eEF2 Thr56 phosphorylation level (b) of broiler chickens exposed to chronic heat stress during 21–42 days of age. The phosphorylation level of eEF2 (P-eEF2) was normalized on the base of total eEF2 (T-eEF2) on the Thr56 site. A representative blot is shown in the inset. Bars express the mean ± SEM (n = 10). Bars with different letters are significantly different (P < 0.05)

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Stress indicators

The effect of chronic heat stress on the H/L ratio and plasma corticosterone concentration is represented in Fig. 2. Results show that the H/L ratio was significantly (P < 0.05) increased in HS broilers compared to control. Likewise, heat stress caused a significant (P < 0.05) elevation in the plasma corticosterone concentration in HS group vs. control group.

Fig. 2
figure 2

The H/L ratio (a) and plasma corticosterone concentration (b) of broiler chickens exposed to chronic heat stress during 21–42 days of age. Bars express the mean ± SEM (n = 10). Bars with different letters are significantly different (P < 0.05)

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Immune function

The results demonstrate that chronic heat stress during 21–42 days of age negatively affected all studied parameters of immune function in broilers (Fig. 3). The heat stress significantly (P < 0.05) reduced both IgG and IgM concentrations in plasma of HS broilers compared to their controls. In addition, the antibody titer against SRBC was decreased from 7.20 in control group to 4.80 in HS group (P < 0.05). Furthermore, heat stress significantly (P < 0.05) suppressed the lymphocyte proliferation in the HS broilers compared to the control ones.

Fig. 3
figure 3

The immune function presented by plasma IgG (a) and IgM (b) concentrations, antibody titer against SRBC (c) and lymphocyte proliferation (d) of broiler chickens exposed to chronic heat stress during 21–42 days of age. Bars express the mean ± SEM (n = 10). Bars with different letters are significantly different (P < 0.05)

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Broiler production performance

Results of the production performance of broilers that were exposed to chronic heat stress during 21–42 days of age are illustrated in Fig. 4. The results revealed that heat stress significantly (P < 0.05) reduced body weight gain and feed consumption in broilers, while the feed conversion ratio was significantly increased by heat stress. Furthermore, broilers exposed to chronic heat stress during 21–42 days of age expressed a significant increase (P < 0.05) in the total percent mortality than that found in broilers from the control group.

Fig. 4
figure 4

The body weight gain (a), feed consumption (b), feed conversion ratio (c), and total percent mortality (d) of broiler chickens exposed to chronic heat stress during 21–42 days of age. Bars express the mean ± SEM (n = 100). Bars with different letters are significantly different (P < 0.05)

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Discussion

The main protein synthesis steps are initiation, elongation, and termination (Alberts et al. 2008). The regulation of mRNA-translation elongation as an important stage of protein synthesis via eEFs in eukaryotic cells was described in previous reports (Browne and Proud 2002; Kaul et al. 2011; Taha et al. 2013). However, other reports focused on the role of eEF2 phosphorylation pathway on the defect in protein synthesis under different stressors in human cells (Kakigi et al. 2011; Leprivier et al. 2013; Hizli et al. 2013; Gismondi et al. 2014) and rodent cells (Patel et al. 2002; Hong-Brown et al. 2007; Hong-Brown et al. 2008; Leprivier et al. 2013). In chickens, early studies have shown that cells undergoing active differentiation in developing chick embryos need a large amount of chicken eEF2 for the prolific synthesis of a variety of proteins (Kim et al. 1993). The protein synthesis eEF2 was then purified from chicken livers (Riis 1996), and the functional characterization of the chicken eEF2 gene was later determined (Lim and Kim 2007). To the best of our knowledge, very limited recent research was conducted regarding the role of eEF2 on protein synthesis in chicken cells (Hazard et al. 2011; McBride et al. 2013; Zheng et al. 2016), and the current study is the first particular report on leukocyte protein synthesis and eEF2 Thr56 phosphorylation in broilers under chronic heat stress condition.

The phosphorylation of eEF2 was reported to control many metabolic pathways and may likewise inhibit the elongation phase of protein translation process (Kaul et al. 2011; Hizli et al. 2013). The current results demonstrate that heat stress significantly minimized leukocyte protein synthesis by 38% compared to control (Fig. 1a). In contrast, broilers exposed to chronic heat stress expressed a significant threefold higher level of leukocytes eEF2 phosphorylation when compared to control (Fig. 1b). These results are consistent with that obtained by Hazard et al. (2011) who found an apparent down-expression of eEF2 protein under restraint and transport stressful situation in chicken muscles. In addition, the phosphorylated eEF2 negatively affects cell ribosomes, slowing or stopping translation and overall protein synthesis (Hershey 1989; Redpath et al. 1993). Likewise, we found that broilers exposed to heat stress expressed a significant increase in the p-eEF2 Thr56 accompanied with a significant decrease in protein synthesis in leukocytes (Fig. 1). Consequently, these events lead to reduce leukocyte cells viability and increase its apoptosis (White-Gilbertson et al. 2008), which may cause the immunosuppression occurred in HS broilers in the current study.

It is well-known that high environmental temperatures alter the activity of the neuroendocrine system of poultry, resulting in activation of the hypothalamic-pituitary-adrenal axis (HPA), and consequently, elevated plasma corticosterone concentrations (Garriga et al. 2006; Quinteiro-Filho et al. 2010; Quinteiro-Filho et al. 2012). The elevated plasma corticosterone and H/L ratio are the two most utilized indicators of the stress condition in birds (Siegel 1995). Substantially, the lower protein synthesis in leukocytes of HS broilers was accompanied with threefold increase in the plasma corticosterone concentration as compared to the control broilers. Also, the H/L ratio in the HS group was twofold higher than the control group (Fig. 2). In a recent study (Mehaisen et al. 2017), it has been reported that daily corticosterone injection as stress simulator in broiler chickens during 21–28 days of age resulted in an increase in the H/L ratio with a global decrease in the circulating leukocytes number; assuming that corticosterone may directly cause a redistribution of leukocyte components in blood and a proportional change in the H/L ratio to bring the cells required for the nonspecific immune response such as heterophils (Cohen 1972). Further studies concluded that the increase in circulating corticosterone level and/or H/L ratio is a necessary physiological acclimation process in response to heat stress in broilers and other animals (Altan et al. 2003; Borges et al. 2004; Renaudeau et al. 2012; Lara and Rostagno 2013). In addition, Fragala et al. (2011) reported that, under exercise stress, glucocorticoids regulate muscle protein content by inhibiting muscle protein synthesis and stimulating protein degradation.

Previous studies were conducted to elucidate how heat stress depresses the immune function and growth performance in chickens (Bartlett and Smith 2003; Mashaly et al. 2004; Niu et al. 2009; Quinteiro-Filho et al. 2010; Lara and Rostagno 2013; Ohtsu et al. 2015). In the present study, the higher p-eEF2 level together with the lower protein synthesis in leukocytes of the HS broilers compared to controls may explain the global depression of the immune function and growth under heat stress. As shown in Fig. 3, the exposure of broilers during 21–42 days of age to chronic heat stress significantly suppressed all studied parameters of immune function. When compared to the control group, the plasma IgG and IgM concentrations were decreased by 47 and 40%, and the antibody titer against SRBC was decreased by 33% in the HS group, respectively. Bartlett and Smith (2003) observed that broilers subjected to heat stress had lower levels of total circulating antibodies, as well as lower specific IgM and IgG levels, both during primary and secondary humoral responses. These findings are in line with our results concerning both IgG and IgM concentrations. Moreover, our results of antibody response to SRBC are supported by those of Niu et al. (2009) who reported a reduced antibody response and phagocytic ability of macrophages in broilers under heat stress.

On the other hand, the low protein synthesis in leukocytes may directly induce the decreased lymphocyte proliferation in broilers exposed to chronic heat stress. Indirectly, it may be due to the higher plasma corticosterone concentration in the HS group compared to the control group, as was recently reported (Mehaisen et al. 2017). In addition, it was reported that exogenous corticosterone treatment increased the plasma corticosterone levels and negatively affected the lymphocyte proliferation (Shini et al. 2010). Particularly, it has been shown that lymphocytes exhibit receptors for many neuroendocrine products of the hypothalamic-pituitary-adrenal (HPA) axis, such as cortisol, which may affect cellular trafficking, proliferation, cytokine secretion, antibody production, and cytolytic activity (Lara and Rostagno 2013). The low number of lymphocytes in HS group (inducing high H/L ratio, as shown in Fig. 2a) may also be a reason of low lymphocyte proliferation in the HS group.

The low productive performance in HS broilers observed in the present study is expected and agrees with the negative effect of heat stress on broiler performance as previously reported (Temim et al. 2000; Niu et al. 2009; Vale et al. 2010; Purswell et al. 2012; Sohail et al. 2012; Quinteiro-Filho et al. 2012). Remarkably, the elongation phase of protein translation requires a substantial amount of metabolic energy (Browne and Proud 2002). Hence, the low feed consumption in HS broilers may be a reason of eEF2 phosphorylation and, in turn, the low protein synthesis in leukocytes during heat stress. These findings can easily explain the low body weight gain and the low immune function observed in HS broiler group in our study. Moreover, Leprivier et al. (2013) stated that the inhibition of protein elongation by eEF2 phosphorylation is one of the adaptation strategies that body can make during nutrient deprivation. In this context, the current study suggests that eEF2 phosphorylation is often the main pathway resulting in low protein synthesis in leukocytes, affecting the immune function of broilers under heat stress condition. The low performance of heat-stressed broilers could also be attributed to the covalent modification of proteins by the same pathway inducing an inhibition of eEF2 in other body tissues unlike leukocyte cells.

Conclusion

To our knowledge, this research introduced the first report that chronic heat stress in broiler chickens during 21–42 days of age significantly increases the phosphorylation level of eEF2 on Thr56 site and suppresses the protein synthesis in leukocytes. Also, the present study showed that the production performance was negatively affected. In addition to the high mortality rate in HS group, a substantial increase was also observed in stress indicators including plasma corticosterone level and H/L ratio. Moreover, heat-stressed broilers expressed lower immune function presented by a depressed antibody titer against SRBC, lowered immunoglobulin IgG and IgM concentrations in plasma, and reduced lymphocyte proliferation compared to control broilers. Collectively, these results indicate that heat stress in broilers decreases the growth rate and immune function, at least in part, via inhibition of protein synthesis. This appears to be regulated by the inhibition of elongation phase of protein translation process which may be directly induced by the eEF2 Thr56 phosphorylation pathway.